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a 6-year - old , 25 kg boy was scheduled for strabismus surgery in both eyes . neither the patient nor his family had any history of neuromuscular disease or a special family history . the preoperative vital signs were blood pressure : 90/50 mmhg , heart rate : 92 beats / min , respiratory rate : 24/min and axillary temperature : 36.4. the patient received ketamine 50 mg iv for sedation before induction in the waiting room . anesthesia was induced with sevoflurane 2.5 vol% by mask ventilation in a mixture of nitrous oxide and oxygen ( fio2 0.5 ) . about 2 min after injection of rocuronium bromide , a size 5.0 cuffed endotracheal tube was inserted without any difficulty under direct laryngoscopy . then 1 min after intubation , the heart rate of patient was increased from 160 to 195 beats / min . at first , the tachycardia was considered to be due to stimulation by the tracheal intubation , but the end tidal carbon dioxide concentration ( etco2 ) was concurrently increased from 35 to 65 mmhg within 5 min . the patient was hyperventilated with 100% o2 through a new anesthetic circuit , and consequently the etco2 was decreased to 45 mmhg . the patient was administered midazolam 1 mg and sufentanyl 15 g intravenously for sedation after discontinuing the sevoflurane and we started propofol infusion as maintenance because propofol is known to be a safe anesthetic agent in patients with mh . for decreasing the body temperature , active cooling was immediately initiated by ice water massage and applying ice packs on the chest , back and axillary area . arterial cannulation was done to continuously monitor the blood pressure and for the arterial blood gas analysis . this was done via external jugular cannulation for rapid infusion of cold iv fluid and central administration of drugs for resuscitation if it was need , and we placed a foley catheter for checking the hour urine output . the arterial blood gas analysis was ph : 7.294 , paco2 : 41.2 mmhg , pao2 : 435.2 mmhg and the base excess : -10.1 thirty minutes min after anesthetic induction , the patient showed an oral temperature of 38 , a pulse of 160 beats / min , a blood pressure of 140/70 and we detected newly occurring ventricular premature beats ( 4 - 5/min ) on the echocardiogram . but we did n't have dantrolene , so we requested dantrolene from the korea orphan drug center . approximately 90 min after the onset of mh , the patient recovered consciousness and the tracheal tube was extubated . the patient showed an oral temperature of 37.5 , a heart rate of 140 beats / min and a blood pressure of 140/80 mmhg , and he maintained the ventricular premature beats ( 4 - 5/min ) . the arterial blood gas analysis showed a ph of 7.390 , a paco2 of 38.0 mmhg , a pao2 of 89.5 mmhg and a base excess of -2.5 mm / l . thereafter , the arrhythmia disappeared , and the patient maintained an oral temperature of 36.5 - 37.4 and a normal blood pressure and heart rate . but about 2 h after the administration of dantrolene , the patient again showed arrhythmia ( ventricular premature beats ) and an increased oral temperature of 38 , a blood pressure of 130/100 mmhg and a heart rate of 130 beats / min . after the second administration of dantrolene , the arrhythmia , oral temperature , heart rate and blood pressure were all normalized , and the laboratory data and arterial blood gas analysis were normalized 2 days after anesthesia induction . the patient was discharged from the hospital four days after the onset of mh without any problem . many early signs of a mh episode can present in various ways and mh may be confused with other medical conditions such as an insufficient depth of anesthesia , hypoxia , hypercarbia , thyrotoxicosis , pheochromocytoma and neuroleptic malignant syndrome . a clinical grading scale helps to establish the likelihood of mh in specific problematic cases . it is based on weighted scores for muscle tone , muscle breakdown , acid - base parameters , temperature , tachycardia or other arrhythmias , and the response to dantrolene . we were able to diagnose mh on the basis of the clinical symptoms and the clinical grading scale by larach et al . . according to this clinical grading scale , when the raw score range is 35 - 48 , the mh rank is 5 and the likelihood of mh is high . the patient in this case received the score of 43 because the petco2 was > 55 mmhg with appropriately controlled ventilation ( 15 points ) , there was an inappropriately rapid increase in temperature ( 15 points ) , inappropriate sinus tachycardia ( 3 points ) and an arterial base excess more negative than -8 meq / l ( 10 points ) . in this case , we used ketamine , sevoflurane , n2o and rocuronium bromide during induction . ketamine and n2o are not triggers for mh [ 6 - 8 ] and rocuronium bromide , which is a non - depolarizing muscle relaxant , is also safe for mh . since shulman et al . first reported that sevoflurane triggers mh in mh - susceptible swine , there have been some reports of mh during sevoflurane anesthesia in human . generally , sevoflurane and desflurane have been reported to be less potent triggers , they produce a more gradual onset of mh and the onset of mh with sevoflurane in humans has been reported to occur both at an early period and after prolonged anesthesia . kinouchi et al . reported mh emerged about 30 minutes after anesthetic induction with sevoflurane in 4-year - old girl who was without a family history of mh and the preoperative laboratory studies did not suggest she was susceptible to mh . reported that mh emerged about 20 minutes after anesthetic induction with sevoflurane in a 56-year - old man who was undergoing cardiopulmonary bypass and moderate hypothermia . . reported that mh emerged about 150 minutes after general anesthesia with sevoflurane in a 24-year - old man who was undergoing bilateral sagittal split ramus osteotomy . as compared with the other case reports , our patient 's etco2 and body temperature were increased simultaneously within 5 minutes after anesthetic induction with sevoflurane . we know that the time of onset of a fulminant episode of mh is unpredictable , and it can vary from within minutes to within several hours of induction , and the fulminant mh episodes are apparently the result of a rapid , sustained rise in myoplasmic ca . however , we could not determine the reason for the rapid onset of mh in this case . during the preoperative evaluation , our attention was drawn to the patient ' strabismus , and strabismus has been observed in mh susceptible patients . however , neither the family history nor the preoperative laboratory studies suggested the patient was susceptible to mh the halothane and caffeine contracture tests are bioassays and they currently remain the most reliable indicators , but they are not generally used in our country , and so a diagnostic contracture test was not performed . this case report demonstrates that sevoflurane can trigger mh within few minutes after exposure and so the patients who are susceptible to mh must be carefully monitored even during anesthetic induction .

we experienced a case of malignant hyperthermia ( mh ) in 6-year - old boy during anesthesia induction for strabismus surgery . it has been generally reported that sevoflurane can induce the delayed onset of mh in the absence of succinylcholine . our case of mh was elicited after about 2 - 3 min of sevoflurane administration with n2o , o2 and rocuronium . however , we successfully treated the patient by early recognition of his condition and administering symptomatic treatment and dantrolene .

malaria has been man 's problem since the 14th century ( the era of the black death in europe ) , and now in the 21st century , its effect has again reached an alarming proportion . malaria remains a major threat to health and economic development of local communities and nations [ 2 , 3 ] . almost half of the world 's population is at risk of the disease [ 46 ] . africa south of the sahara records about 90% of the world 's all malaria deaths for two main reasons . the first is that infections are caused by plasmodium falciparum ( p. f. ) ( the most dangerous of the four human malaria parasites ) and the second is because the mosquito anopheles gambiae ( the most effective malaria vector and also the most difficult to control ) is most widespread in africa . malaria infection during pregnancy is a major public health problem in tropical and sub - tropical regions throughout the world . children under 5 of years of age and pregnant women are recognised by the world health organization as the vulnerable groups to malaria infection . the disease causes about 300500 million clinical cases and 1.5 to 2.7 million deaths worldwide each year ( who in ) even though there is a downward trend in these figures . the prevalence of malaria in africa is high with an estimated average prevalence of p. f. of 63% in west africa and 39% in eastern and southern africa . reports of outpatient visits due to malaria for some africa countries such as malawi , ur tanzania , uganda , and zambia vary from 18% in 1985 to 46% in 2000 while hospital admission due to malaria in these countries varies from 20% in 1985 to 60% in 2000 according to rbm . child mortality due to malaria seems to have been higher in eastern / southern africa countries than in west african countries between 1990 and 1998 . malaria accounts for 63% of the diseases reported in healthcare facilities across the six geopolitical zones of nigeria , while it accounts for 11% of maternal deaths and its prevalence among pregnant women was put at insecticide - treated nets ( itns ) are known to be highly effective in the reduction of morbidity and mortality in malaria rbm . in recent times , their usage in the african continent has been vigorously scaled up for coverage and in the introduction of long - lasting insecticide nets ( llins ) . itns have been scaled up in many african countries among which are tanzania , zambia , kenya , mali , and malawi ( rbm ) . in these countries , itns are usually distributed freely to parents of children and pregnant women ( the vulnerable groups ) while , in some countries , nigeria inclusive , they are made available at retail shops and/or are supplied at subsidised prices or under the voucher scheme [ 12 , 13 ] . rbm programme is put in place to assist endemic countries to conduct national needs assessments from which strategic plans could be developed . it also intended to serve as a forum to match country 's plans with international donors . in view of the complications of malaria in pregnancy , especially in this period of drug resistant malaria parasites , roll back malaria ( rbm ) programme was launched in 1998 with at least three new or refined tools to combat the disease . the tools are artemisinin - based combination therapies ( acts ) , insecticide - treated nets ( itns ) , and intermittent preventive treatment ( ipt ) for pregnant women . besides , other rbm measures used for malaria control are irs , larval control , quick diagnosis , administration of artemisinin - based combination therapies ( acts ) ( freely given in some countries for all age groups ) , and adoption of chloroquine , mefloquine , and sulphadoxine - pyrimethamine ( sp ) ( fansidar ) and primaquine . in spite of the efforts galvanised to combat malaria in africa , there is still a high burden of malaria of 20%40% with an average of 30% of all outpatient visits to the clinics in all african countries where malaria is endemic ( rbm ) . in many of these countries , the report indicated that between 20% and 50% of all hospital admissions are due to malaria . reports ( rbm ) indicate that less than 40% of malaria morbidity and mortality occur in formal health facilities which constitute a small fraction of the total burden but many of the reports do not include nongovernmental facilities like the faith clinics . by 2005 , it was expected that 60% of pregnant women in nigeria would sleep under an itn and 60% of pregnant women would receive ipt using sulphadoxine - pyrimethamine ( sp ) at least twice during anc . these targets were to gradually rise to a value between 75 and 80% by 2010 and coverage was sustained thereafter . a national malaria situation survey conducted in 2000 reported by indicated that preventive health behaviour in malaria in terms of net use among pregnant women in nigeria seems to be generally low in all the six geopolitical zones . also , reported a generally poor usage of nets among all categories of people in nigeria with only 20% of households owning nets , some of which are not necessarily itns , whereas observed a higher coverage of the usage of nets in urban areas . the need to further confirm current state of usage of rbm tools among this vulnerable group and the influence of location in this regard was crucial . in view of this , the present study had the impetus to investigate the acceptability and utilisation of rbm tools , their influencing factors , and the influence of location on the behaviour outcomes among pregnant women who access government and faith clinics in oyo state , nigeria , which seem to be usually sidelined by most health programmes initiated even when a sizeable number of pregnant women patronise them . what is the level of acceptance of intermittent preventive treatment ( ipt ) of malaria in pregnancy and itns by pregnant women who attend different health facilities in oyo state?is there a difference in acceptance of ipt and itns by pregnant women in terms of the health facilities accessed and location?to what extent are ipt and itns utilised by pregnant women who access different health facilities in oyo state?is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location?is there a relationship between acceptance and utilisation of ipt and itns by these pregnant women?what are the factors influencing ( i ) acceptance and ( ii ) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state ? what is the level of acceptance of intermittent preventive treatment ( ipt ) of malaria in pregnancy and itns by pregnant women who attend different health facilities in oyo state ? is there a difference in acceptance of ipt and itns by pregnant women in terms of the health facilities accessed and location ? to what extent are ipt and itns utilised by pregnant women who access different health facilities in oyo state ? is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location ? is there a relationship between acceptance and utilisation of ipt and itns by these pregnant women ? what are the factors influencing ( i ) acceptance and ( ii ) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state ? the study adopted the survey and causal comparative types of research as none of the variables were manipulated ; they were studied as they occurred and inferences made on the basis of the findings obtained . the levels of acceptance and utilisation of sulphadoxine - pyrimethamine ( fansidar ) , the drug of choice for intermittent preventive treatment ( ipt ) in pregnancy , and insecticide - treated nets ( itns ) between government clinics and faith clinics or mission homes were compared . oyo state is located in the southwest geopolitical zone of nigeria and is one of the malaria endemic areas of the country . the disease afflicts children under five and pregnant women two of the most vulnerable groups resulting in high morbidity and mortality in the state . for instance , data from puts the trend of malaria prevalence in the state from a total of 358,780 cases in 2006 to 403,468 in 2009 . the study population consisted of all pregnant women in oyo state , nigeria , who were attending antenatal ( anc ) clinics at both the primary healthcare centres ( phc ) and faith clinics , respectively , in 2009 - 2010 when the study was conducted . the 33 local government areas in the state were stratified into urban and rural composition and purposive sampling technique was employed to select three local government areas , respectively , from both urban and rural locations to take care of the location of the faith clinics / mission homes which were dominant in urban locations . all the phc clinics and mission homes ( faith clinics ) in the selected local government areas were used for the study , while purposive sampling technique was used to select a total of 650 pregnant women but 582 who accessed government clinics and 50 who accessed faith clinics in these lgas who had complete information on them formed the final sample . a structured questionnaire that was valid and reliable pregnant women acceptance and utilisation questionnaire ( pwauq , = 0.81)used for data collection was administered once to the participants . pwauq consisted of background information about the pregnant women and their spouses ' occupations and educational levels , locations , and number of pregnancies and their outcomes among others . the instruments on acceptance of rbm tools , utilisation of itns , and factors influencing acceptance and utilisation were closed - ended questions administered once to the participants . data collection was carried out by the researchers with the assistance of fourteen trained assistants ( six rbm programme managers in the six lgas and eight others trained for the purpose of data collection ) . the researchers explained the purpose of the research work to all pregnant women that were present including the health workers at each phc or faith clinic or mission home visited . pregnant women in rural locations who had low educational qualification filled their questionnaire through the assistants who interpreted the contents to them in the local language yoruba without influencing their responses . the data obtained were coded and analysed using the statistical package for the social sciences ( spss ) version 20 . positive responses were coded with one ( 1 ) while negative responses were coded with zero ( 0 ) . percentages were computed for the responses relating to levels of acceptance and utilisation of rbm tools . inferential statistics such as t - test and analysis of variance ( anova ) were computed to determine significant levels of acceptance and utilisation of rbm tools between pregnant women who accessed government and faith - based health facilities and those in urban and rural locations . pearson product moment correction was used to analyse the relationship between acceptance and utilisation of rbm tools . multiple regressions were computed to establish the factors that influenced the acceptance and utilisation of the rbm tools . what is the level of acceptance of intermittent preventive treatment ( ipt ) of malaria in pregnancy and itns by pregnant women attending different health facilities in oyo state ? what is the level of acceptance of intermittent preventive treatment ( ipt ) of malaria in pregnancy and itns by pregnant women attending different health facilities in oyo state ? pregnant women who accessed government clinics ( 49.8% ) tended to accept itns even though 72% of them claimed to like sleeping under a mosquito net , while 52.2% claimed they a total of 28% and 30% of pregnant women who accessed faith clinics reported that they accepted these rbm tools , respectively , though 72% of them indicated they like sleeping under a mosquito net and 64% react to anti - malarial drugs ( table 1).(2)is there a difference in the acceptance of ipt and itns by pregnant women in terms of the health facility accessed and location ? is there a difference in the acceptance of ipt and itns by pregnant women in terms of the health facility accessed and location ? there was a significant difference in acceptance of ipt and itns by pregnant women in the two health facilities in table 2 , t(630 ) = 3.51 , p 0.05 , and t(630 ) = 4.15 , p 0.05 , respectively . pregnant women in government clinics with m = 4.74 , sd = 1.08 and m = 4.66 , sd = 1.15 seem to accept ipt and itns compared to their counterparts in faith clinics , mean = 4.16 , sd = 1.59 ; m = 3.96 , sd = 0.93 . the effect sizes ( cohen 's d ) of 0.14 or 14% and 0.16 or 16% , respectively , were obtained for the variance in acceptance in the two health facilities . significant influence of location was observed on the extent to which pregnant women in government health facility only accepted ipt ( table 3 ) . pregnant women in rural locations with mean 4.92 , sd = 1.00 were better in the acceptance of ipt than those in the urban areas , mean 4.55 , sd = 1.13 , t(580 ) = 4.12 , p 0.05 , with an effect size ( cohen 's d ) of 0.17 or 17% . the influence of location on acceptance of itns was not significant , t(580 ) = 0.58 , p 0.05 . faith clinics were not found in rural locations.(3 ) to what extent are ipt and itns utilised by pregnant women attending different health facilities in oyo state ? to what extent are ipt and itns utilised by pregnant women attending different health facilities in oyo state ? pregnant women who accessed government clinics ( 60.8% ) utilised itns in spite of the claim that 62.9% reported sleeping under an itn before their current pregnancy and 73.2% claimed it is comfortable sleeping under an itn . among pregnant women who accessed faith clinics , 18% utilise itns even though 48% claimed they find sleeping under an itn comfortable . more women ( 66.8% ) in government clinics than their counterparts in faith clinics ( 38.0% ) used ipt to the expected two doses . as many as 79% of the pregnant women in government clinics and 62% in faith clinics indicated their willingness to use ipt in their subsequent pregnancies ( table 4).(4 ) is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location ? is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location ? a significant difference in the utilisation of ipt and itns as presented in table 5 was observed between these women , t(630 ) = 5.81 , p 0.05 , and t(630 ) = 3.99 , p 0.05 . pregnant women in government clinics utilised ipt and itns more than those who accessed faith clinics . the effect sizes ( cohen 's d ) of 0.23 or 23% and 0.16 or 16% , respectively , were obtained for the variance in the utilisation of ipt and itns , respectively . a significant difference in the utilisation of ipt by the pregnant women who accessed government clinics by location , t(580 ) = 641 , p 0.05 , was observed with an effect size of 0.26 or 26% but the itns utilisation was not significant , t(580 ) = 1.170 , p > 0.05 , with an effect size of 0.05 or 4.86% ( table 6 ) . faith clinics were not found in rural locations.(5 ) is there a relationship between acceptance and utilisation of rbm tools by these pregnant women ? is there a relationship between acceptance and utilisation of rbm tools by these pregnant women ? as presented in table 7 , the utilisation of rbm tools which was found to be significantly related with the acceptance of rbm tools among pregnant women in the state government clinics ( r = 0.398 , n = 582 , p 0.05 ; r = 0.16 ) and pregnant women in faith clinics ( r = 0.379 , p 0.05 ; r = 0.14 ) . this explained 16% and 14% of the variation between acceptance and utilisation among these women , respectively.(6 ) what are the factors influencing ( i ) acceptance and ( ii ) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state ? what are the factors influencing ( i ) acceptance and ( ii ) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state ? the multiple regression model summary of the nine influencing variables ( table 8) significantly explained and predicted the acceptance of rbm tools among pregnant women in government clinics ( f(9,572 ) = 6.320 , p 0.05 ) but not in faith clinics . the variables accounted for 8% of variance in the pregnant women 's acceptance of rbm tools whereas other variables not investigated accounted for the remaining 92% . with respect to the t - test results in relation to the significant multiple regression coefficient obtained , the nine variables significantly predicted the acceptance of rbm tools through number of pregnancies ( t = 5.172 , = 0.217 ) . four other variables , educational qualification of the pregnant woman ( t = 2.871 , = 0.120 ) , marital status ( t = 2.928 , = 0.117 ) , age of pregnant woman ( t = 2.805 ; = 0.115 ) , and husband 's occupation ( t = 0.2212 , = 0.093 ) , also contributed significantly but inversely to the acceptance of rbm tools ( table 8) . the multiple regression of the nine influencing independent variables of the utilisation significantly explained and predicted the utilisation of rbm tools among pregnant women in government clinics ( f(9,572 ) = 3.607 , p 0.05 ) but not in faith clinics ( table 9 ) . the nine variables accounted for 3.9% variance in the utilisation of rbm tool whereas other variables not investigated accounted for the remaining 96.1% . concerning the t - test results in relation to the significant effect of the multiple regression coefficient obtained , the nine variables significantly predicted the utilisation of rbm tools by pregnant women in government clinics through the number of pregnancies had ( t = 2.818 , = 0.121 ) . marital status and educational qualification of the pregnant women also contributed significantly but inversely to the utilisation of rbm tools among these women ( t = 3.248 , = 0.132 and t = 2.496 , = 0.106 , resp . ) . the findings with respect to the acceptance of rbm tools ( ipt and itns ) by pregnant women indicated that pregnant women who accessed government clinics accepted these rbm tools better than those who accessed faith clinics . between 50% and 72% of the pregnant women who accessed government clinics accepted ipt and itns whereas between 30% and 72% of pregnant women who accessed faith clinics accepted these rbm tools . the overall acceptance by these women ( even when pregnant women who accessed faith clinics claimed they engage in dual registration ) could be as a result of adequate enlightenment on the knowledge of rbm programme provided to the pregnant women in government clinics through health education at anc visits , which is a regular practice of primary healthcare in the clinics . the targets of over 82% in the use of rbm tools by pregnant women were set by the african heads of state to be achieved by year 2010 . the observed trend in this study the level of education of an individual generally dichotomises one into an ignorant or informed person . according to , health education helps people understand health and how their behaviour may affect their health . since such education encourages people to make their own choices for a healthy life , it will ensure the success of the prevention , treatment , and control of malaria programme put in place in order to reduce to the barest minimum maternal and child morbidity and mortality , in the state . other findings in this study indicate that health facility accessed by the pregnant women significantly influenced acceptance : government clinics : t(630 ) = 3.51 , p 0.05 ; faith clinics : t(630 ) = 4.15 , p 0.05 . acceptance was also location based as women in the rural locations significantly accepted ipt in pregnancy compared to those in urban locations ( t = 4.12 , p 0.05 ) but location did not significantly influence the acceptance of itns for this group of women . this implied that rbm programme in oyo state is a healthcare focused programme geared towards the rural people through the primary healthcare . thus , the rural dwellers see rbm programme as their own programme which they embrace wholeheartedly more than the urban dwellers . personal experiences of the researchers show that dwellers in the urban areas take malaria for granted and probably see it as a common disease occurrence . findings indicate a modest level of the utilisation of ipt and itns by the pregnant women . more of the pregnant women in government clinics ( between 58% and 73% ) utilised ipt and 67.2% used itns whereas between 18% and 60% and 38.0% of those who accessed faith clinics used these tools , respectively . the observed difference could be attributed to the religious beliefs and practices of the women in faith clinics who do not believe in the use of drugs even though they engage in dual registration . the study of finds support in respect of this finding . although researchers like revealed that factors such as availability of nets , cost of nets , and inadequate information on where to obtain the nets and the attitude of users towards the use of net had great influence on the poor and improper utilisation of nets ( itns ) , the findings of this present study seem to have indicated a considerable improvement in the utilisation of itns among the pregnant women . findings differed markedly from the findings of that reported less than 10% usage of nets among pregnant women and children in uganda but are in consonance with the study which reported 58% usage of itns among women attending anc and delivery units in burkina faso and that reported a variation between 32% and 69% usage of itns among pregnant women in six african countries , nigeria inclusive . findings equally find support in the study of which found that itns use in niger improved after a nationwide integrated campaign . the influence of location on the utilisation of rbm tools by the pregnant women in this study was important as pregnant women in rural locations who accessed government clinics utilised ipt compared to those in urban areas , significant at p 0.05 . in this regard , findings in nigeria [ 22 , 23 ] which corroborated are in support of the fact that the use of nets was very variable among urban and rural communities in oyo and ekiti states , respectively . a national malaria situation survey conducted in 2000 reported by also indicated that net use among pregnant women in nigeria seems to be generally low in all the six geopolitical zones . it also indicated a generally poor usage of nets among all categories of people in nigeria with only 20% of households owning nets , some of which are not necessarily itns , whereas observed a higher coverage of the usage of nets in urban areas . there is therefore need to further reach pregnant women in urban locations to improve their attitude to ipt and itns utilisation as well as those who access faith clinics . in addition , a positive but low correlation seems to exist between acceptance and utilisation of ipt and itns among pregnant women who accessed government clinics ( r = 0.398 ) accounting for 16% variance in utilisation compared to those who accessed faith clinics ( r = 0.379 ) accounting for 14% variance in utilisation . this implied that , all things being equal , high acceptance implies reliable utilisation of ipt and other rbm programme packages . the acceptability of and receptiveness to rbm tools by some of the pregnant women could have facilitated their utilisation of the tools with ease . the implication of this finding is that any effective public enlightenment and sensitisation carried out on rbm programme and tools will make the right impact on reducing the menace of malaria on pregnant women irrespective of the health facility accessed . from the findings in this study , pregnant women in the state have a high acceptance of rbm tools . it appears , however , that the acceptance of these tools by pregnant women who accessed government clinics depended on factors such as number of pregnancies had ( = 0.217 ) which significantly and positively influenced these women 's acceptance of these tools , but educational qualification of the pregnant woman ( = 0.120 ) , marital status ( = 0.117 ) , age of the pregnant women ( = 0.115 ) , and husband 's occupation ( = 0.093 ) , though significant , inversely influenced acceptance . what these findings imply is that as the age increased or educational qualification and marital status or husbands ' occupations of these women improved , their acceptance of rbm tools became low . on the issue of the utilisation of the tools by pregnant women who accessed government clinics , the findings revealed that utilisation was influenced by such factors as the number of pregnancies had ( = 0.121 ) which contributed significantly and positively to utilisation and marital status ( = 0.132 ) and educational qualification of the pregnant women ( = 0.106 ) which contributed significantly and inversely to the utilisation of rbm tools among these women . these variables did not influence both the acceptance and utilisation of pregnant women in faith clinics . three variables . number of pregnancies , educational qualification of the pregnant women , and marital status which were observed to significantly and consistently influence the acceptance and utilisation of rbm tools in this study seem to be contiguously related to one another and portend strong indications that the observed finding about these women has grave implications for their purchasing powers . precisely , decisions about spending money are predominantly and traditionally made by men in many african cultures . also , a large number of the pregnant women who participated in the study were housewives who were with no adequate means of livelihood of their own and who had to depend on their husbands ' incomes to be able to purchase the nets and the drugs . some of the women may , therefore , not be able to make independent financial decisions since they do not have much income of their own and as such , their acceptance and utilisation of these tools could have been influenced not only by the number of pregnancies had , but also by marital status and husbands ' occupations . thus , the spouse and the nature of occupation are central to the women 's responses to these tools . findings also corroborate pulford , herzel , bryant , siba , and mueller in who reported that lack of affordability is an important barrier to ownership of itns . with this kind of findings , the attainment of the two main objectives of rbm programme which are reducing the burden of malaria particularly in the two most vulnerable groups ( pregnant women and children under 5 years of age ) and contributing to the positive health and socio - economic development of the nation is feasible and possible , all things being equal . among the pregnant women who accessed faith clinics , the findings indicated that all the nine variables did not predict their acceptance and utilisation of the rbm tools primarily because these women are not necessarily inclined to accept or utilise these tools due to their religious beliefs and practices . the study established the current status of the acceptance and utilisation of rbm programme tools ( itns and sp in pregnancy ) among pregnant women who accessed two models of healthcare facilities in oyo state from 2001 to 2009 , an initiative implemented with the purpose of stemming of malaria - related morbidity and mortality among these pregnant women and their unborn children . though the acceptance and utilisation of rbm tools were higher among pregnant women who accessed government phc clinics and in rural areas than among pregnant women who accessed faith clinics and in urban areas , these were far from meeting the set targets . pregnant women 's marital status and husbands ' occupations significantly and statistically influenced acceptance while husbands ' occupations influenced the utilisation of rbm tools implying that spouses ' statuses play significant roles in achieving set targets . we recommend that intensive effort should be made by healthcare providers to encourage especially more of the educated urban women to utilise rbm programme tools , especially the itns . rbm programme should not focus only on the utilisation of rbm tools but the scope should be expanded to include information dissemination on behavioural change towards malaria and its consequences . malaria consumables such as insecticide - treated nets ( itns ) and medications such as sulphadoxine - pyrimethamine should be made available at the various health centres and given freely . they should also be available at the open chemist stores at cheap and affordable prices to the average person .

the study is an investigation of the acceptance and utilisation of sulphadoxine - pyrimethamine ( fansidar ) , the drug of choice for intermittent preventive treatment in pregnancy , and insecticide - treated nets among pregnant women who access different health facilities in oyo state , nigeria . pregnant women ( 582 ) attending government primary healthcare antenatal clinics and 50 attending faith clinics purposively selected responded to structured instruments that were analysed using percentages , t - test correlation , and multiple regression . acceptance and utilisation of rbm tools were higher in government clinics than faith clinics and in rural areas . pregnant women in government clinics , 60.8% and 66.8% , and faith clinics , 18% and 38.0% , utilised roll back malaria tools , significant at t(630 ) = 5.81 , p 0.05 , and t(630 ) = 3.99 , p 0.05 , respectively . pregnant women in rural locations who accessed government clinics utilised roll back malaria tools more than those in urban areas , t(580 ) = 641 , p 0.05 . number of pregnancies , educational qualification of the pregnant women , and marital status significantly and consistently influenced acceptance and utilisation of these tools . to ensure that set targets are met , the utilization of rbm tools among the two categories of pregnant women can be improved by increasing the supply of the tools and ensuring that treatment is free .

western health services are characterized by increasing demands for effectiveness , production , and financial profit and economizing . this has an impact on professional standards and clinical nursing values and constitutes a source of job - related stress and unrest for the nurses . norwegian studies from hospitals and local health services show that lack of resources and heavy work pressure lead to ethically difficult prioritizations and a lowering of nursing standards . european studies demonstrate that professional ethical codes become unattainable ideals for many nurses due to the lack of resources and heightened effectiveness in the healthcare environment . lack of resources and the lowering of priorities cause considerable strain on healthcare professionals both in hospitals and the local health services . this strain can be understood as moral distress , a concept associated with the ethical dimension in practice and with issues related to difficulties in practice in terms of maintaining professional values , responsibility , and duties [ 711 ] . moral distress , even though it is understood differently in different studies , has shown to have negative consequences and contributes to emotional discomfort , such as , for example , anger , frustration , withdrawal , insecurity or poor quality in patient care , reduced job satisfaction , and occupational fatigue [ 1115 ] . we took our point of departure in the national core values in the norwegian specialist health services : respect , quality , and safety . the norwegian national core values for the specialist health services were introduced with the health reform in 2002 . we wanted to explore whether these values after ten years are present and of importance in nurses ' daily practice and how this affects the nurses ' daily practice and professional wellbeing . the object of this study was to examine nurses ' experience of how ethical values are expressed in daily practice in a norwegian hospital where health reforms have been introduced as well as market thinking . the collection of data was carried out by conducting focus group interviews with a total of 20 nurses from different somatic and psychiatric bed units in a medium large hospital in norway . the ages of the participants were from 27 to 60 years , with 2 to 38 years of experience as nurses , the majority around 20 years . the request for participants for the study was sent to the administration in each clinic , and the participants were then selected and asked by their immediate superior if they were willing to take part in the study . the selection of participants was convenient as the request to participate was made to all the nurses in the units without any criteria due to , for example , gender or work experience . focus groups are a qualitative method where a group of people with certain common qualities are gathered together to discuss a given , well - defined topic , in permissive and nonthreatening surroundings . focus groups should be informal and the questions should be direct , comfortable , and simple but not guiding . one of the foremost advantages of focus groups is the interaction between the participants which may generate more profound and richer data than individual interviews . three interviews were conducted with nurses from different somatic units and one interview with nurses from different psychiatric units . after three interviews in the somatic units , we reached a point of saturation in the data and decided to conduct a last interview with participants from psychiatric units to look for comparable results . the second author was moderator during three of the interviews , and this provided both continuity and experience in the moderator role . the interviews lasted 90 minutes , they were tape recorded , and ongoing notes were taken by the assistant moderator . the focus groups took place in the hospital 's meeting room during the participants ' working hours . a semistructured topic guide ensured that the same key topic was under discussion in the different focus groups . the topic guide 's starting point was the national core values in the norwegian specialist health service : quality , safety , and respect , and how these values were expressed or challenged in everyday work . other topics were whether the core values or other ethical values functioned as guidance in daily practice and whether reflection about ethics and values took place in the daily work . the aim of the study was to explore attitudes , thoughts , feelings , experiences , and knowledge among nurses about core values and other ethical values . the method must be adapted to the research question and thus is a qualitative method chosen [ 19 , 20 ] . the main key to content analysis is that the words in the text are classified into smaller content categories . we experienced that the participants were greatly committed and candid in the focus groups , with the result that the material obtained was highly informative and comprehensive . in the analysis we emphasized the findings that generated most engagement and discussion among the participants . the analysis started after each interview with listening to the tape and making a verbatim transcription of each interview . then the results from the three interviews from the somatic units were considered across and analyzed as a whole . the interview from the psychiatric units was separately analyzed similarly to the three others , and one looked for comparable results . we chose to consider the manifest content , that is , what the text says [ 21 , 22 ] . the next step was to find meaning units , condense , and then encode them . the codes were analyzed and systematized , and five main categories appeared , and the codes were sorted into these main categories . ( 2 ) the system . ( 3 ) management . ( 4 ) patient . ( 5 ) nursing . the codes and the content in all the main categories were analyzed , condensed , and placed into subcategories , and a brief abstract was written summarizing the main content from the different subcategories . these abstracts were considered across , and further meaning compacting was done in order to systematize and further abbreviate the text . in this process we identified two main themes containing and presenting the results : values and reflection are important for the nurses , time pressure and nursing frustrations . values and reflection are important for the nurses , time pressure and nursing frustrations . the validity , or authenticity and trustworthiness of a study , is seen in two dimensions , internally and externally . this enabled us during the entire process to discuss and verify perceptions and conclusions which enhanced the internal validity of the study . the study was conducted with a homogeneous group , nurses , and special nurses without leader responsibility and with daily patient contact . if the study had been carried out with nurse managers in the group , the results might have been different . the study 's preliminary findings have been presented for experienced nurses and other researchers who found the results recognizable . the external validity of the study could have been strengthened if corresponding studies had been carried out in several hospitals . the study was carried out in a medium sized hospital in norway with nurses from different somatic and psychiatric units . it represents diversity both in terms of fields of study and nurses ' previous experiences and can thus be transferable to other nurses in other hospitals . the project has been reported to the norwegian social science data services ( nsd ) . the participants received verbal and written information about the study and agreed in writing to voluntary participate . as earlier described , two main themes were identified in the analysis , and the results are presented with sub - themes under each main theme : values and reflection are important for the nurses , time pressure and nursing frustrations in daily work . values and reflection are important for the nurses , time pressure and nursing frustrations in daily work . the nurses from all units describe how ethics , values , and reflection on values are of great importance for the quality in nursing . the informants see the need for values that are formulated in writing and the need for specific aims for their workplace . yet they feel that the values are solidly rooted even though they are not formulated in writing . the informants from somatic units say that they are aware of when the values are absent in practice and that it is their task to speak up about undignified conditions.it would have been fine if it was like that , that we all went along the same road with values at the bottom . i feel , and can see it among my colleagues , that these values are solidly rooted . it has surely to do with the training and what one sort of has learnti would say that there is often more focus on the hustle and bustle and lack of space , so one would like to be reminded of the values . one needs it even though it lies deeply in us it would have been fine if it was like that , that we all went along the same road with values at the bottom . i feel , and can see it among my colleagues , that these values are solidly rooted . it has surely to do with the training and what one sort of has learnt i would say that there is often more focus on the hustle and bustle and lack of space , so one would like to be reminded of the values . the informants say that being nurses they need to have basic values and that what is most important is to see the patient and to treat him with respect . the informants say that their work is based on the basic nursing values learned in their training and from colleagues with good values . the nurses in somatic units experience that values are repressed and may be neglected in their daily work due to time pressure , lack of resources , and great challenges . nurses from somatic units prioritize helping patients in grave and acutely life - threatening conditions . with limited resources , this can result in undignified conditions for other patients whose illnesses are not life - threatening . these patients , who , for example , have to wait a long time without anyone keeping an eye on them , are given insufficient or unsatisfactory nursing . in these cases , the nurses feel that the job they are doing is not good enough . these values vanish into thin air when it is a matter of whether the patient breathes or does not breathe , and so the prioritizing becomes tough and at that point i can not exactly manage to see these values , well , and even if they are there in the tiny moment when you are with the patient , i do believe that there are many of us who think that we could have done a better job it has often to do with resources , unfortunately . at times these values vanish into thin air when it is a matter of whether the patient breathes or does not breathe , and so the prioritizing becomes tough and at that point i can not exactly manage to see these values , well , and even if they are there in the tiny moment when you are with the patient , i do believe that there are many of us who think that we could have done a better job the quality varies . the nurses in the somatic units experience the quality in nursing as varying on account of lacking resources and time pressure in their daily work . the informants state that the quality is good when patients ' basic needs are taken care of , when they do not have to lie in the corridor but are given a room and when one has time to talk with them and their families . if the nurse finds time to do this little extra for the patient and does not have to run from room to room , she will feel that she has done a good job which will make her feel good inside . the informants from the psychiatric units report that good quality in nursing depends on evidence - based knowledge and a flexible and individually adjusted program for psychiatric treatment , with client participation . the informants tell us that the quality is totally dependent on the composition of the staff and that experienced nurses can help to create an atmosphere of quiet and security in the milieu and also , for example , in a coerced treatment situation.it is about offering flexible psychiatric treatment . that 's what we are preoccupied with , the fact that no one is alike that 's what we are preoccupied with , the fact that no one is alike the informants from somatic units inform us that they will go to great lengths for quality and that they feel it is important to adapt nursing measures to the patient 's needs rather than focusing on procedure . nurses from all the units emphasize that procedures can not be followed in all situations and that one can not produce recipes or procedures for interpersonal relationships.it has to do with interpersonal relationships . we can not write it , provide a recipe or a procedure to be followed in all situations . because things can really go wrong then , since every situation is unique and there are so many facets in what we do . experience and basic values and ethical principles are required , and good child rearing , that is always welcome it has to do with interpersonal relationships . we can not write it , provide a recipe or a procedure to be followed in all situations . because things can really go wrong then , since every situation is unique and there are so many facets in what we do . experience and basic values and ethical principles are required , and good child rearing , that is always welcome safety is not always there . respect , genuine presence , and good communication with patients and relatives generate safety and is described as decisive for quality as a whole . at times the informants from the somatic units experience insecurity and lack of control in their work situation . they feel that it all is haphazard and that they have to rely on sheer luck when there is a lack of both time and resources.i'm thinking a little about it in terms of safety and procedures . one can of course say that when they are busy developing it , it is a form of safety , but it happens quite often that when i am working i feel that things are not safe . when it is too much for you to cope when there are and there can be long periods between each time you are with a patient , so there is not much safety to speak about one can of course say that when they are busy developing it , it is a form of safety , but it happens quite often that when i am working i feel that things are not safe . when it is too much for you to cope when there are so many you should give care to . and there can be long periods between each time you are with a patient , so there is not much safety to speak about in psychiatry , safety was described as a safe workplace which will not be closed down , where there is enough staff , and the technical and administrative systems are functioning . a candid and honest environment where nothing is too stupid to say among colleagues all the nurses confirm that professional and ethical reflection is important for nursing professionals , but in the somatic units formal arenas and time for reflection are lacking . the informants in the somatic units often have informal talks about reflection with other nurses and physicians in the course of their workday but are continually interrupted because things are hectic and one certainly can not stop the running of the ward.well , the things that we know would be good for the quality we do not get done in practice because one has to keep things going . we haven't got the time to stop and even if the hospital maybe has taken a patient 's life , we do not have time for reflection and certainly not together with a surgeon well , the things that we know would be good for the quality we do not get done in practice because one has to keep things going . we haven't got the time to stop and even if the hospital maybe has taken a patient 's life , we do not have time for reflection and certainly not together with a surgeon only two of the informants from somatic units have been offered clinical nursing supervision . the nurses from the somatic units have requested clinical nursing supervision , but regular offers have neither been made nor has there been a tradition for it . the nurses say that one can ventilate ideas , get feedback , and have one 's values with you to the supervision , and this they believe may turn you a better nurse.i'm in clinical nursing supervision and that is very useful since there are many cases that touch one deeply . it is mostly a place for letting it all come out , and then you get feedback from the others about what they are thinking , because they too have often been in the same situation i 'm in clinical nursing supervision and that is very useful since there are many cases that touch one deeply . it is mostly a place for letting it all come out , and then you get feedback from the others about what they are thinking , because they too have often been in the same situation the informants from the psychiatric units had experienced formal clinical nursing supervision . in addition , they described a culture and an organizing of the daily work which provided adequate time for reflection and supervision . the nurses from the psychiatric units say that it is important to be observant and available to new staff who need clinical supervision and that clinical supervision has been important and essential for their own choice to continue in this profession.i am thinking about the report between the shifts , well , it becomes partly a kind of clinical supervision plus that when one is at work one keeps the discussions going well , it becomes partly a kind of clinical supervision plus that when one is at work one keeps the discussions going the nurses from the psychiatric units describe a reflective workday with discussions about right or wrong , values , and reflection on values in most situations . the nurses from the psychiatric units confront many ethical dilemmas and say that it is important and necessary that several people take part in these discussions.i do not think we say that today we are evaluating values in our unit , but it goes on all the time . a lot of it in the staff room , but also at morning meetings , team meetings , where things are taken up and discussed whether this is right , or should we do it differently i do not think we say that today we are evaluating values in our unit , but it goes on all the time . a lot of it in the staff room , but also at morning meetings , team meetings , where things are taken up and discussed whether this is right , or should we do it differently constant guilty conscience . the informants from the somatic units are often feeling frustrated and worn out on account of considerable work and time pressure . they say they always have to go to greater lengths and run faster and that this work pressure gives them a feeling of mass production and of having to calm things down in their daily work which they feel are both unsatisfactory and tiring.the pressure is huge , and there is quite simply too much to do . one does what one can , but it is perhaps not quite what one actually wants to do or manages to do . and one has a constant bad conscience , well , one feels that one should have done more and that 's a strain the pressure is huge , and there is quite simply too much to do . one does what one can , but it is perhaps not quite what one actually wants to do or manages to do . and one has a constant bad conscience , well , one feels that one should have done more and that 's a strain rock - hard priorities . some of the informants from the somatic units fear that they might turn into indifferent and superficial nurses because of the frequent rock - hard priorities given to basic needs in their daily work that as much as possible should be done in the shortest time . the great number of corridor patients also contributes to the fact that patients have to wait for a long time before the nurses can find time for them.in my opinion there is rock - hard prioritizing in our daily work . first of all , one must prioritize the things that are most important for the patient , be it shaving , taking a shower or medicine , but then it has also to do with this great number of corridor patients , the corridors have actually been filled up , and then there are patients with newly discovered cancer diagnoses who have to lie out there in the corridor . one does not feel like going home and knowing that this is where she will be lying tonight , and she is 50 years old and has pulmonary cancer . even if the patient seems to feel alright about it , it is clear that she only does so because she has cancer after all and that is much worse than lying in the corridorthe ethical values are actually challenged according to the way our employer organizes us within the existing staff resources . when there is sickness during the day one must n't call anyone for the first one who is sick , but we are then supposed to go to greater lengths , run faster , we have to be everywhere at once and that is at the expense of safety and quality and care and in my opinion there is rock - hard prioritizing in our daily work . first of all , one must prioritize the things that are most important for the patient , be it shaving , taking a shower or medicine , but then it has also to do with this great number of corridor patients , the corridors have actually been filled up , and then there are patients with newly discovered cancer diagnoses who have to lie out there in the corridor . one does not feel like going home and knowing that this is where she will be lying tonight , and she is 50 years old and has pulmonary cancer . even if the patient seems to feel alright about it , it is clear that she only does so because she has cancer after all and that is much worse than lying in the corridor the ethical values are actually challenged according to the way our employer organizes us within the existing staff resources . when there is sickness during the day one must n't call anyone for the first one who is sick , but we are then supposed to go to greater lengths , run faster , we have to be everywhere at once and that is at the expense of safety and quality and care and the informants from the surgical units report that patients with cancer or chronic wounds have to wait because the picking up and bringing of patients to surgery are prioritized , and this is experienced by nurses as an ethically difficult prioritizing . the informants from the somatic units speak about the great gap between theory and practice , and they feel frustrated when reality is so far from the ideals they were taught in their training . the nurses also state that these conditions affect their professional pride and that they are discouraged and feel that they barely can keep their heads above water.i feel like taking care of the poor person who was admitted to our hospital six months ago and is getting more and more depressed . at the same time . you must pick up someone from post - op . all the time , while you are standing there confused , you actually want to go to that patient but you did get that phone - call . i can never manage to do what i ought to do with the cancer patients . they see all the names on the list but they do not quite understand what it feels like to be in the centre of the anthill i feel like taking care of the poor person who was admitted to our hospital six months ago and is getting more and more depressed . at the same time you have this pressure all the time . you must pick up someone from post - op . all the time , while you are standing there confused , you actually want to go to that patient but you did get that phone - call . i can never manage to do what i ought to do with the cancer patients . they see all the names on the list but they do not quite understand what it feels like to be in the centre of the anthill when time pressure became the topic in the psychiatric units , the informants pointed out that in psychiatry there should be sufficient time . in psychiatry , the nurses claim that a possible demand from the management for greater efficiency is not consistent with the way they are working.it is all right to be busy , but never so busy that you feel that you are now losing your grip on someone . on the whole we need that in psychiatry because we can not go fast here , that is obvious . it is obvious that if the management comes with demands for far greater efficiency , then it will go all wrong , there will be a collision it is all right to be busy , but never so busy that you feel that you are now losing your grip on someone we need that in psychiatry because we can not go fast here , that is obvious . it is obvious that if the management comes with demands for far greater efficiency , then it will go all wrong , there will be a collision they feel that they do not have a say in decisions , are not being heard , and know that they would have much to contribute with if they were permitted to do so.we would have welcomed being permitted to take some part in the decisions . because it feels like we have no influence on anything , it would have been absolutely fantastic if we were permitted to take part and speak up about some matters . i do not believe they are aware of what we are doing during the day . i feel that there wo n't be much quality in the long run , i really do we would have welcomed being permitted to take some part in the decisions . because it feels like we have no influence on anything , it would have been absolutely fantastic if we were permitted to take part and speak up about some matters . i do not believe they are aware of what we are doing during the day . i feel that there wo n't be much quality in the long run , i really do the nurses from somatic units describe how the management does not set aside sufficient time for talking about values or focusing on them , for example , in staff meetings . the leaders are especially responsible for creating targets in their units and what should be focused on but as said before , we are all responsible for supporting and complying with it the leaders are especially responsible for creating targets in their units and what should be focused on but as said before , we are all responsible for supporting and complying with it the system can not be changed . the informants from the somatic units place the responsibility for present conditions on the system without defining more specifically who or what the system is . the nurses say that , when the system does not function , it is to the patient 's detriment . the system and its financial limitations are stated as the reason why ethical values are challenged by structural issues . one has the impression that the system is almost impossible to change or do anything about.we talk about quality and about what we can do about it , like with procedures , and deviations , but there is a lot in this system that has to do with quality that we can not do anything about . . we could certainly have sat in a group discussing values , how to put them to best use , but a lot has to do with having more staff in order to be able to include quality and safety in our work , and respect that we hopefully have in any case we talk about quality and about what we can do about it , like with procedures , and deviations , but there is a lot in this system that has to do with quality that we can not do anything about . like economy , that there is too little funding and all that . we could certainly have sat in a group discussing values , how to put them to best use , but a lot has to do with having more staff in order to be able to include quality and safety in our work , and respect that we hopefully have in any case all the nurses in this study experience that ethics , values , and reflecting on values are of great importance for the quality in nursing . the ability to be compassionately affected and empathic is anchored in ethics , as the basis for value susceptibility and depth of intentionality and motivation behind the nurses ' actions . emotions are of importance for the normative demarcation between impartial justice and utility maximization on the one hand and compassion on the other . the nurses in our study say that it becomes obvious to them when the ethical values are absent in practice and that it is their duty to speak up about unworthy conditions . the fact that they are aware of the lack of values and unworthy conditions confirms their moral and ethical sensitivity which again rests on sensibility and the ability to be accessible in human encounters . our study demonstrates that the nurses ' ability to be accessible in human encounters in their work is threatened by time pressure and lack of staff . the nurses in our study give expression to this by stating that they fear becoming superficial and indifferent nurses because their work load is too heavy for satisfactory nursing . this is a challenge involving professional healthcare education , the attitude of the entire national health service , politics , and the financial structures which have an impact on this setting . they witness that there is a big difference between theory and practice and this gives rise to frustration . it is consistent with research around this phenomenon which shows that reality shock is traumatic ; it is a reaction in newly trained nurses who can not practice what they have been trained to do and wish to do . in the opinion of our informants , what is most important is to see the patient and have respect for him , and they consider this to be a fundamental nursing value which they learnt in their training and from colleagues who have good values . none of the nurses refer to or mention codes for professional ethics as a basis for values . this is consistent with european studies that show that nurses lack knowledge about ethical codes and do not use them to evaluate moral aspects in practice . nurses from the somatic units state that there is little focus on ethical values on the part of the management , and they maintain that it is the management that is mainly responsible for what is brought into focus . bondas has done research on nursing management and taken part in the development of caritative leadership theory . caritative leadership ( cl ) has been developed from a caring science perspective with the caritas motive as its point of departure , human love , and mercy , focused on ministering to the patient . cl strives for an effective and sustainable development of nursing and caring , with a holistic view on patients , relatives , and staff . together with the staff , the leader creates a caring culture with the patient at its centre . in this type of culture the ethical values must be included and focused on , both by staff and leaders . the nursing management 's focus on human dignity , human love , and mercy may be set aside by the introduction of a market oriented leader style derived from industry and business . in her research on first line nursing management bondas found that these leaders , together with the staff , have to prepare the ground for nursing care . preparing the ground for nursing care is a continuous process for the nursing leaders which is important for the unit 's core function , for creating direction and quality in nursing , and with the patient as main focus . in our study , the nurses in somatic units repeatedly describe a culture where it seems that the managers do not prepare the ground for nursing care but are concentrating on production objectives , cost savings , and effectiveness instead of on human dignity and compassion . this is illustrated by the fact that the nurses have to prioritize transport of remunerative elective surgery patients , that is , part of the hospital 's production , rather than giving care to cancer patients and other patients who are not part of the same production . they also experience varying support from the managers and that the management does not understand what is going on or what it feels like to be a nurse such practice is not compatible with a caritative , nurturing culture which should prepare the ground for good nursing care in the best interest of the patient , relatives , and the nurse [ 1 , 27 ] . the nurses from the somatic units have little opportunity to get formal clinical supervision or to reflect on their practice , not even after serious incidents . in most somatic units the nurses who do have this offer are extremely pleased with it ; they find it useful , and say that it can turn you into a better nurse . the nurses from the psychiatric units described a reflective workday where the ethical values and reflection on them were present in most situations . it was an internalized way of working which facilitated good conditions for the patients as well as for the nurses . the nurses in psychiatry said that they were caught up in many ethical dilemmas and that it was important and correct that several people took part in the discussion of these issues . confronting the suffering of the depressed and mentally ill patient activates the nurses own feelings of pain , and they seek help from colleagues when they experience the relationship to the patient as painful . they need a forum which enables them to verbalize and explore their own feelings , where they are met with acceptance and response where feelings are set into a professional context . the nurses from the psychiatric units had all had clinical nursing supervision , and their workday was organized in such a manner that there were time and room for constructive reflection on the practice . this contributed to an improvement of the practice and prevented the nurses from taking incidents and problems from their practice home with them . some of the informants said that supervision had been decisive for their choice to continue in their profession . to be able to discuss feelings and experiences from the practice with other nurses will help the nurse to understand her own moral stance in terms of other nurses and the organization . clinical supervision can have a positive effect on the nurses ' experience of their own wellbeing in as much as psychic symptoms such that anxiety is reduced and the feeling of being in control grows . summarized research on clinical supervision demonstrates that clinical supervision can be both advantageous for professional practice and also for the patients . it is the health organizations who are responsible for developing and maintaining clinical supervision for nurses . clinical supervision is a reflective practice where nurses are given support and supervision on practice , which then will contribute to the nurses ' professional and personal development . the nurses from the somatic units told about high work pressure , rock - hard prioritizations , varying quality , and sometimes a lack of safety . they experienced that the values vanished into thin air , that is , became insignificant in what they referred to as rock - hard prioritizations of basic needs . as a result of their work situation , the informants felt frustrated , worn - out , and feared becoming superficial . they describe a busy day with constant bad conscience and a feeling of not being adequate . several studies from norwegian hospitals and nursing homes show that lacking resources lead regularly to suboptimal professional standards for nursing and medical treatment . the tendency is to lower the standard in order to safeguard the patient 's vital needs , and this is at the expense of good practice and all - round care . the nurses have to reduce their professional obligations and adapt to reality , and this creates a state in the nurses that can be understood as moral distress . the nurses in our study tell about crowded corridors and patients with newly diagnosed cancer who must lie in the corridor . this is in accordance with studies showing that crowded rooms and corridors make it difficult to safeguard the patient 's dignity and private life , and that the nurse feels moral unease for not being able to give the patient care in accordance with professional ideals . research on moral distress shows that it can arise on the basis of clinical situations and internal factors in the nurse , for example , a sense of powerlessness and lack of control over the situation , as well as external factors in the culture or organization , for example , inadequate staff . research shows that moral distress is linked to stress of conscience , while moral stress is seen in a more psychological perspective . moral distress and related concepts are linked to moral and ethical sensitivity and the ethical climate which forms the basis for the nurse 's moral freedom of action . compromises with ethical values or duties may have prolonged and negative effects causing the staff to become insensitive to the moral aspect of their work , or they may leave their profession . the nurses in our study experienced that they did not have a say in the matter and that there was nothing to be done with the system which caused these difficult conditions . they describe a feeling of powerlessness and lack of hope in terms of being able to influence and improve the conditions . none of our informants talked about leaving the profession , but they were discouraged and felt that they were barely keeping their head above water . research shows that the concern about nurses leaving their profession as a result of moral distress has created a greater awareness among health leaders and that the need for changes in the system is necessary as a partial response to the increasing level of moral distress in the health service [ 9 , 11 ] . as shown before , the nurses in the psychiatric units had access to clinical nursing supervision and constructive professional reflection , and none of these expressed frustration or a guilty conscience as a result of time pressure and lacking resources . as shown before , reflective practice promotes moral practice and we therefore believe that formal clinical nursing supervision and reflection , in addition to changes in the system , will also promote the values in practice and thus contribute to a reduction in moral distress . recent studies show that there is a high degree of moral distress among health professionals in many countries . health professionals themselves need to reaffirm the values and convictions that are important for their practice . the healthcare environment must be organized in a way that supports and promotes the health staff 's moral considerations . if the healthcare environment is not seen as a moral community but rather as simulated market places , then the health staff 's moral freedom of action will be reduced and make them more vulnerable to moral distress . the results of the study demonstrate that all the nurses believe that ethical values are of decisive importance for the quality in nursing . the nurses from the psychiatric units have a more reflective workday where the values are internalized in their work . the nurses from the somatic units experience that the values are repressed and become meaningless words in a busy workday with time pressure and resource shortage . these nurses have to lower their professional standard , thus compromising their clinical nursing values and ideals . the nurses from somatic units are worn out and feel frustrated ; they are constantly troubled by a guilty conscience and a sense of powerlessness . financial and organizational structures contribute to the fact that the values are repressed in the nurses ' practice and that this , together with insufficient time for clinical supervision and reflection , can be the basic reason for moral distress . we suggest a change of system based on the caritative leadership theory which may contribute to a combining of nursing and administration in such a way that it prevents suffering in the patient and will also further the development of nursing and care , humane and professional , on all levels in the organization . we also consider formal and regular clinical nursing reflection and supervision as crucial for safeguarding moral practice and thus preventing the development of moral distress .

the objective of this study was to examine how nurses experience ethical values as they are expressed in daily practice in a norwegian hospital . a growing focus in western healthcare on effectiveness , production , and retrenchment has an influence on professional nursing standards and nursing values . lack of resources and subsequent ethically difficult prioritizations imply a strain on nurses . this study is qualitative . data collection was carried out by conducting 4 focus group interviews . the data was analyzed using content analysis . the results are presented in two main themes : ( 1 ) values and reflection are important for the nurses ; ( 2 ) time pressure and nursing frustrations in daily work . the results demonstrate that nurses believe the ethical values to be of crucial importance for the quality of nursing ; however , the ethical values are often repressed in daily practice . this results in feeling of frustration , fatigue , and guilty conscience for the nurses . there is a need for changes in the system which could contribute to the development of a caring culture that would take care of both patients and nurses . in an endeavour to reach this goal , one could apply caritative leadership theory , which is grounded on the caritas motive , human love , and mercy .

cassava ( manihot esculenta crantz ) is one of the most important crops worldwide in terms of production and it is considered the most important food source for people living in tropical regions with arid soils . world production of cassava was estimated to be 276 millions of tons in 2013 and therefore it is considered to be the eighth most important product . cassava is euphorbiaceae and belongs to the genus manihot , which contains 90 species approximately . cassava is a high - yielding crop and its roots constitute a major food source for over 800 million people [ 46 ] mainly from africa , asia , and south america . this starch is transformed to be used in different industrial processes , in derivates such as alcohol and fructose - glucose syrups . this crop has high conversion rates of solar energy into carbohydrates and shows high tolerance to adverse abiotic stress . cassava plants can survive after long periods of drought in arid and low fertility soils . microsatellites ( also known as short tandem repeats ( strs ) , simple sequence repeats ( ssrs ) [ 11 , 12 ] , simple sequence length polymorphism ( sslp ) , and sequence tagged microsatellite site ( stms ) ) are sequence motifs of one to six bp , repeated in tandem . ssrs have been employed for studies of diversity [ 10 , 1618 ] , phylogeny , and evolution , as molecular markers in marker - assisted selection and have contributed significantly to the construction of genetic linkage maps [ 15 , 22 ] . ssrs are considered allele specific , highly polymorphic , codominant , heterozygous , reproducible , economic , and multiallelic molecular markers . the ssrs offer the opportunity to be employed in different studies given that they are under neutral selection when located in noncoding regions . ssrs can be classified in two classes : class i is composed of those with 20 bp repeats and class ii grouped ssrs from 12 to 20 bp . this classification is based on the observation that larger ssrs ( class i ) are demonstrated as more polymorphic than the shorter ssrs ( class ii ) . changes in length are due to a replication phenomenon known as slippage , although the unequal crossover in recombination also has a significant influence . these markers are relatively easy to automate and are generally considered more informative than other markers such as the single nucleotide polymorphisms ( snps ) due to the number of alleles that can be detected . ssrs have the advantage of being pcr - based markers because the flanking sequences are suitable for primer design . ssrs could be functionally implicated in chromatin organization , gene expression , and recombination hotspots and could affect dna replication . furthermore , in some cases position and changes in ssrs are associated with phenotypic changes . despite their importance and wide usefulness , ssrs genomic distribution studies in plant species are relatively scarce . with the advent and new advances in sequencing technologies , it is possible to analyze whole plant genomes for ssr discovery . genomic studies of ssrs distribution have been conducted on arabidopsis where it was first found that coding regions have a low frequency of ssrs and that these regions are highly rich in trinucleotides and hexanucleotides . these analyses also led to the conclusion that 5 sequences had higher frequencies of ssrs than other genome sequences [ 20 , 33 ] and that selective pressure acts differentially across genomic regions . an important feature is that in arabidopsis there is high prevalence of a - rich repeats . molecular markers have been of paramount importance in cassava for genetic diversity [ 17 , 3439 ] , evolution , and molecular systematic studies . ssrs in cassava have been favored over darts ( diversity arrays technology ) due to their codominant and multiallelic nature . strategies for ssrs identification in cassava have included enriched dna libraries [ 25 , 34 ] and the pursuit in ests sequences [ 10 , 23 ] . given that multiple groups have identified ssrs markers independently , it is highly probable that the same markers have been found several times and named differently . genomic analyses of cassava ssrs would contribute to the understanding of cassava genome architecture and evolution and possibly correlate ssr 's frequency , distribution , and sequence motifs , with genomic localization and function . we searched the cassava genome near - complete sequence ( http://www.phytozome.com/ ) to gain an insight into genomic composition of cassava 's ssrs . we carried out ssrs identification and characterization on the cassava 's genome and their distribution in exons , introns , and utr ( untranslated regions ) . a gene ontology ( go ) annotation was conducted for the ssrs present in the gene regions . cassava whole genome sequence ( version cassava4 ) was obtained from the phytozome database available at http://www.phytozome.net/cassava . this 532.5 mbp cassava genome sequence belongs to the genotype am560 - 2 , an inbred lined derived from the cultivar mcol1505 . ssrs identification was made with the pearl script misa ( microsatellite identification tool , http://pgrc.ipk-gatersleben.de/misa ) . the parameters established for misa were adjusted for the identification of class i ssrs ( length 20 bp ) of di- , tri- , tetra- , penta- , and hexanucleotides . class i ssrs were chosen because they have proven to be more polymorphic than ssrs of 12 to 20 bp . mononucleotides were not considered because of the possibility of sequencing or assembly errors . for compound ssrs ( distinct and adjacent ssrs ) , the maximum difference between two ssrs was set as 100 bp or less . for comparative purposes , a genomic identification of ssrs in other species the genomes of the related species selected were : populus trichocarpa ( poplar ) and linum usitatissimum ( flax ) which belongs to the order malpighiales ; ricinus communis ( ricinus ) and jatropha curcas ( jatropha ) that belong to the euphorbiaceae family . coding , 3 utr , and 5 utr cassava sequences were extracted using the biomart tool and introns were extracted using a pearl script . altogether we obtained ~40 mbp of coding sequences , ~50 mbp of intron sequences , and ~2 mbp and ~4 mbp of 5 and 3 utr sequences , respectively . ssrs density , ssr types , and motif distribution in cassava were assessed , analyzed , and compared through information stored in excel files . with the aim of obtaining the codon usage in cassava coding sequences we used the cusp program of emboss ( the european molecular biology open software suite , cambridge , uk ; http://emboss.sourceforge.net/ ) . for the purpose of assigning functional categories to the sequences from the different gene regions containing ssrs , we searched for the classes to which each sequence belonged and were grouped according to go categories . the functional classes for each gene were obtained using the biomart data mining tool hosted in phytozome . the categorizer tool ( http://www.animalgenome.org/bioinfo/tools/countgo/ ) was used to count go classes and group them into functional categories . the go_root classification method and single counting method were set as parameters for the go terms counting , to obtain a classification based on the three main categories : molecular function , biological process , and cellular component . plant_goslim classification method and single counting method were used as parameters to group the sequences in the different subcategories . in order to conduct an exploratory analysis of the ssrs present in the complete cassava genome and to make comparisons with genomes of related species , we detect 26.579 class i ssrs in the cassava genome , using the misa tool . considering the whole genome sequence length ( 536 mbp ) , the density of ssrs present in cassava was estimated to be ~50 ssrs per mbp ( figure 1 ) . in ricinus a density of 71,7 ssrs / mbp was identified , while in poplar we found 99 ssrs / mbp . the ssrs density was 30 ssrs / mbp for flax and 87,7 ssrs / mbp in jatropha . in general , the ssrs density in cassava was less than the average found in the assessed species ( 67,7 ssrs / mbp ) . based on the type of repetition we found that 37,4% of all ssrs found in cassava correspond to dinucleotides , 24% are trinucleotides , 8,6% are tetranucleotides , 24,2% pentanucleotides , and 5,8 correspond to hexanucleotides ( figure 2 ) . most ssrs in cassava genome are dinucleotides as have been observed in most species . indeed in most of the evaluated species we observed that the most common ssr type is dinucleotide with the exception of flax , which has a higher number of trinucleotides . according to this , in flax dinucleotides just accounted for 24% , while trinucleotide accounted for 47,3% of all ssrs identified . a high proportion of pentanucleotides was found in cassava ( 24,2% ) in contrast to the other species , which have an average of 9% of this type of ssr . to determine the distribution of ssrs in the cassava genome , we carried out an ssr search in coding , utrs , and intron sequences . we obtained coding sequences corresponding to 34.151 annotated genes , 3 utr sequences from 15.420 genes and 5 utr sequences from 14.111 genes . a pearl script allowed the extraction of 122.806 intron sequences corresponding to 24.309 genes . following the ssrs search on each of these regions , as we expected , coding regions were found to have the lowest density of ssrs ( figure 3 ) . we found that the average density of ssrs in the whole genome is higher than in coding regions . for example we identified 49,9 ssrs / mbp ssrs in the whole genome while only 15,5 ssrs / mbp were found in coding sequences . of 34.151 coding sequences analyzed , we found that 587 contained at least one ssr and 32 had more than one ssr . the density of ssrs in the whole genome was lower than in noncoding regions ( introns and utrs ) ( figure 3 ) . according to a previous report , which have indicated that utr regions are ssr rich , we observed that in cassava 5 utrs contain the greatest amount of ssrs ; we identified 434 ( 196,1 ssrs / mbp ) ssrs in 5 utrs and 202 ( 50,5 ssrs / mbp ) ssrs in 3 utrs . we expected to find more ssrs in the 3 utr than in the 5 utrs as it has been reported previously . however , we observed that 5 utr sequences have between 2,4- and 12,6-fold higher ssr density than other regions and almost fourfold higher density than in the whole genome . higher ssrs densities in 5 utrs were also observed in arabidopsis and a similar proportion to that we found was identified in rice . the existence of more than one ssr in a single sequence was found to be scarce . only 0,09% of coding sequences , 0,39% of introns , 0,16% of 5 utr , and 0,02% of 3 utr have more than one ssr in a single sequence . a comparison of the motifs in different genomic regions was done because the motifs proportion changes across the genome in a similar manner to the ssrs number . as a result of selection pressure it has been noted that most of the ssrs found in coding regions are tri- or hexanucleotides avoiding frame shifts in this way [ 20 , 49 , 50 ] . this situation was also found in cassava coding sequences where tri- and hexanucleotides account for 95,6% of the ssrs and almost no tetra- and pentanucleotides were identified on these regions . the results also suggest that noncoding sequences , as observed in the whole genome , have a high proportion of pentanucleotides ( figure 4 ) . this type of ssr may have evolved from polya stretches and could generate important secondary structures . in the entire cassava genome there is prevalence of the at / at motif represented by approximately 22% of the ssrs identified ( figure 5(a ) ) . this is the most abundant motif found in several plant genomes [ 12 , 15 , 26 , 48 , 51 ] . in cassava coding sequences we found a prevalence of aag / ctt and agc / ctg ( figure 5(b ) ) . ssrs in coding regions could give an indication of codon usage preference . to determine if the ssrs identified in coding regions correspond to the most used triplets in cassava the motif aag which is the most commonly found in coding sequences is in fact the third most used codon in cassava with a percentage of 3,2% among all the nucleotide combination triplets ( table 1 ) . the ssr motifs agc / ctg , agg / cct , atc / atg , and acc / ggt that were also found in high frequencies are used in percentages between 0,9 and 1,4% indicating that these codons are not used frequently . in noncoding sequences the most common ssr motif is ag / ct ( figures 5(c ) , 5(d ) , and 5(e ) ) similar to previous reports for several plant species [ 27 , 48 , 53 ] . in 5 utr sequences , most of the ssrs were of the ag / ct and aag / ctt type . similar observations were reported in other dicotyledonous species like arabidopsis and soybean , but not in monocot plants such as rice or maize . these differences in motif distribution in upstream gene sequences often lead to differences in genomic structure and gene regulation on both groups of plants [ 20 , 49 , 54 ] . the gc type was not identified in any of the cassava sequences groups . in order to gain some insight into the putative function of the genes containing ssrs , we classified those genes according to go categories ( figure 6 ) . putative molecular function was attributed to 55,4% of gene coding sequences that contain ssrs , 51,3% of intronic sequences , and 54,7% and 44,3% of 3 and 5 utr sequences , respectively . about 35,7% of the sequences containing ssrs belonged to genes classed in biological processes , while 10,2% , 12,5% , 9,9% , and 18,7% of the coding , intronic , and 3 and 5 utr sequences , respectively , corresponded to genes grouped in the cellular component category . a detailed categorization for each different go category was made ( supplementary figures 1 , 2 , 3 , and 4 in supplementary material available online at http://dx.doi.org/10.1155/2014/471461 ) . when the subcategories comprised in biological process were compared , we observed that although there were some evident differences , the majority of genes containing ssrs belonged to the cellular process subcategory . the metabolic , biosynthetic , and protein metabolic processes were the categories with more ssr - containing genes . in terms of the cellular component , a common feature was that sequences containing ssrs belonged to genes that encoded proteins located frequently inside cell , cell membrane , and nucleus . regarding the molecular function , the ssrs - containing genes were mostly related to catalytic , binding , hidrolase , transferase , and transporter activity . interestingly we found that cassava genes coding for proteins located in thylakoid , vacuole , and golgi apparatus contain ssrs exclusively on intron sequences . a similar situation was observed for genes related to carbohydrate binding , pollen - pistil interaction , pollination , regulation of gene expression , epigenetic process , and reproduction . we also observed that there is just one gene with ssr associated with embryonic development and its ssr is located on the 5 utr of the gene . in addition some genes contain ssrs in their coding and intron sequences but not in the utr regions ( related to carbohydrate metabolic processes , lipid binding , motor activity , and genes that encode proteins located on the cell wall , external encapsulating structures , and peroxisomes ) . in a similar manner , genes related to response to biotic stimulus have ssrs exclusively on the coding and intron sequences . on the other hand genes related to response to abiotic stimulus had ssrs exclusively on their utr sequences . for genes associated with response to endogenous stimuli and stress response , ssrs were detected in all their regions ( intron , coding , and utrs sequences ) . we found that , as expected , cassava has high frequencies of dinucleotides and that a unique feature of this plant was its unusual high frequency of pentanucleotides . the predominance of a specific ssr class has been observed in rice . in terms of gene analysis , coding sequences are the regions with the lowest density of ssrs while the 5 utrs are the counterpart with the highest content . in general we identified 621 ssrs ( 15.5 ssrs / mbp ) in coding sequences , 4.120 ssrs ( 82,3 ssrs / mbp ) in introns , and 434 ( 196,1 ssrs / mbp ) and 202 ( 50,5 ssrs / mbp ) ssrs in 5 and 3 utr sequences , respectively . it is estimated that in cassava there are approximately 1.000 ssrs previously identified . after following the methodology proposed here we identified 26.579 ssrs in the cassava genome . previous studies on ssr detection in cassava genomic libraries reported the identification of 12 , 32 , 545 , or 1.576 ssrs . previous efforts in searching cassava ssrs in sequences yielded the identification of 531 , 49 , 836 , 1.889 , 431 , 7.270 , or 163 ssrs in cassava ests . the low number of ssrs previously found could be explained for the low number of genome sequences reported at that moment . with the recent cassava genome release a global and genomic analysis of ssrs is possible . additionally , no study had focused earlier on the distribution of ssrs along different gene regions in cassava . the identification of ssrs in several gene sequences is not only informative but also useful to develop makers to map the genes in which they reside . however it is important to note that some of these could correspond to anonymous , with an unknown function , type of markers and although they have been useful for developing genetic maps and for diversity studies most of them have no specific known function . through ssrs data comparison we determined that cassava has only 49,9 ssrs / mbp being one of the species with lower ssrs densities in its genome compared with phylogenetically closer species . based on cassava nuclear dna quantity it has been estimated that the cassava genome is 772 mbp and nonetheless the sequenced genome is 533 mbp . the lacking sequences of about ~240 mb could consist of repetitive dna which has not been assembled . although it has been observed that ssrs are preferentially found in nonrepetitive dna , the low quantity of ssrs in cassava could be associated with the possibility that the nonassembled sequences in the genome would have a considerable amount of ssrs . it is important to note that ssrs identified in gene sequences are potential powerful molecular markers for use in breeding programs . due to their location inside genes , these markers save effort and resources in the early stages of searching for markers closely linked to particular genes . the ssr markers identified in this work would be an important resource for genetic mapping analysis of the genes in which they are located . in addition , they could help to make phylogenetic analysis to understand the diversity of those genes . owing to their mutation properties , these ssrs would give hints about evolutionary changes on the cassava genome . the prevalence of dinucleotide ag / ct in cassava has been observed in previous studies where the search was made through the development of ssrs libraries [ 25 , 34 ] or by in silico ssr search in ests [ 10 , 56 ] . here we observed that this is the second most observed motif in the genome and it is the most frequent in noncoding sequences . the latest result is consistent with the statement that the ag / ct is in fact the most common dinucleotide in vascular plant ests [ 52 , 53 ] and in coding regions according to morgante et al . . we observed that in cassava the dinucleotide ac / gt or ga / ct accounted only for 1,5% of the ssrs in all the evaluated sequences , while the other dinucleotides added up to 38% . compared to the genomes of animals previous studies highlighted the absence of this motif in sequences of several plant species [ 51 , 53 ] and are indeed the least frequent ssr in almost every assessed organism with the exception of escherichia coli . the availability of cassava genome sequences enabled a more effective assessment of ssr marker distribution in this study . . this will be quite useful in cassava since the genome sequence is highly fragmented . finally the go categories assignment of the genes where the ssrs were identified can be useful in studies where the objective is to map a specific group of genes corresponding to a functional category such as abiotic or biotic stress .

we conducted a ssrs density analysis in different cassava genomic regions . the information obtained was useful to establish comparisons between cassava 's ssrs genomic distribution and those of poplar , flax , and jatropha . in general , cassava has a low ssr density ( ~50 ssrs / mbp ) and has a high proportion of pentanucleotides , ( 24,2 ssrs / mbp ) . it was found that coding sequences have 15,5 ssrs / mbp , introns have 82,3 ssrs / mbp , 5 utrs have 196,1 ssrs / mbp , and 3 utrs have 50,5 ssrs / mbp . through motif analysis of cassava 's genome ssrs , the most abundant motif was at / at while in intron sequences and utrs regions it was ag / ct . in addition , in coding sequences the motif aag / ctt was also found to occur most frequently ; in fact , it is the third most used codon in cassava . sequences containing ssrs were classified according to their functional annotation of gene ontology categories . the identified ssrs here may be a valuable addition for genetic mapping and future studies in phylogenetic analyses and genomic evolution .

all animals ( charles river , yokohama , japan ) were housed under controlled temperature ( 25 1c ) and 12 hr : 12 hr light : dark conditions ( lights on at 07:00 hr ) with food and water available ad libitum . all of the procedures were performed in accordance with the guidelines for animal care stipulated by the japanese physiological society and approved by the ethics committees of miyazaki university . all efforts were made to minimize animal pain and suffering , and the number of animals used . in the first set of experiments , adult female wistar rats ( approximately 10 weeks old ) were used . from 1 week prior to sample collection , vaginal smears were taken daily from the rats for determination of the estrous cycle , and accordingly , the animals were subdivided into 4 groups : diestrus ( n=9 ) , proestrus ( n=7 ) , estrus ( n=9 ) and metestrus ( n=16 ) . we showed the plasma amino acid concentration with supplemental table , and the percentage concentration ratios for the various amino acids were also presented as circular profiles relative to the amino acid concentrations of diestrus . in the next experiment , adult female rats were mated on the day of proestrus , and the day after mating was counted as day 1 of pregnancy ( p1 ) . blood samples were taken every 5 days ( p5 , 10 , 15 and 20 ; n=4 ) . after delivery and during lactation , blood samples were taken from the dams in the same way ( l1 , 5 , 10 , 15 and 20 ; n=4 ) , and we take the samples from the female pups just after weaning ( n=6 ) . the percentage concentration ratios for the various amino acids were also presented as circular profiles relative to the concentrations determined just before pregnancy ( proestrus ) . all blood samples were collected into heparinized capillary tubes by the tail tip incision method , and these were centrifuged immediately at 4c and 14,000 rpm for 4 min . we think that the possibility of contamination of amino acids derived from erythrocyte by hemolysis is almost none , since we paid scrupulous attention not to hemolyze at the blood sampling and hemolyzed sample was excluded from the assay . the resulting plasma samples were mixed with 2 volumes of 5% ( w / w ) trichloroacetic acid and centrifuged immediately at 4c and 10,000 rpm for 20 min to remove the precipitated protein . ufc5010bk , millipore , billerica , ma , u.s.a . ) , and the plasma amino acid concentrations were measured using an automatic amino acid analyzer ( l-8800a ; hitachi , tokyo , japan ) . we focused on the 20 amino acids that are the components of proteins : valine ( val ) , leucine ( leu ) , isoleucine ( ile ) , alanine ( ala ) , methionine ( met ) , proline ( pro ) , tryptophan ( trp ) , phenylalanine ( phe ) , tyrosine ( tyr ) , threonine ( thr ) , glutamine ( gln ) , asparagine ( asn ) , serine ( ser ) , glycine ( gly ) , cysteine ( cys ) , lysine ( lys ) , arginine ( arg ) , histidine ( his ) , aspartic acid ( asp ) and glutamic acid ( glu ) . briefly , amino acids were separated by cation - exchange chromatography and detected spectrophotometrically after post - column reaction with ninhydrin reagent . all sample collections were performed at the same time ( 11:3012:30 hr ) to minimize any influence of circadian rhythm . all data were expressed as the mean standard error of the mean ( sem ) . in the first experiment , differences between groups at the various stages of the estrous cycle were analyzed by one - way anova and post hoc tukey s test . in the other experiment , data for amino acid levels were analyzed by one - way repeated anova , and changes between time points during the experimental period were analyzed by tukey s test . the sexual difference ( fig . 3d ) were analyzed by student s t - test . to visualize the results , the concentrations of amino acids among the experimental groups for the 2-dimensional circular plane figures , we plotted the percentage concentration of each amino acid relative to the control level ( at diestrus or proestrus ) . in the three - dimensional figures , principal components analysis ( pca ) was performed to reduce the dimensionality of the data set and to identify new meaningful underlying variables . to obtain a more accurate result in the latter analysis , we used 40 kinds of amino acids and their metabolic intermediates , including taurine , sarcosine , citrulline , ornithine and hydroxyproline , for pca analysis . the actual concentrations of the various amino acids in plasma are shown in tables 1table 1alterations in plasma amino acid concentrations during the estrous cycle in female rats and age - matched male ratsamino acids(mol / l)diestrus(n=9)proestrus(n=7)estrus(n=9)metestrus(n=16)age - matched male(n=4)valine175.8 6.2182.0 12.1168.9 5.6189.6 4.3157.8 4.8leucine124.4 3.8129.3 7.5126.0 4.0135.2 4.6110.5 4.2isoleucine72.8 2.077.7 5.174.7 2.378.9 1.465.9 3.4alanine408.9 10.7459.5 27.3453.2 24.7444.0 13.4349.1 31.2*methionine66.6 3.363.4 4.964.1 1.774.5 2.956.3 3.4proline223.7 8.9217.6 17.0215.1 9.1238.4 5.9170.5 6.5*tryptophan141.9 6.8129.9 7.0130.1 6.3145.3 3.6106.1 7.4*phenylalanine67.0 2.171.4 4.170.9 2.073.0 1.866.7 1.8tyrosine67.6 2.574.4 5.669.3 2.874.6 3.091.2 4.7*threonine320.4 18.0357.3 40.1332.4 25.5363.9 15.4265.5 5.0glutamine650.5 30.4637.5 25.6619.1 20.5689.8 18.4556.3 16.6asparagine51.0 3.248.2 4.244.4 2.153.1 2.244.5 2.8serine261.6 11.2275.4 19.4267.8 9.8283.9 9.8197.5 7.9*glycine281.1 13.1283.1 17.5263.8 7.9282.3 8.8255.2 3.8cysteine1.5 0.41.4 0.41.7 0.71.4 0.3ndlysine491.2 19.2479.6 30.4461.9 21.9521.9 20.7336.8 21.5*arginine178.5 12.6176.5 4.8196.5 9.0251.7 28.1125.8 14.0*histidine58.3 2.359.8 3.157.0 1.662.3 1.851.3 0.6aspartate10.8 1.711.8 1.312.7 1.714.6 1.67.0 1.0glutamate91.3 8.286.4 7.894.3 6.999.7 7.681.6 13.4the data represent the mean standard error of the mean ( sem , n=716 ) . * ; p<0.05 vs. diestrus by student s t - test . , 2table 2alterations in plasma amino acid concentrations during pregnancyamino acids ( mol / l)proestrus ( p0)p5p10p15p20valine172.3 17.9247.3 83.1305.8 85.3211.1 61.1201.1 64.6leucine123.6 11.0124.5 6.0119.6 4.0107.5 2.6103.1 9.6isoleucine73.3 7.674.9 3.671.6 2.266.3 1.068.5 7.4alanine414.3 26.3361.6 14.6348.2 15.7373.8 49.7510.6 33.6methionine59.3 4.360.3 4.167.5 4.457.8 5.654.0 3.2proline191.6 12.3186.3 13.0171.7 8.8175.2 12.6184.1 9.1tryptophan119.3 6.2100.6 1.2106.2 6.5101.8 4.362.4 5.5*phenylalanine67.8 3.661.1 2.364.5 3.258.8 3.159.8 4.9tyrosine74.8 5.264.0 3.274.4 6.751.9 5.8 * 50.3 4.9*threonine276.0 16.1235.1 13.9240.6 21.0268.7 13.8292.1 23.9glutamine601.1 11.4614.6 20.7623.2 36.4631.1 39.5598.8 24.3asparagine43.1 5.040.1 1.637.4 1.637.7 2.550.9 3.2serine246.4 17.6230.7 6.2207.5 5.3 * 202.8 8.8 * 221.8 6.3glycine272.2 21.2259.5 9.3232.2 14.0158.1 7.9 * 127.6 3.6*cysteinendndndndndlysine435.8 19.8422.1 48.3418.6 31.2527.4 30.9623.4 60.3*arginine160.8 20.0174.6 9.4166.2 4.4162.1 24.8140.4 8.7histidine59.3 4.658.9 1.557.8 3.144.8 3.2 * 36.6 2.1*aspartate11.9 2.310.3 2.18.3 0.89.2 1.19.0 0.6glutamate87.6 12.582.8 15.478.1 10.480.2 10.183.3 10.6the data represent the mean standard error of the mean ( sem , n=4 ) . * ; p<0.05 vs. non - pregnant female rats group . , 3table 3alterations in plasma amino acid concentrations during lactation in dams and the pups just after weaningamino acids(mol / l)proestrus ( p0)l1l5l10l15l20pupsvaline172.3 17.9130.5 32.2154.6 12.0182.8 1.4168.7 9.8129.2 24.4206.8 10.2leucine123.6 11.097.5 10.9115.6 8.4133.6 3.5129.6 6.5117.7 18.2146.5 8.8isoleucine73.3 7.666.1 4.273.6 5.985.5 1.679.8 4.074.2 11.297.0 3.8alanine414.3 26.3739.3 50.2 * 621.8 41.0585.1 35.2608.9 61.9572.1 44.2738.9 33.5methionine59.3 4.370.0 6.682.5 3.7 * 98.7 4.2 * 99.3 5.3 * 90.5 6.4 * 86.1 2.7proline191.6 12.3288.6 27.7 * 268.7 18.7 * 259.0 8.9 * 268.7 14.9 * 239.4 15.3358.2 39.7tryptophan119.3 6.2106.1 12.8113.3 2.5118.1 7.2115.4 9.9113.7 8.999.5 4.9phenylalanine67.8 3.666.3 4.867.7 4.770.9 2.171.8 6.465.0 6.476.4 5.6tyrosine74.8 5.285.0 8.387.4 10.681.9 6.384.4 7.569.0 9.8148.1 17.3threonine276.0 16.1386.4 60.6390.9 21.2377.4 20.5347.2 17.1316.8 23.4219.5 12.7glutamine601.1 11.4818.9 40.7 * 704.7 43.1711.7 37.1752.6 35.3 * 688.8 25.6626.0 46.6asparagine43.1 5.090.5 9 * 71.4 2 * 84.3 3.1 * 91.7 1.9 * 76.5 2.2 * 75.7 12.9serine246.4 17.6422.5 29.7 * 372.0 30.4 * 366.3 12.4 * 353.0 12.1 * 321.4 15.1300.6 24.0glycine272.2 21.2400.6 34 * 421.6 41.6 * 465.5 20.6 * 461.7 22.7 * 381.3 14.7613.2 25.6cysteinendndndndndndndlysine435.8 19.8686.5 100.8 * 521.1 34.9550.1 30.8489.6 2.6459.8 32.2232.9 16arginine160.8 20213.8 20.8196.5 17.3194.9 8.6182.5 5.5178.7 13.9240.2 26.6histidine59.3 4.656.3 4.566.2 3.378.6 3.4 * 72.3 3.263.2 6.7119.4 7.7aspartate11.9 2.311.4 2.013.4 5.85.5 1.47.2 0.76.8 1.216.9 2.2glutamate87.6 12.576.8 1.793.1 16.766.1 5.865.7 4.871.1 6.6119.3 7.8the data represent the mean standard error of the mean ( sem , n=4 , 6 ) . * ; p<0.05 vs. non - pregnant female rat group , ; p<0.05 vs. dams on day 20 of lactation group .. to compare the types of amino acid changes occurring during the estrous cycle , pregnancy or lactation , we displayed the ratios of the respective amino acids at each stage in the form of circular profiles relative to the corresponding values for diestrus or proestrus . the data represent the mean standard error of the mean ( sem , n=716 ) . * ; p<0.05 vs. diestrus by student s t - test . the data represent the mean standard error of the mean ( sem , n=4 ) . * ; p<0.05 vs. non - pregnant female rats group . the data represent the mean standard error of the mean ( sem , n=4 , 6 ) . * ; p<0.05 vs. non - pregnant female rat group , ; p<0.05 vs. dams on day 20 of lactation group . the levels of the individual amino acids in plasma were almost the same among proestrus , estrus , metestrus and diestrus , and their circular profiles showed no significant differences ( fig . 1.alterations in plasma amino acids concentrations ( a c , f ) , body weight ( d ) and daily food intake ( e ) at various stages of the estrous cycle in female rats and also in age - matched male rats . the concentrations of individual plasma amino acids are shown as a percentage ratio relative to the concentrations in diestrus females . a : results for proestrus ( red line ) , b : estrus ( yellow line ) , c : metestrus ( green line ) and f : age - matched males ( blue line ) . the data for body weight and food intake represent the mean standard error of the mean ( sem ) . d , diestrus ( white bar ) ; p , proestrus ( black bar ) ; e , estrus ( gray bar ) ; m , metestrus ( diagonal bar ) . amino acids indicated by red shading and asterisks showed a significant difference vs. diestrus females ( p<0.05 ) . , table 1 ) . on the other hand , the shapes of the circular profiles changed during the course of pregnancy ( fig . 2.temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight ( b ) and food intake ( c ) during pregnancy in female rats ( n=4/group ) . the concentrations of individual plasma amino acids are shown as a percent ratio relative to the concentrations in non - pregnant female rats ( proestrus ; n=4 , black line ) . amino acids indicated by red shading and asterisks showed a significant difference vs. non - pregnant females ( p<0.05 ) . , table 2 ) . the plasma ser level decreased significantly in mid and late ( p10 and 15 ) pregnancy , and tyr , gly and his decreased significantly at the late and end stages ( p15 and 20 ) of pregnancy , whereas trp and lys significantly decreased and increased at the end of pregnancy , respectively . alterations in plasma amino acids concentrations ( a c , f ) , body weight ( d ) and daily food intake ( e ) at various stages of the estrous cycle in female rats and also in age - matched male rats . the concentrations of individual plasma amino acids are shown as a percentage ratio relative to the concentrations in diestrus females . a : results for proestrus ( red line ) , b : estrus ( yellow line ) , c : metestrus ( green line ) and f : age - matched males ( blue line ) . the data for body weight and food intake represent the mean standard error of the mean ( sem ) . d , diestrus ( white bar ) ; p , proestrus ( black bar ) ; e , estrus ( gray bar ) ; m , metestrus ( diagonal bar ) . amino acids indicated by red shading and asterisks showed a significant difference vs. diestrus females ( p<0.05 ) . temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight ( b ) and food intake ( c ) during pregnancy in female rats ( n=4/group ) . the concentrations of individual plasma amino acids are shown as a percent ratio relative to the concentrations in non - pregnant female rats ( proestrus ; n=4 , black line ) . amino acids indicated by red shading and asterisks showed a significant difference vs. non - pregnant females ( p<0.05 ) . much larger changes in the circular profiles of plasma amino acids were observed during the lactation period ( fig . 3.temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight ( b ) and food intake ( c ) during lactation in female rats ( n=4/group ) . the concentrations of individual plasma amino acids are shown as a percentage ratio relative to the concentrations in non - pregnant females ( proestrus ; n=4 , black line ) . d shows the results of the pups just after weaning ( n=6 , blue line ) compared with the dams on the day 20 of lactation ( n=4 , red line ) . amino acids indicated by red shading and asterisks showed a significant difference vs. each control group ( p<0.05 ) . , table 3 ) : many amino acids showed significant increases , and none showed a significant decrease . plasma pro , ser and gly levels increased continuously from day 1 until day 15 of lactation . the levels of asn and met increased significantly from days 1 and 5 respectively until the end of lactation , ala and lys were increased significantly only on day 1 of lactation , his was increased on day 10 , and gln was increased on days 1 and 15 . 3d shows the results of the pups just after weaning compared with the dams on day 20 of lactation . many amino acids ( val , ala , pro , tyr , gly , his , asp and glu ) showed significant increases , and only 2 amino acids ( thr and lys ) were decreased . temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight ( b ) and food intake ( c ) during lactation in female rats ( n=4/group ) . the concentrations of individual plasma amino acids are shown as a percentage ratio relative to the concentrations in non - pregnant females ( proestrus ; n=4 , black line ) . d shows the results of the pups just after weaning ( n=6 , blue line ) compared with the dams on the day 20 of lactation ( n=4 , red line ) . amino acids indicated by red shading and asterisks showed a significant difference vs. each control group ( p<0.05 ) . pca analysis revealed clear differences in the 3d plots among the groups ( fig . 4a and 4bfig . 4.principal component analysis ( pca ) plots from amino acid concentration data of individuals ( a ) and median ( b ) in each experiment . black plot ; male rats age - matched with females in the estrous cycle experiment , blue plot ; estrous cycle experiment , red plot ; pregnancy period and green plot ; lactation period . ) . on the pca plot , we added the plasma amino acid levels for an age - matched male group ( n=4 ) to confirm whether or not a sex difference was evident . the positions of each sample were plotted against the axes of the first three components ( pc1 , pc2 and pc3 ) in a 3d space and were colored according to each experimental group ( black plot ; male rats age - matched with the females used in the estrous cycle experiment , blue plot ; estrous cycle experiment , red plot ; pregnancy period and green plot ; lactation period ) . pca analysis allowed visual identification of data patterns and highlighted similarities and differences among the reproductive stages in female rats . the dispersion of the points assembled in each group . principal component analysis ( pca ) plots from amino acid concentration data of individuals ( a ) and median ( b ) in each experiment . black plot ; male rats age - matched with females in the estrous cycle experiment , blue plot ; estrous cycle experiment , red plot ; pregnancy period and green plot ; lactation period . presentation of the amino acid profiles in a 2-dimensional circular plane , using the levels of amino acids during diestrus or proestrus as controls , demonstrated that the profiles changed during pregnancy and lactation in dams , but not during the estrous cycle . in addition , 3-dimensional pca analysis revealed a different distribution of plasma amino acid levels among reproductive stages or sex , especially in late pregnancy and early lactation in dams . these observations suggest that the levels of amino acids in female rats differ from those in males and that they change according to reproductive stage . generally , it has been considered that protein metabolism differs between males and females , since the sexes differ considerably in muscle mass and energy consumption . some of these differences may be correlated with sex steroid hormones : testosterone and estrogen stimulate and inhibit protein synthesis , respectively [ 14 , 27 ] . also , it has been reported that ovariectomy causes an increase of bcca and a decrease of ala in plasma . changes in the circular amino acid profiles in dams during pregnancy or lactation may result from transitional changes in the plasma levels of steroid . on the other hand , in rats during the estrous cycle , there were no significant differences in the circular profiles , even though plasma steroid hormone levels do vary during the cycle . although the reason for this unexpected result is unknown , it may be results of short term change of steroid levels in estrus cycle . another possibility is that pregnant or lactating dams have a higher nutritional demand and food intake in comparison with normal rats . the present study also demonstrated an increase of food intake in pregnant and lactating dams . in spite of the increase of food intake during pregnancy , most of the significant changes in plasma amino acid levels were falls , rather than increases . in addition , most of the amino acids that showed reduction during pregnancy were glycogenic amino acids . in pregnant rats , increase of insulin and ketone body , and decrease of glucose are observed in peripheral blood [ 5 , 13 ] . therefore , decrease of glycogenic amino acid during pregnancy may be due to promoting the gluconeogenesis for supply of glucose to the fetus . another possible reason for the reduction in many plasma amino acid levels may have been an increase in the plasma progesterone level . it has been reported that administration of progesterone to humans causes a marked reduction of many amino acids in plasma . however , if progesterone is the main reason for the decrease of amino acids , this does not explain why many amino acids showed increases in their levels in lactating dams , whose plasma progesterone level is high . the only amino acid that showed an increased level in plasma at the end of pregnancy was lys ( a ketogenic amino acid ) . although the reason for this is unclear , lys is utilized for acetyl - coenzyme a , some of which is employed for cholesterol synthesis . furthermore , it has been reported that dietary lys and arg are related to plasma cholesterol levels and that a decrease in the ratio of arg to lys is associated with an increase in the plasma cholesterol level . cholesterol may be necessary for the rapid increase in the level of estrogen at the end of pregnancy for initiation of lactation . in contrast with pregnant dams , all of the amino acids that showed significant changes in lactating dams increased . all of these , except for lys and met , were non - essential and glycogenic amino acids . during the lactation in comparison with pregnancy , therefore , these non - essential and glycogenic amino acids may be for supplying sufficient glucose or amino acid to product the milk in the mammary gland . the increase in the lys level on the initial day of lactation may have been due to the increase observed at the end of pregnancy . ala was significantly increased in early lactation ( l1 ) and showed a tendency to increase as lactation proceeded . it has also been reported that the expression of amino acid transporter ( aat ) and lipid synthesis - related genes in the mammary gland is increased more during lactation than during pregnancy in the mouse . in addition , the amino acid concentrations in secreted milk during lactation are different from those of maternal plasma . in the fetus immediately before delivery , amino acid levels are known to be higher than in the mother . on the basis of these observations , we expected that the plasma amino acid levels in rats just after weaning might be higher than in mature rats , and measured the plasma amino acid levels in pups just after weaning . as expected , many kinds of amino acid in plasma showed levels markedly higher than in the dam ( fig . it might be generally believed that the increase of food intake results in increase of amino acid levels in blood . certainly , increase of food intake brings about increase of plasma amino acid levels in hyperphagic animals like the ob / ob mouse . however , this correlation between food intake and plasma amino acid levels did not apply to pregnant rats , since main change in amino acid level in pregnant rats was decrease , but not increase , nevertheless , food intake increased . in lactating dam , however , some amino acids concomitant with food intake increased in the present study also . but , the rises of bcaa were not recognized in lactating dam . therefore , we supposed that the plasma amino acid levels in lactating dam were influenced by not only food intake , but also metabolic change . in the present study , we showed that imbalance of amino acid profiles strongly reflects the metabolic changes occurring in vivo and that such data would be applicable to various fields , such as diagnosis , treatment with infusion to patient , nutrition for the pregnant women and production of artificial milk .

we measured the plasma levels of amino acids at various reproductive stages in female rats , including the estrous cycle , pregnancy and lactation , and compared the resulting amino acid profiles using two- or three - dimensional figures . these figures revealed that the amino acid profiles of pregnant and lactating dams differed considerably from those during the estrous cycle or in male rats . the plasma levels of individual amino acids were almost the same between proestrus , estrus , metestrus and diestrus , and their profiles did not differ significantly . however , the amino acid profiles changed during pregnancy and lactation in dams . the plasma ser level decreased significantly in mid and late pregnancy , whereas tyr , gly and his decreased significantly in the late and end stages of pregnancy , and trp and lys significantly decreased and increased at the end of pregnancy , respectively . much larger changes in amino acid profiles were observed during lactation , when the levels of many amino acids increased significantly , and none showed a significant decrease . plasma pro , ser and gly levels increased continuously from day 1 until day 15 of lactation , whereas asn and met increased significantly from days 1 and 5 respectively until the end of lactation . these results suggest that the profiles of plasma amino acids show characteristic changes according to reproductive stage and that it may be necessary to consider such differences when performing amino acid - based diagnosis .

rabies diagnosis is based on fluorescent antibody testing ( fat ) in brain smears and inoculation of brain suspension either in mouse neuroblastoma cell cultures or intracerebrally in mice as confirmatory assays with high sensitivity and specificity for postmortem diagnosis [ 13 ] . these techniques are well suited for rapid and reliable routine diagnosis , if brain material is available . for other diagnostic specimens sampled intra vitam in case of clinical suspicion , for example , saliva , cerebrospinal fluid , or skin biopsies , more suited molecular techniques with excellent sensitivity have been developed and validated , mostly targeting the conserved nucleoprotein gene of the lyssavirus genome , for example , [ 410 ] . among these , there are protocols both for classical rt - pcr and for real - time rt - pcr , which adds speed , efficiency , contamination safety , and reliability to the technique combined with the potential to quantify the viral load [ 11 , 12 ] . a major advantage offered by these molecular techniques is the characterisation of viral isolates by sequencing the given amplification products followed by phylogenetic or phylogeographic analysis [ 1315 ] . the greatest challenge for these powerful novel techniques is the ever growing spectrum of known lyssavirus species / genotypes ( gt ) , probably all of which having the potential to cause animal and human rabies fatalities [ 16 , 17 ] . so far , 12 lyssavirus species and three as yet unclassified species have been identified . the development of classical simple or ( hemi)nested rt - pcr methods with particular emphasis on the diagnosis of a broad spectrum of lyssaviruses species has been published in a number of studies [ 1823 ] . also several real - time rt - pcr protocols suited for the broad detection or differentiation of several genotypes have been developed [ 9 , 2428 ] . is of particular interest for the rabies epidemiology in europe concerning the european bat lyssaviruses types 1 and 2 [ 2932 ] apart from classical rabies [ 33 , 34 ] . this assay uses genotype ( gt ) specific probes and fluorophore signals for direct differentiation of gt1 , gt5 , and gt6 in a single - tube reaction . the goal of this work was , based on a comprehensive review of the rich literature on the theme and adapting thereof , to develop , establish , and validate classical and real - time rt - pcr protocols for ( intravitam ) diagnosis of rabies and molecular - epidemiological characterisation of viral strains with the main emphasis on the growing number of known species / genotypes . all operations with potentially infectious material apart from centrifugation in closed tubes were performed in a class ii biological safety cabinet . frozen cell culture supernatants of rabies virus strains propagated previously on bhk-21 cells ( baby hamster kidney cells , atcc , manassas , usa ) were thawed , centrifuged at 2,000 g for 10 min at 4c , and filtrated using the millex - ha 450 nm filter ( millipore , cork , ireland ) . approximately 1 g of cerebellum , medulla oblongata , and hippocampus from unfixed , freshly obtained brain material was homogenized to a 20% brain suspension using a mortar and pestle , after addition of approximately 1 g of quartz sand ( merck kgaa , darmstadt , germany ) and 5 ml of modified eagle medium with earle 's salts with 2.2 g / l nahco3 ( bioswisstec ag , schaffhausen , switzerland ) supplemented with 5% penicillin 10,000 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) and 20% foetal calf serum ( fcs ; paa laboratories gmbh , pasching , austria ) . the suspension was then decanted into a 5 ml tube ( 5 ml polystyrene round - bottom tube ; bd biosciences , erembodegem , belgium ) to let it sediment for 1 hour at 4c and subsequently centrifuged at 1,400 g for 10 min and filtrated as above . frozen brain suspensions from former mouse inoculation tests were handled like frozen cell culture supernatant . 500 l of rna storage solution ( ambion , foster city , usa ) was added to 100200 l of fresh or previously frozen saliva samples or swabs , which were subsequently vortexed ( vortex genie 2 , tewis laborbedarf ag , berne , switzerland ) for 1 min and centrifuged in a biofuge pico ( heraeus holding gmbh , hanau , germany ) at 5,000 rpm for 10 min . cerebrospinal fluid ( csf ) samples were diluted 1 : 2 to 1 : 4 with rna storage solution ( ambion , foster city , usa ) . skin biopsies were shaved , cut into small pieces with a sterile pair of scissors , and further processed as described for brain suspension . a panel of 31 lyssaviruses ( including 4 species ) was used to test the diagnostic performance of our pcr ( table 1 ) . stocks of viral strains were produced in neuroblastoma cells ( mna 42/13 ) as described for the rabies tissue culture infection test ( rtcit , described below ) . the maintenance medium used for the first passage was dulbecco 's mem supplemented with 0.5% neomycin 50 iu / ml ( bioswisstec ag , schaffhausen , switzerland ) , 5% tryptose phosphate ( bioconcept ltd . amimed , allschwil , switzerland ) , 1% nonessential amino acids ( bioswisstec ag , schaffhausen , switzerland ) , 3% foetal calf serum , and 1% l - glutamine 200 mm ( bioswisstec ag , schaffhausen , switzerland ) to which 1% diethylaminoethyl - dextran ( deae - dextran ; sigma - aldrich corporation , st . dulbecco 's mem with 10% foetal calf serum was used for the 3 consecutive passages . staining of the cells was performed after each passage using rabies dfa reagent ( millipore , livingston , uk ) as a conjugate . the standard fluorescent antibody test ( fat ) was performed with brain tissue samples as previously described . rabies tissue culture infection test ( rtcit ) using four consecutive passages on murine neuroblastoma cells [ 4143 ] was applied to clinical specimens like brain specimens , liquor , saliva , or skin biopsies . primers , probes , and synthetic dna were obtained from microsynth ( microsynth gmbh , balgach , switzerland ) . the location of suitable n - directed primers and probes for heminested rt - pcr and real - time rt - pcr , respectively , was chosen and evaluated based on published work [ 8 , 18 , 22 , 25 ] . using multiple sequence alignment ( clustalx 2.0.3 program [ 4446 ] ) of the n gene region of the available lyssavirus species rabv ( 33 sequences ) , lbv ( 4 sequences ) , mokv ( 5 sequences ) , duvv ( 4 sequences ) , eblv-1 ( 17 sequences ) , eblv-2 ( 14 sequences ) , ablv , aravan , khujand , irkut , and west caucasian bat ( each one ) from genbank ( national center for biotechnology information and national library of medicine , rockville pike , usa ) , variable positions of primers and probes were adjusted with wobble positions for a potentially broader match . several probes for real - time rt - pcr for broadening the spectrum of detectable species were designed in this work ( table 2 ) . as internal control for conventional rt - pcr , amplification of gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was used . as internal inhibition control for real - time rt - pcr , primers and probes for the amplification of sendai virus ( atcc , manassas , usa ) , which was added to the samples before rna isolation , were used ( table 2 ) . synthetic dna with a length of 125 bases encompassing positions 48172 according to the pasteur virus genome ( x03673 ) of the following lyssavirus species were obtained from microsynth : aravan virus ( arav ) ef614259 , khujand virus ( khuv ) ef614261 , bokeloh bat lyssavirus ( bblv ) jf311903 , australian bat lyssavirus ( ablv ) af418014 , irkut virus ( irkv ) ef614260 , lagos bat virus ( lbv ) eu293110 , mokola virus ( mokv ) 293117 , shimoni bat virus ( shiv ) gu170201 , and west caucasian bat virus ( wcbv ) ef614258 . rna was isolated from 140 l of sample supernatant using the qiaamp viral rna mini kit ( qiagen , germantown , usa ) according to the manufacturer 's instructions , except the usage of rna storage solution ( ambion , foster city , usa ) for elution . heminested rt - pcr was performed as previously described using the onestep rt - pcr kit . briefly , for the first round 3 l of extracted rna was amplified in the rt - pcr mix prepared according to manufacturer 's instructions , supplemented with 0.6 m of each primer ( jw12-f , jw6as1-r1 , and jw6as2-r1 ; table 2 ) and 5 u rnase inhibitor ( rnasin plus , 40 u/l ; promega , madison , usa ) , with the following cycling conditions : 50c for 30 min and 95 for 15 min for reverse transcription and subsequent activation of the polymerase , followed by 10 cycles of 95c for 20 s , 60c for 30 s ( 1c decrease / cycle ) , followed by 72c for 30 s and 35 cycles of 95c for 20 s , 52c for 30 s , followed by 72c for 30 s. for the second round , the taq dna polymerase kit ( qiagen , germantown , usa ) was used with 2 l of the first round product , 2.5 l 10x pcr buffer , 1.2 mm final dntp concentration ( invitrogen , foster city , usa ) , 0.4 m of heminested primers ( jw12-f , jw10as1-r2 , and jw10as2-r2 ; table 2 ) , 0.5 u of taq dna polymerase , and 19.35 l nuclease free water ( ambion , foster city , usa ) . cycling conditions were as follows : 95c for 5 min followed by 35 cycles of 94c for 20 s , 52c for 20 s , followed by 72c for 30 s. amplifications were performed in a 2720 thermal cycler . internal controls using gapdh primers , as described in the one - round pcr , were run in parallel in the first round . gel electrophoresis was performed using a 1.5% tae agarose gel ( agarose le , analytical grade ; promega , madison , usa ) stained with ethidium bromide ( eurobio , courtaboeuf , france ) . for sequencing , the desired band was excised under uv light . real - time rt - pcr ( ndwd ) adapted from nadin - davis et al . and wakeley et al . [ 8 , 25 ] was performed using the quantitect probe rt - pcr kit ( qiagen , germantown , usa ) . the 25 l reaction volume consisted of 9.5 l rnase - free water , or 9 l in the multiplex mix , 12.5 l 2x quantitect rt - pcr master mix , 0.25 l of the rabvd1 forward and reverse primer ( 0.4 m final concentration each ) , 0.25 l of the probes rabvd1 , lysgt5 , and lysgt6 together in the multiplex mix or 0.25 l of the probes ivvwcbv - p , ivvmok - p , ivvablv2n - p , ivvlbv - p , ivvkhujand - p , and ivvsblv2-p ( table 2 ) alone in a mix ( all 0.2 m final concentration ) , 0.25 l of quantitect rt mix , and 2 l of sample rna . additionally , for the internal sendai virus control , 5 l of sample rna was added to a mix containing 6.625 l of rnase - free water , 12.5 l 2x quantitect rt - pcr master mix , 0.625 l of sendai forward primer , reverse primer , and probe mix ( sendai - f , sendai - r , and sendai - p , 8 m ; table 2 ) , and 0.25 l of quantitect rt mix . the reactions were carried out in microamp fast optical 96-well reaction plates with barcode 0.1 ml ( applied biosystems , foster city , usa ) in a 7500 fast real - time pcr system v1.3.1 ( applied biosystems , foster city , usa ) using the following cycling conditions : 1 cycle of 50c for 30 min and 95c for 15 min followed by 45 cycles of 95c for 15 sec and 50c for 1 min . amplification curve analysis was performed using the 7500 software v2.0.6 ( applied biosystems , foster city , usa ) . for cloning , a segment of viral genome covering a part of the n gene ( positions 1933 according to the pasteur virus genome accession number x03673 ) of cvs-11 was generated using primers designed for this purpose ( twclon - f and twclon - r , table 2 ) . amplification was performed using the onestep rt - pcr kit with the master mix consisting of 29.25 l of rnase - free water , 10 l 5x qiagen onestep rt - pcr buffer , 0.4 mm of each dntp , 0.6 m of the cloning primers , 10 u rnasin ( 40 u/l ) , and 2 l of qiagen onestep rt - pcr enzyme mixed with 5 l of extracted rna to a total volume of 50 l . amplifications were performed in a veriti 96-well thermal cycler ( applied biosystems , foster city , usa ) using the following cycling conditions : 30 min at 50c , followed by 15 min at 95c and 40 repetitive cycles of 1 min at 94c , 1 min with a temperature gradient from 56c to 46c in steps of 2c , and 1 min at 72c . after gel electrophoresis excised pcr fragments with a length of 933 bp were eluted with the qiaquick gel extraction kit ( qiagen , germantown , usa ) and cloned into the pcr - ii - topo vector using the topo ta cloning kit ( invitrogen , foster city , usa ) according to the manufacturer 's instructions . sequencing reactions were performed by microsynth ag , balgach , switzerland . for this purpose , reaction mixtures containing 22.5 ng dna per 100 bp , 30 pmol of each primer used for the amplification ( table 2 ) , and depc treated water ( ambion , foster city , usa ) to a final volume of 15 l were prepared . sequences were edited using the seqman ii v5.01 software ( dnastar , madison , usa ) and subsequently evaluated using the clone manager 9 software ( scientific & educational software , cary , usa ) . the 543 bp nucleotide sequences of the nucleoprotein gene obtained from the heminested pcr reaction , together with additional sequences retrieved from genbank , were saved in fasta file format . the sequences were then aligned using the clustalx 2.0.3 software and the genedoc software v2.5.0 . thirty - one lyssaviruses ( 19x rabv , 7x eblv-1 , 4x eblv-2 , and 1x duvv ) were analysed in rtcit . all isolates were viable in murine neuroblastoma cells and could be visualized using rabies dfa reagent as a conjugate . for the comparison with infectious titres , rna of the classical rabies virus strain cvs-11 was extracted from cell culture supernatants ( bhk-21 cells or neuroblastoma cells mna 42/13 ) and serially diluted from 10 to 10 . the infectious titre was determined according to the spearman - karber formula [ 49 , 50 ] in fluorescent focus forming doses 50% ( ffd50 ) . in the heminested rt - pcr ( hnrt - pcr ) , the amplification product of 582 bp was visible up to a dilution of 10 corresponding to a detection limit of 0.4 ffd50 ( 10 ffd50 ; figure 1 ) . in the real - time rt - pcr ( ndwd ) the limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive . using this definition , cvs-11 was detectable up to a dilution of 10 ( once at a dilution of 10 ) . the amplification plot showed regular intervals of the curves with ct - values from 18.1 ( dilution 10 ) to 40.0 ( dilution 10 ) . relating to infectious titre real - time rt - pcr reached a detection limit of 0.003 ffd50 ( 10 ) for cvs-11 . the efficiency of the real - time rt - pcr ( ndwd ) was determined using linear regression of the ct - values and the titres of the samples ( figure 2 ) . on the basis of the slope coefficient the limit of detection in terms of dna copy numbers was determined using 10-fold serial dilutions of a cloned 933 bp segment of n of cvs-11 containing 10 to 10 dna copies / pcr reaction . in the hnpcr , the limit of detection was 100 dna copies/3 l of starting material of the cvs-11 plasmid . in the real - time pcr ( ndwd ) , the 10-fold serial dilution of the cvs - plasmid was tested in triplicate . the limit of detection was 10 dna copies/2 l of the cvs-11 plasmid with ct - values between 38.6 and 47.5 . the efficiency was at 82.8% with a slope coefficient of 3.82 ( not shown ) . the intra - assay repeatability of the real - time pcr ( ndwd ) proved to be excellent with very low coefficients of variation up to 10 dna copies ( 0.181.37% ) with and outlier at 10 copies ( 12.05% ; table 3 ) . a total of 31 lyssaviruses ( 19 identified as classical rabv , 7 as eblv-1 , 4 as eblv-2 , and 1 as duvv ) were used to test the diagnostic performance of the pcr ( table 1 ) . all viruses used were detected with the expected band size in the hnrt - pcr technique ( table 4 ) . the specificity of the amplified product was confirmed as rabv , eblv-1 , eblv-2 , or duvv by sequencing in each case . host gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control , indicating that sample material was present and rna isolation , reverse transcription , and amplification were not inhibited . all samples were also tested positive by the real - time rt - pcr assay ( table 4 ) . sendai virus as internal control for inhibition was run in parallel using the same cycling conditions . in the real - time rt - pcr ( ndwd ) with an annealing temperature of 50c , some samples were detected with more than one probe . with the exception of the sample derived from a raccoon dog from poland , the probe for the homologous genotype always yielded the lowest ct - value . in this exceptional case , the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 ( 15.0 versus 16.1 ; table 4 ) . comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the rabvd1-p and lysgt6-p probe , respectively ( figure 3 ) . duvenhage virus , also designated as genotype 4 , was detectable with the probe adapted for genotype 6 in spite of 3 mismatches ( lysgt6-p ) . the probe lysgt6-p was able to detect synthetic dna fragments of bblv , arav , and irkv , although containing one or two mismatches , respectively . the synthetic dna fragments of khuv , ablv , lbv , mokv , shibv , and wcbv , which exhibited from four ( ablv / khuv ) up to ten ( wcbv ) mismatches to the best fitting probe for gt1 , gt6 , or gt5 , respectively , were all detected with the corresponding species - specific new probe ( table 2 ) . 10 copies of the synthetic dna were detected at ct - values of 27.933.5 for ablv , mokv , shibv , and wcbv ( not shown ) . the efficiency of detection of khuv , which showed an atypical amplification plot at the highest number of 10 copies of the synthetic dna , and that of lbv was lower with ct - values of 40.0 and 40.5 at a copy number of 10 , respectively ( not shown ) . multiplexing of combinations of these probes ( ivvlbv - p ( fam : 6-carboxyfluorescein ) , ivvkhujand - p ( yy : yakima yellow ) , and ivvsblv2-p ( cy5 : cyanine 5 ) , as well as ivvwcbv - p ( fam ) , ivvmok - p ( yy ) , and ivvablv2n - p ( cy5 ) ) as for the species 1 , 5 , and 6 was not successful . the 543 bp amplification products of the nucleoprotein gene obtained in the heminested pcr reactions were sequenced ( table 1 ) and analysed phylogenetically with additional sequences retrieved from genbank representing all known lyssaviruses . the similarity of the 543 bp nucleotide sequences of the nucleoprotein gene ( positions 74616 according to the pasteur virus genome ) among the lyssaviruses included ranged from 64.6% to 90.1% ( hamming distance ) . the resulting phylogenetic tree obtained by the neighbor - joining method implemented in the mega5 software is presented in figure 4 . clinical specimens like brain suspensions ( 1x human , 12x mouse ) , skin biopsies from the nape of the neck ( 3x human , 1x mouse ) , saliva ( 4x human ) , and cerebrospinal fluids ( 6x human ) were used for the establishment of the assays . both hnrt - pcr and real - time rt - pcr ( ndwd ) were shown to work properly on all these samples without significant inhibition using gapdh as internal control for conventional rt - pcr and sendai virus for real - time rt - pcr ( ndwd ) , which was mixed to the samples before rna isolation . skin biopsies taken from the neck and lip of a mouse ( white swiss mouse at an age of 3 weeks ) euthanized 2 weeks after intracerebral infection with sad bern virus 20 years ago were positive with the hnrt - pcr and real - time pcr ( ndwd ) . skin biopsies taken from other locations on the head were positive with the real - time pcr ( ndwd ) and weakly positive in hnrt - pcr whereas samples taken around the vibrissae were weakly positive in real - time pcr ( ndwd ) only . sequencing of amplification products excluded a contamination with the positive cvs-11 control ( not shown ) . brain suspension from an imported human rabies case in 2012 was already strongly positive after one round of the hnrt - pcr . phylogenetic analysis of the 543 bp nucleotide fragment revealed its close relationship to the classical rabies virus strains circulating in the insectivorous mexican free - tailed bat , tadarida brasiliensis , a species common in the southern united states and mexico . this allowed identification of the origin of the patient 's infection , who had travelled extensively and did not report any previous biting incident [ 51 , 52 ] . based on a large amount of published work , we were able to establish and quantitatively characterise rt - pcr protocols for the detection of lyssaviruses in clinical samples . well suited real - time protocols [ 8 , 25 ] for the detection of classical rabies virus and the genotypes 5 and 6 ( eblv-1 and eblv-2 ) were adapted and extended for the detection of at least 13 lyssavirus species . to this goal , 6 species - specific probes ( khuv , ablv , lbv , mokv , shibv , and wcbv ) were designed and verified on synthetic dna fragments encompassing the targeted sequence of the lyssaviral nucleoprotein gene . multiplexing the probes in a single - tube reaction as described for the genotypes 1 , 5 , and 6 was not possible with the newly designed probes , probably due to false priming and/or interference within the mix of reagents and synthetic target sequence . as far as evaluated with the multiplexed probes for the genotypes 1 , 5 , and 6 using 31 viral isolates , which were all detectable with high sensitivity , direct differentiation of targeted genotypes on the base of the quantitative reaction ( ct - values ) was mostly possible . furthermore , a single probe for genotype 6 ( species eblv-2 ) was able to detect up to 7 genotypes / species . we consider this type of cross hybridization as an advantage of the technique using degenerate primers and probes in terms of a broad detectability of lyssavirus rather than a lack of accuracy of discrimination . in an approach using sybr green qpcr with similarly degenerate primers spanning the same part of n as in this work , the detection of all lyssavirus species known at that time was achieved . using several genotype - specific probes in a taqman real - time protocol , we were able to add more intrinsic confidence to the specificity of the analysis . quantitative characterisation of the assay using the probe for genotypes 1 , 5 , and 6 on cvs-11 showed excellent sensitivity and repeatability with a detection limit of as low as an infectious dose of 0.003 ffd50 combined with an analytical sensitivity of 10 dna copies at efficiencies of 94.5% and 82.8% , respectively . considering the amount of degeneration in both primers and probes , the efficiencies determined must be considered satisfactory . as far as the absolute detection limit in terms of target copies is concerned , the usage of plasmid dna rather than rna must be kept in mind . since reverse transcription can not be assumed as 100% efficient and reproducible , the limit for rna might be somewhat lower . for further characterisation of samples with a positive reaction in real - time rt - pcr , excellent simple and ( hemi)nested rt - pcr protocols are available . with the protocol used for this work , all 31 viral strains used could be amplified and sequenced , confirming the diagnostic broadness of the assay . phylogenetic analysis of these partial nucleotide sequences belonging to genotypes 1 , 4 , 5 , and 6 along with known sequences confirmed the suitability of this relatively conserved genomic region for molecular - epidemiological characterisation of lyssaviruses circulating worldwide [ 5658 ] . this was also confirmed in the context of a human rabies case imported to switzerland in 2012 , which could be attributed unequivocally to an exposure to a bat of the species tadarida brasiliensis in california , usa . in this work we could show and validate the suitability of an adapted and further developed real - time rt - pcr protocol for the rapid , efficient , and highly sensitive intravitam diagnosis of a wide spectrum of lyssavirus species followed by rapid molecular - epidemiological characterisation of viral isolates respective to origin of the virus and source of exposure , using heminested rt - pcr . this new tool is also particularly promising for active surveillance of european bat lyssaviruses using oral swabs in live - captured bats .

the causative agents of rabies are single - stranded , negative - sense rna viruses in the genus lyssavirus of rhabdoviridae , consisting of twelve classified and three as yet unclassified species including classical rabies virus ( rabv ) . highly neurotropic rabv causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome . rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health . due to growing variety of the genus lyssavirus observed , the development of suitable molecular assays for diagnosis and differentiation is challenging . this work focused on the establishment of a suitable real - time rt - pcr technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation . the real - time rt - pcr was adapted with the goal to detect the whole spectrum of lyssavirus species , for nine of which synthesized dna fragments were used . for the detection of species , seven probes were developed . serial dilutions of the rabies virus strain cvs-11 showed a 100-fold higher sensitivity of real - time pcr compared to heminested rt - pcr . using a panel of thirty - one lyssaviruses representing four species , the suitability of the protocol could be shown . phylogenetic analysis of the sequences obtained by heminested pcr allowed correct classification of all viruses used .

intraocular pressure ( iop ) is one the most important modifiable risk factors for glaucoma progression.13 iop measurements in the ophthalmology clinic are often limited to office hours , which do not fully represent the nychthemeral iop peaks and fluctuations.4,5 previously , 24-hour iop monitoring studies have reported fluctuation as high as 8.21.4 mmhg , but many of these studies measured iop in the sitting position , overnight in a hospital environment , and over a few sampling periods , which does not truly reflect the physiological and normal setting of one s daily living , especially when the subjects are awakened at night for iop measurements.610 various studies have reported the significance of iop fluctuation on glaucoma progression.11,12 while there are studies that report otherwise,13,14 much of the existing literature has only documented inter - visit or daytime iop variability over a limited number of hours . this is at least partly due to limitations of how frequently repeated tonometric iop measurements can be taken . a contact lens - based sensor ( cls ) is nowadays available for the recording of ocular dimensional profiles for up to 24 hours.15 this device has been shown to be safe and tolerable in healthy subjects and glaucoma patients as well as to provide reproducible recording of 24-hour profiles.1618 the aim of this study was to analyze the 24-hour ocular dimensional profile in normal - tension glaucoma ( ntg ) patients on medical treatment . informed patient consent and approval by the institutional review board of the hospital authority of hong kong were obtained prior to study commencement . this was a prospective cohort study from july 2012 to june 2013 , conducted at a university hospital in hong kong . the study recruited consenting adults ( age > 18 years old ) with unilateral or bilateral ntg who were currently on topical antiglaucoma medications . ntg was defined as open angle on gonioscopy ; progressive thinning of the retinal nerve fiber layer ( rnfl ) on optical coherence tomography , with corresponding glaucomatous visual field changes on the humphrey visual field analyzer ; and an iop 21 mmhg on all clinical visits based on previous medical records . cases with previous glaucoma surgery or laser treatment , active or previous corneal disease , and subjects with only one functional eye were excluded . the sensimed triggerfish ( sensimed ag , lausanne , switzerland ) is a soft silicone cls that enables recording of the ocular dimensional profile over a 24-hour period with minimal disturbance to one s daily routines and sleep cycles . such 24-hour profiles are related to the 24-hour iop profiles.18 dimensional changes are recorded in the corneoscleral area for 30 seconds every 5 minutes over 24 hours and each recording burst represents 300 data points , the medians of which are plotted as a single graph which makes up the 24-hour ocular dimensional profile measured in sensory output units of millivolt equivalents ( mveq ) . the device was ce - marked and thus approved for clinical use in 2009 . in healthy volunteers and glaucoma patients , the cls was found to be tolerable in normal activities of daily living and during sleep . a contact lens with base curve 8.7 mm was also found to be well adapted in most eyes , although the device also exists in steeper ( 8.4 mm ) and flatter ( 9.0 mm ) base curves.1618 the cls was placed on the subject s eye by an ophthalmologist in the outpatient clinic after a slit - lamp examination of the anterior segment and goldmann applanation tonometry ( gat ) by a single investigator . for those with unilateral disease , the cls was placed on the eye with ntg . for those with bilateral disease , a random eye assignment by card shuffling was used to determine the eye for the cls placement . the subject then returned home with lubricating eyedrops and carried on his or her daily activities ( both indoor and outdoor ) , apart from showering or swimming ( as the device can not be in contact with water ) . subjects continued their same regimen of antiglaucoma eyedrops and slept in their habitual position at night . after 24 hours , the subject returned to the clinic to have the cls removed followed by a slit - lamp examination and gat . cls profile parameters were extracted following smoothing of the profile using locally weighted polynomial regression . cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) . diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) . sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively . number of peaks : a peak is defined as a local maximum point in the smoothed curve . the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak . the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test . whitney u - test : nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers . nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers . fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications . cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) . diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) . sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively . number of peaks : a peak is defined as a local maximum point in the smoothed curve . the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak . the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test . whitney u - test : nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers . nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers . fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications . in the 18 subjects enrolled in the study , there were seven males and eleven females . gat iops before and after the cls wear were 15.32.2 and 13.81.7 mmhg , respectively , while on the same antiglaucoma medications ( p=0.05 ) . the mean rnfl thickness was 72.99.8 m and the mean deviation and pattern standard deviation on humphrey visual field analysis were 6.34.5 decibels ( db ) and 6.14.1 db , respectively . in the 18 subjects , 38.9% ( 7/18 ) used a single prostaglandin eyedrop administered in the evening , 44.4% ( 8/18 ) used a twice - daily non - prostaglandin eyedrop , and 17.6% ( 3/17 ) used both a nocturnal prostaglandin eyedrop plus a twice - daily antiglaucoma eyedrop . the mean 24-hour variability from the mean cls signal was 75.921.5 ( range : 35.7120.9 ; 95% confidence interval [ ci ] : 62.886.7 ) mveq . the mean nocturnal variability from the mean was 52.120.3 ( range : 23.9105.1 ; 95% ci : 41.662.5 ) mveq , which was 48.9% less than the mean diurnal variability from the mean ( 77.620.6 ) ( range : 43.9118.4 ; 95% ci : 66.988.2 ) mveq ( p=0.002 ) . there were no statistically significant differences in the mean diurnal , nocturnal , or 24-hour variability between those with and without prostaglandin analog treatment ( all p>0.1 ) ( table 1 ) . the mean number of peaks during sleep and daytime was 6.42.3 and 9.32.4 , respectively . the number of peaks was 54.7% less during the nocturnal period than the diurnal period ( p=0.001 ) . there was no significant difference between the frequencies of nocturnal peaks among those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications ( table 2 ) . the mean sleep time of subjects was from 8.53 pm 12.6 minutes to 7.06 am 3.6 minutes . the study population had a mean positive linear slope on their cls profile from wake - to - sleep with a mean of 53.242.9 mveq / hour , signifying an increase in the ocular dimensional profile when going to sleep . similarly , the population had a mean negative linear slope from sleep - to - wake with a mean of 42.547.6 mveq / hour , signifying a decrease in the ocular dimensional profile upon waking . on comparing the two slopes , the rate of increase when going to sleep was significantly greater than the rate of decrease upon waking ( p<0.001 ) ( table 3 ) . the ocular dimensional profiles of all 18 ntg subjects were unique , with individual peaks occurring at different time intervals throughout the day and night ; no two tracings were identical . the mean ocular dimensional profile of the study population increased during sleep ( positive wake - to - sleep slope ) and decreased upon waking ( negative sleep - to - wake slope ) , with the mean rate of increase during sleep being significantly greater than the rate of decrease upon waking ( p<0.001 ) . we are inclined to agree with previous postulations , wherein the increase in nocturnal iop was thought to be related to the gravitational pull of fluids to the eye and an increase in episcleral venous pressure during the supine sleep posture.19 through the use of the 24-hour cls , we affirm that the phenomenon of increased iop with supine posturing was consistent among ntg subjects under medical treatment . other authors have also observed the persistence of posture - induced iop changes despite treatment of ntg patients with iop - lowering medication.20 similarly , a previous japanese study reported , in a population of ntg subjects , that the iop spikes recorded from the habitual positions ( sitting up during the day and supine at night ) were positively correlated to the spikes induced from a postural - change test and after a water - drinking test.21 in contrast , renard et al reported that , in 27 subjects with suspected ntg , 24-hour iop monitoring revealed that 54.5% exhibited a diurnal acrophase and 36.4% exhibited a nocturnal acrophase , while 9.1% had no nychthemeral rhythm in the absence of medical treatment.22 the existence of concomitant obstructive sleep apnea syndrome in 80% of assessed patients could have influenced the observed iop rhythm in their cohort.22 before the availability of continuous 24-hour ocular dimensional profile recording , it has been reported that , in many glaucoma patients , the iop peaks occurred outside of office hours23,24 and that two - thirds of iop peaks in untreated glaucoma patients occurred during the nocturnal period.25 in our ntg population , we noted that there were 55% less peaks occurring nocturnally than diurnally ( p=0.001 ) . the reduction in the number of peaks at night in our ntg population could be related to the use of prostaglandin antiglaucoma eyedrops , since 57% of our subjects were at least on prostaglandin antiglaucoma eyedrops nocturnally . however , it may also be simply due to the absence of physical activity , eye blinking , and saccadic movements at night , as these parameters are known to cause iop spikes and fluctuations.26 other authors have suggested that the nocturnal iop and iop spikes may be related to visual field progression in ntg subjects.21 study on the effect of medical treatment on iop fluctuations has shown that prostaglandin antiglaucoma eyedrops offer the best sustained 24-hour iop reduction , while brimonidine offers the least.27 in our study , the mean nocturnal variability was significantly lower than the mean diurnal variability by 49% ( p=0.002 ) . our findings are in agreement with a previous study by pajic et al28 reporting higher coefficients of variation in cls data during the daytime than at nighttime in ntg patients . pajic et al reported reduced variability in nocturnal cls data independent of whether or not patients were on antiglaucoma medication.28 we have not recorded cls profiles in our patients without medical treatment and therefore do not know whether the same is valid for this patient cohort ; however , it seems plausible that the lower level of activity during sleep provides a natural reduction in variability , which iop - lowering medical treatment may further influence . holl et al recorded cls profiles in primary open - angle glaucoma and ocular hypertension patients and found no difference in 24-hour , diurnal , or nocturnal standard deviation of cls values in the presence of prostaglandin analog treatment as compared to without treatment.29 the variability of diurnal versus nocturnal cls values was not evaluated . thus , in this patient cohort , it seems that , while the overall ocular dimensional profile was higher at night , the number of peaks and variability from the mean were significantly reduced at night for ntg subjects on medical treatment . it was interesting to note that the gat iop after cls wear ( 13.81.7 mmhg ) was lower ( p=0.05 ) than the iop before the cls wear ( 15.32.2 mmhg ) , although the readings were measured exactly 24 hours apart . this seemingly lower iop after the cls use was short of statistical significance and can be explained by day - to - day variations in iop . there was no sample size calculation , and the relatively small sample size of the study did not allow for evaluation of differences in iop - related pattern nor stratification by the type of treatment . furthermore , only one 24-hour measurement with the cls was performed , therefore we could not ascertain the reproducibility of the 24-hour iop profile . at present , the ocular dimensional profile is something that can not be translated into iop and therefore has no validation for use in clinical practice . additional clinical research needs to be done before we can state ( based on evidence ) that the cls can be useful in optimizing the timing of medical therapy for individual patients . nevertheless , this is one of the few publications in the literature analyzing the 24-hour ocular dimensional profile on a continuous basis in ntg subjects receiving medical treatment . continuous 24-hour ocular dimensional profiles recorded in ntg patients on medical treatment revealed that the profile increased during sleep and decreased upon waking . there was a 50% reduction in both the variability from the mean and number of peaks during sleep as compared to daytime .

purposeto analyze the 24-hour ocular dimensional profile in normal - tension glaucoma ( ntg ) patients on medical treatment.methodsconsecutive , medically treated ntg subjects were recruited from a university eye center . subjects were on a mean of 1.70.7 types of antiglaucoma medications and 56.6% were on a prostaglandin analog . a contact lens - based sensor device was worn in one eye of ntg patients to record the intraocular pressure ( iop)-related profile for 24 hours , recording the following : variability from mean over 24 hours , nocturnally and diurnally , as well as the number of peaks and troughs diurnally and nocturnally.resultsin 18 ntg subjects , the nocturnal variability around the mean contact lens - based sensor device signal was 48.9% less than the diurnal variability around the mean . the number of peaks was 54.7% less during the nocturnal period than during the diurnal period . the rate of increase in the ocular dimensional profile when going to sleep was significantly greater than the rate of decrease upon waking ( p<0.001).conclusionin medically treated ntg subjects , there was more variability in the iop - related pattern during the daytime and there were fewer peaks during sleep .

high - throughput techniques have become common in the study of these alterations . however , the analysis of this type of data is challenging . one of the main difficulties is in sorting out the alterations that drive tumorigenesis from those that are only byproducts of the high number of divisions cancer cells undergo and have no effect on the cancerogenic phenotype . moreover , the existence of different types of alteration makes the detection of causative ones even more difficult . hence , it is clear the need for approaches to analyze and integrate cancer genomics data . intogen integrates high - throughput data related to different types of alterations taking place in cancer such as copy number alterations , point mutations and transcriptomic changes from many independent studies to identify the genes and modules ( e.g. kegg pathways , go terms ) more significantly altered in different tumor types ( 1 ) . the data in intogen consists of publicly available cancer genomic studies collected from databases such as gene expression omnibus ( geo ) ( 3 ) , arrayexpress ( 4 ) , cosmic ( 5 ) , progenetix ( 6 ) , the sanger cancer genome project ( http://www.sanger.ac.uk/genetics/cgp/ ) and the data portal of the cancer genome atlas ( 7 ) . each study contains results from high - throughput analyses of a number of human primary tumor samples compared to normal cells ( normal cells of the same tissue in the case of expression ) related to one or more types of cancer for a specific alteration . at the first step , all the samples in the study are annotated with appropriate terms from international classification of disease for oncology ( icd - o ) ( 8) : a topography term indicating location in human body and , if available , a morphology term describing histological classification . studies are also annotated with the platform(s ) used for the experiment . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units , which correspond to a set of assays in one experiment annotated with the same topography and morphology . analysis units are also created with assays in one experiment annotated with the same topography and any morphology ( figure 1 ) . analysis units. table 1 summarizes the number of studies , experiments and analysis units included in the current version of intogen ( v03 ) . each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units in two ways , 1 ) according to the topography and the morphology , and 2 ) according to the topography ( with the morphology annotated as any morphology ) . table 1.summary of the data content in intogen ( v03)alteration typemain data sourcesnumber of independent studiesnumber of experimentsnumber of analysis unitstranscriptomicgeo118122243arrayexpresstcgagenomic ( copy number)progenetix188188343sanger cancer genome projecttcgatotal306310586 data annotation and classification . each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units in two ways , 1 ) according to the topography and the morphology , and 2 ) according to the topography ( with the morphology annotated as any morphology ) . summary of the data content in intogen ( v03 ) in intogen framework , the analysis is performed at different levels : on one side each experiment is analyzed independently ( experiment level ) and those experiments classified with the same topography and morphology terms are combined ( combination level ) . also on the other side , the analysis is performed at the level of genes ( gene level ) and at the level of modules ( module level ) . a module is defined by a set of genes with some biological property in common , we currently analyze gene ontology ( go ) modules , kegg pathway modules , modules derived from genes sharing a transcription factor - binding side ( tfbs ) in their promoter and genes sharing microrna target motifs in their 3-utr [ see ref . first , within each analysis unit , we identify genes altered in more samples than expected by chance using oncodrive [ see ref . the results for the same gene are combined statistically across the analyzed experiments classified with the same topography and morphology terms using the weighted z - method ( 9 ) . if the study contains enough samples ( at least 20 ) for which morphology type information is known , then detection of significant alterations can be done at the level of topography and the level of morphology . the limit of 20 samples was setup to increase the reliability of results , as we consider that smaller number of replicates in a large - scale study can lead to anomalous conclusions ( 1 ) . in this way , alterations specific to certain morphology type can be identified as well as those common to the topography of the cancer in general . after significantly altered genes are detected , enrichment analysis is done to find significantly altered modules ( e.g. biological processes or pathways ) per experiment . in the same way as before , the results for the same module are combined across studies that analyze the same cancer type . each analysis unit ( set of assays from the same study using the same platform and annotated with the same icd - o terms ) is analysed to detect the significantly altered genes . the experiment results with the same icd - o terms are combined both at the gene level and at the module level . for methods details each analysis unit ( set of assays from the same study using the same platform and annotated with the same icd - o terms ) is analysed to detect the significantly altered genes . the experiment results with the same icd - o terms are combined both at the gene level and at the module level . for methods details see ( 1 ) . as can be expected , the interpretation of these highly inter - related results requires powerful visualization methods . the browser of intogen facilitates the exploration and intuitive visualization of results at different levels ( available at : http://www.intogen.org ) , while the biomart portal ( 2 ) ( available at : http://biomart.intogen.org ) allows complex queries and facilitates the bulk download of the all analysis results . in intogen biomart portal , users can query for three types of data . for each type , there is a database ; intogen experiments , intogen combinations and intogen oncomodules ( table 2 ) . table 2.databases and data sets in the biomart of intogendatabasesdata setsdescriptionexperimentsgene genomic alterationsrecurrence and significance of genomic alteration ( gain and loss ) for each gene at the level of experimentsgene transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each gene at the level of experimentskegg genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each kegg pathway at the level of experimentskegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each kegg pathway at the level of experimentsgo genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each go term at the level of experimentsgo transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each go term at the level of experimentstfbs genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each tf at the level of experimentstfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each tf at the level of experimentsmirna genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each mirna at the level of experimentsmirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each mirna at the level of experimentscombinationsgene genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each gene at the level of combinations ( tumor types and subtypes)gene transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each gene at the level of combinations ( tumor types and subtypes)kegg genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each kegg pathway at the level of combinations ( tumor types and subtypes)kegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each kegg pathway at the level of combinations ( tumor types and subtypes)go genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each go term at the level of combinations ( tumor types and subtypes)go transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each go term at the level of combinations ( tumor types and subtypes)tfbs genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each tf at the level of combinations ( tumor types and subtypes)tfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each tf at the level of combinations ( tumor types and subtypes)mirna genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each mirna at the level of combinations ( tumor types and subtypes)mirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each mirna at the level of combinations ( tumor types and subtypes)oncomodulescombinationssets of genes significantly altered in each cancer type and subtypeexperimentssets of genes significantly altered in each experiment databases and data sets in the biomart of intogen in intogen experiments database , there are data sets for genomic and transcriptomic alterations . users can query the results of the recurrence analysis for these alterations at the level of genes or modules , such as kegg pathways and go categories , for each experiment included in intogen . for both types of data sets , the results can be filtered in many different ways . here are a few examples : genes annotated with a list of go ids , genes in a specific chromosomal band , a list of selected entrez / ensembl gene ids . the user can also filter by significance level and the results can be restricted to experiments done by specific authors , performed on a specific platform type , etc . the columns in the results can be determined by the selections done in attributes section . for experiment - level data , there are a number of statistics derived from the analysis that can be retrieved such as the number of samples in the experiment , the expected / observed number of alterations and p - values etc . finally , a table that contains the selected attributes for the genes or modules is retrieved . in intogen combinations database , users can query the results for combinations , that is , the integration of results from experiments annotated with the same icd - o terms . this database includes data sets for genomic and transcriptomic alterations for genes and modules . the filters and attributes works in a similar way as in experiment database but without publication nor platform attributes and filters , and including the results attributes specific to the combination method . in intogen oncomodules database , there are two data sets , one for combinations and one for experiments . each data set contains lists of genes that are significantly altered in a specific combination of icd - o terms or in a specific experiment . again the user can filter the results in a variety of ways , with the significance level he / she likes , according to certain characteristics of genes , for a cancer type and , in the case of oncomodules at the level of experiments , for author or platform type . the data in intogen consists of publicly available cancer genomic studies collected from databases such as gene expression omnibus ( geo ) ( 3 ) , arrayexpress ( 4 ) , cosmic ( 5 ) , progenetix ( 6 ) , the sanger cancer genome project ( http://www.sanger.ac.uk/genetics/cgp/ ) and the data portal of the cancer genome atlas ( 7 ) . each study contains results from high - throughput analyses of a number of human primary tumor samples compared to normal cells ( normal cells of the same tissue in the case of expression ) related to one or more types of cancer for a specific alteration . at the first step , all the samples in the study are annotated with appropriate terms from international classification of disease for oncology ( icd - o ) ( 8) : a topography term indicating location in human body and , if available , a morphology term describing histological classification . studies are also annotated with the platform(s ) used for the experiment . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units , which correspond to a set of assays in one experiment annotated with the same topography and morphology . analysis units are also created with assays in one experiment annotated with the same topography and any morphology ( figure 1 ) . analysis units. table 1 summarizes the number of studies , experiments and analysis units included in the current version of intogen ( v03 ) . each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units in two ways , 1 ) according to the topography and the morphology , and 2 ) according to the topography ( with the morphology annotated as table 1.summary of the data content in intogen ( v03)alteration typemain data sourcesnumber of independent studiesnumber of experimentsnumber of analysis unitstranscriptomicgeo118122243arrayexpresstcgagenomic ( copy number)progenetix188188343sanger cancer genome projecttcgatotal306310586 data annotation and classification . each sample assay in a study is annotated for the platform and the icd - o topography and morphology terms . an experiment in intogen consists of a set of assays coming from the same study that have been performed with the same platform . analysis units in two ways , 1 ) according to the topography and the morphology , and 2 ) according to the topography ( with the morphology annotated as in intogen framework , the analysis is performed at different levels : on one side each experiment is analyzed independently ( experiment level ) and those experiments classified with the same topography and morphology terms are combined ( combination level ) . also on the other side , the analysis is performed at the level of genes ( gene level ) and at the level of modules ( module level ) . a module is defined by a set of genes with some biological property in common , we currently analyze gene ontology ( go ) modules , kegg pathway modules , modules derived from genes sharing a transcription factor - binding side ( tfbs ) in their promoter and genes sharing microrna target motifs in their 3-utr [ see ref . first , within each analysis unit , we identify genes altered in more samples than expected by chance using oncodrive [ see ref . the results for the same gene are combined statistically across the analyzed experiments classified with the same topography and morphology terms using the weighted z - method ( 9 ) . if the study contains enough samples ( at least 20 ) for which morphology type information is known , then detection of significant alterations can be done at the level of topography and the level of morphology . the limit of 20 samples was setup to increase the reliability of results , as we consider that smaller number of replicates in a large - scale study can lead to anomalous conclusions ( 1 ) . in this way , alterations specific to certain morphology type can be identified as well as those common to the topography of the cancer in general . after significantly altered genes are detected , enrichment analysis is done to find significantly altered modules ( e.g. biological processes or pathways ) per experiment . in the same way as before , the results for the same module are combined across studies that analyze the same cancer type . each analysis unit ( set of assays from the same study using the same platform and annotated with the same icd - o terms ) is analysed to detect the significantly altered genes . the experiment results with the same icd - o terms are combined both at the gene level and at the module level . for methods details each analysis unit ( set of assays from the same study using the same platform and annotated with the same icd - o terms ) is analysed to detect the significantly altered genes . the experiment results with the same icd - o terms are combined both at the gene level and at the module level . for methods details as can be expected , the interpretation of these highly inter - related results requires powerful visualization methods . the browser of intogen facilitates the exploration and intuitive visualization of results at different levels ( available at : http://www.intogen.org ) , while the biomart portal ( 2 ) ( available at : http://biomart.intogen.org ) allows complex queries and facilitates the bulk download of the all analysis results . in intogen biomart portal , users can query for three types of data . for each type , there is a database ; intogen experiments , intogen combinations and intogen oncomodules ( table 2 ) . table 2.databases and data sets in the biomart of intogendatabasesdata setsdescriptionexperimentsgene genomic alterationsrecurrence and significance of genomic alteration ( gain and loss ) for each gene at the level of experimentsgene transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each gene at the level of experimentskegg genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each kegg pathway at the level of experimentskegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each kegg pathway at the level of experimentsgo genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each go term at the level of experimentsgo transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each go term at the level of experimentstfbs genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each tf at the level of experimentstfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each tf at the level of experimentsmirna genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each mirna at the level of experimentsmirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each mirna at the level of experimentscombinationsgene genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each gene at the level of combinations ( tumor types and subtypes)gene transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each gene at the level of combinations ( tumor types and subtypes)kegg genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each kegg pathway at the level of combinations ( tumor types and subtypes)kegg transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each kegg pathway at the level of combinations ( tumor types and subtypes)go genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for each go term at the level of combinations ( tumor types and subtypes)go transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for each go term at the level of combinations ( tumor types and subtypes)tfbs genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each tf at the level of combinations ( tumor types and subtypes)tfbs transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each tf at the level of combinations ( tumor types and subtypes)mirna genomic alterationsrecurrence and significance of genomic alterations ( gain and loss ) for putative targets of each mirna at the level of combinations ( tumor types and subtypes)mirna transcriptomic alterationsrecurrence and significance of transcriptomic alterations ( upregulation and downregulation ) for putative targets of each mirna at the level of combinations ( tumor types and subtypes)oncomodulescombinationssets of genes significantly altered in each cancer type and subtypeexperimentssets of genes significantly altered in each experiment databases and data sets in the biomart of intogen in intogen experiments database , there are data sets for genomic and transcriptomic alterations . users can query the results of the recurrence analysis for these alterations at the level of genes or modules , such as kegg pathways and go categories , for each experiment included in intogen . for both types of data sets here are a few examples : genes annotated with a list of go ids , genes in a specific chromosomal band , a list of selected entrez / ensembl gene ids . the user can also filter by significance level and the results can be restricted to experiments done by specific authors , performed on a specific platform type , etc . the columns in the results can be determined by the selections done in attributes section . for experiment - level data , there are a number of statistics derived from the analysis that can be retrieved such as the number of samples in the experiment , the expected / observed number of alterations and p - values etc . finally , a table that contains the selected attributes for the genes or modules is retrieved . in intogen combinations database , users can query the results for combinations , that is , the integration of results from experiments annotated with the same icd - o terms . the filters and attributes works in a similar way as in experiment database but without publication nor platform attributes and filters , and including the results attributes specific to the combination method . in intogen oncomodules database , there are two data sets , one for combinations and one for experiments . each data set contains lists of genes that are significantly altered in a specific combination of icd - o terms or in a specific experiment . again the user can filter the results in a variety of ways , with the significance level he / she likes , according to certain characteristics of genes , for a cancer type and , in the case of oncomodules at the level of experiments , for author or platform type . query # 1 use a list of genes to check whether they have been significantly gained or lost in the topology breast . databasedata setsfiltersattributesdatabase : intogen combinationsgene genomic alterationsgenes : i d list limit by a file with ids ( ensembl , entrez etc.)genes > ensembl > gene ensembl idicd - o topography and morphology : breast ; any morphologygenes > ensembl > gene symbolreferences > external references > entrez gene idresults > genomics > gain p - valueresults > genomics > loss p - value the result of high - throughput analysis is usually a list of genes such as genes significantly deregulated in an expression experiment . as resources are limited , for downstream analysis , this gene list must be prioritized . one way to do this is to check if the individual genes are altered in any way in the panel of cancer experiments in intogen . in query 1 , the user can download the combined results for genomic alterations filtering them with their gene list by clicking on the i d list limit box and specifying the type of i d they use . for example , in order to filter using gene symbols such tp53 and rb1 , in the filters sections , the user should check the i d list limit box and select the user can filter using a number of ids such as go i d , refseq , etc . in the figure 3 a screenshot of the web interface selecting the attributes for this query the user should click on the results button on the upper - left black bar , the count button gives the number of rows that match the query and the new button allows to start a new query . the user should click on the results button on the upper - left black bar , the count button gives the number of rows that match the query and the new button allows to start a new query . databasedata setsfiltersattributesintogen oncomodulescombinations : oncomodulestype of alteration : gaingenes > ensembl > gene ensembl idicd - o topography and morphology : lung ; any morphology databasedata setsfiltersattributesintogen combinationsgene transcriptomic alterationsgenes : i d list from the previous querygenes > ensembl > gene ensembl idicd - o topography and morphology : lung ; any morphologygenes > ensembl > gene symbolresults > transcriptomic > upregulation p - valueresults > transcriptomic > downregulation p - value there are different types of alterations taking place in cancer . it is important to cross - check the relative contributions of different alteration types . with query 2 , the user will get a list of ensembl genes gained in lung , nos ; any morphology experiments . the user can also retrieve the identifier he / she chooses such as gene symbols , entrezgene i d , etc by changing the attributes for genes . with query 3 , the user can use the list from the previous query , to filter the results for transcriptomic alterations . query # 4 compare the genomic alterations in brain cancer in general and two specific morphology types ; ependymoma and astrocytoma . databasedata setsfiltersattributesintogen combinationsgene genomic alterationsicd - o topography and morphology : brain ; any morphologygenes > ensembl > gene ensembl idicd - o topography and morphology : brain ; astrocytoma , nosgenes > ensembl > gene symbolicd - o topography and morphology : brain ; epedymoma , nosresults > genomics > gain p - valueresults > genomics > loss p - value since the dissection of brain cancer into intrinsic subtypes has prognostic value , it has been the interest of experimental scientist to find gene list that can distinguish cancer subtypes . with query 4 , the user can download the genomic alterations in brain cancer and those for specific mophologies , epedymoma and astrocytoma . in the filters section icd - o , multiple icd - o terms can be selected by clicking while keeping the control key down for windows machines and the command key down in mac machines . query # 5 compare the expression level of the genes annotated with go cell cycle term in different breast cancer experiments . databasedata setsfiltersattributesintogen experimentsgene transcriptomic alterationsicd - o topography and morphology : breast ; any morphologygenes > ensembl > gene ensembl idfilters : i d list limit by go i d : go:0007049genes > ensembl > gene symbolresults > transcriptomic > upregulation , p - valueresults > transcriptomic > downregulation , p - valueresults > transcriptomic > upregulation : total number of samples while enrichment with modules is informative , the user can also get the results for the genes in a module . by comparing the two results , it is possible to see which genes from the pathway are more likely to determine the activity of the pathway in different experiments of the same cancer type . with query 5 , the user can filter the results from all breast studies for the genes with cell cycle annotation and compare the studies . to filter the results for gene with a specific go i d , in the filters section , activate i d list limit box and select go term id as the type of identifier . databasedata setsfiltersattributesintogen experimentskegg pathway transcriptomic alterationsicd - o topography and morphology : prostate gland ; any morphologykegg pathway idkegg nameresults > transcriptomic > upregulation p - valueresults > transcriptomic > downregulation p - value while cancers from different patients show extensive heterogeneity in terms of the specific genes altered , the set of biological processes / pathway affected by these alterations are similar . enrichment analysis of sets of genes with a specific biological property is very useful to detect such patterns . with query 6 , the user can retrieve the results for the pathways from kegg for different experiments that study breast cancer . query # 7a retrieve a table that lists the analysis units for transcriptomic alterations in intogen . databasedata setsfiltersattributesintogen experimentskegg pathway transcriptomic alterationsnone selectedicd - o : topography and morphologyexperiment : publication authors , publication year , pubmed i d , publication title , experiment idplatform : platform title query # 7b retrieve a table that lists the analysis units for genomics alterations in intogen . databasedata setsfiltersattributesintogen experimentskegg pathway genomic alterationsnone selectedicd - o : topography and morphologyexperiment : publication authors , publication year , pubmed i d , publication title , experiment idplatform : platform title in order to retrieve the list of analysis units in intogen , the user has to perform two queries , one for transcriptomic alterations and the other for genomic alterations . it is important to select appropriate attributes to describe the analysis units , use no filters and retrieve the unique results only ( click on unique results only ) . intogen is a cancer analysis tool designed to facilitate the integration , analysis , exploration and interpretation of oncogenomic data . in addition to its browser , its biomart interface provides access to high - throughput data related to genomic and transcriptomic alterations taking place in different types of cancers . a unique feature of intogen is that it provides analysis at different levels of integration . the user can compare the results for individual experiments to those obtained by merging the experiments studying the same cancer type . a major feature of the biomart interface is that it facilitates bulk download of the data . we will continue adding new data from public databases as well as cancer projects such as tcga ( 5 ) and icgc ( 10 ) . another advantage of using intogen is that the data downloaded can directly be analyzed in gitools ( 11 ) ( http://www.gitools.org ) , which is a stand - alone tool designed for the analysis and visualization of high - throughput data . for example , one can easily perform enrichment analyses on intogen data with modules or gene sets from various biomart portals to explore large - scale patterns in cancer genomics data ( see http://help.gitools.org/xwiki/bin/view/tutorials/ for examples ) . with cheaper and faster sequencing technologies being available continuously , resources like intogen that allow the integration , visualization and interpretation of large amount of oncogenomics data will gain importance . we continuously work on improvements and updates on the system to be able to incorporate the data obtained using sequencing technologies . with more high - quality data and new analysis methods incorporated into intogen , spanish ministry of science and technology ( saf2009 - 06954 ) ; agaur fellowship of the catalonian government ( to g.g . ) . funding for open access charge : spanish ministry of science and technology ( saf2009 - 06954 ) .

recently , we created intogen , a resource to integrate a large amount of cancer genomic data . intogen aims at facilitating the detection of the most recurrent alterations that drive tumorigenesis . it collates , annotates and analyzes high - throughput data about transcriptional , genomic and mutational changes taking place in tumors from different studies annotated with specific cancer types . currently , it contains 118 studies for mrna expression profiling and 188 studies for genomic alterations covering in total 64 different tumor topographies . in this article , we describe the biomart portal for intogen . the portal provides easy access to different types of data and facilitates the bulk download of all the analysis results . here , we describe the general features of intogen and give example queries to demonstrate its use.database url : www.intogen.org .

sotos syndrome is a dysmorphic syndrome characterized by early overgrowth , developmental delay , advanced bone age and characteristic craniofacial appearance . sotos syndrome results from mutation involving the nuclear receptor set - domain - containing protein ( nsd1 ) gene , located on chromosome 5q . the mutational mechanism can be a point mutation in the nsd1 gene or a microdeletion that includes nsd1 . fluorescence in situ hybridization ( fish ) did not detect microdeletion of 5q35 in this patient . nsd1 gene mutations are also found in weaver syndrome where camptodactyly is a common feature . this report describes camptodactyly for the first time in a girl with sotos syndrome and provides further evidence that sotos and weaver syndrome are allelic disorders . we describe a two and half years old girl born of non - consanguineous tamilian parents . milestones were delayed with head holding at 6 months , walking at 2 years and delayed speech . family history was unremarkable . at two and a half years she weighed 15 kg , height was 97 cm and head circumference was 52.5 cm ( all above 90 centile ) . she had a high forehead with frontal bossing , dolichocephaly , large ears , pre - auricular pits , down - slanting palpebral fissures , high arched palate , pointed chin and pectus carinatum [ figure 1 ] . she had three caf au lait spots distributed on the chest and trunk , large hands and camptodactyly of the left hand [ figure 2 ] . fish studies carried out using probe rp11 - 265k23 did not reveal a microdeletion of the 5q35 region . face showing the facial features of sotos syndrome left hand showing camptodactly a clinical diagnosis of sotos syndrome was made based on the criteria comprising of rapid early growth , advanced bone age , developmental delay and characteristic facial appearance . camptodactyly in sotos syndrome has not been previously described in literature to the best of our knowledge . a high frequency of congenital heart defects has been reported in patients with intragenic mutations of the nsd1 gene and phenotypic overlap with other overgrowth syndromes , in particular with weaver syndrome is seen . though this sotos patient is likely to have a point mutation of the nsd1 gene , this could not be confirmed due to lack of facilities . sotos syndrome was first recognized as a distinct clinical syndrome in new england in 1964 . the diagnosis is based on the clinical criteria of rapid early growth ( pre and post natal ) , advanced bone age , developmental delay and characteristic facial appearance . growth is rapid in the first years of life but final height may not be excessive . intellectually , the iq ranges from 21 to 103 with a mean of 74 and almost half of affected children achieve normal schooling . behavioral issues are common and are one of the key areas that can influence the outcome . hypotonia is usually present from birth and although this improves during childhood , subtle evidence may remain even in adults . congenital heart disease is not very common in this condition and overall incidence of cardiac defects is approximately 8% . an association of sotos syndrome with tumor development was documented over 30 years ago and has been a point of debate ever since . gorlin et al . estimated a risk of 3.9% of benign or malignant tumors in sotos syndrome . handicaps in sotos syndrome are fewer than previously believed and tend to improve with age . in patients with sotos syndrome harboring a chromosomal translocation kurotaki et al . isolated the nuclear receptor set - domain - containing protein ( nsd1 ) gene from the 5q35 breakpoint . added noted a large difference between japanese and non - japanese patients in the frequency of microdeletions , which occurred in 49 ( 52% ) of the 95 japanese but in only 1 ( 6% ) of the 17 non - japanese . there was a strong correlation between presence of an nsd1 alteration and clinical phenotype , in that 28 of 37 ( 76% ) patients with typical sotos or sotos - like phenotype had nsd1 mutations or deletions . studied nsd1 gene in a series of typical sotos patients ( 23/39 ) , sotos - like patients ( lacking one major criteria , 10/39 ) and weaver patients ( 6/39 ) . they conclude that nsd1 mutations account for most cases of sotos syndrome and a significant number of weaver syndrome cases . comparing the clinical phenotype of children carrying either a deletion or a mutation , rio et al . failed to detect distinctive features except for the severity of mental retardation . they reported , 4/6 children carrying a nsd1 deletion were extremely severely mentally retarded with no language at all , major delay in motor milestones and autistic features . by contrast , in patients carrying nsd1 mutations , mental retardation was usually mild to moderate with verbal skills being more affected . the major differential diagnoses for sotos syndrome are other conditions with overgrowth including beckwith - weidemann , weaver , nevo and simpson golabi behmel syndromes . . however nsd1 gene mutations have been found in beckwith - weidemann syndrome , weaver syndrome and the 11p15 abnormalities seen in beckwith - wiedemann syndrome have been found in some cases of sotos syndrome . camptodactly has not been previously described in literature to the best of our knowledge with sotos syndrome but is seen in weaver syndrome where nsd1 mutations have been described . these could be due to , allelic heterogeneity , effect of other modifying genes ethnic background and nutritional status adding to the overall expression of a syndrome . microdeletions of chromosome 5 were not detected in our case suggesting a likely point mutation in the nsd1 gene and further evidence of that weaver and sotos syndrome are allelic . further delineation of the phenotype with molecular studies will provide correct genotype - phenotype correlations .

we describe a girl with sotos syndrome presenting at two and a half years age with developmental delay . she has camptodactyly which has not previously been reported in sotos syndrome but is a common finding in weaver syndrome . both these conditions have been reported to have nsd1 gene mutations . this report is consistent with the conditions being allelic .

the human gut harbors trillions of bacteria that contribute to host physiological and immunological functions such as nutrient acquisition , maintenance of the gut immune system , and protection against exogenous pathogens ( 123 ) . one of the recent notable findings is the contribution of gut microbiota to the development and homeostasis of the immune system ( 13 ) . the effects of microbiota on the host immune system have been determined by studies in germ - free ( gf ) animals ( 45 ) . gf mice showed reduced numbers and size of peyer 's patches ( 5 ) , less lamina propria cd4 t cells ( 6 ) , reduced levels of the class - switched antibodies iga and igg ( 78 ) , and they lacked a developed gut - associated lymphoid tissue ( 910 ) . thus , maturation of the gut immune system generally takes place following the colonization of gut microbiota suggesting a mutual relationship between the immune system and the microbiota . the gut microbiota consists of bacterial components and metabolites , both of which are important for the development and maintenance of the host immune system . resident microbiota contains pathogen - associated molecular patterns ( pamps ) such as lipopolysaccharide ( lps ) , peptidoglycan , flagellin , and bacterial dna and rna that are recognized by pattern recognition receptors ( prrs ) . prrs are expressed on immune cells , including antigen - presenting cells ( apcs ) , and they play crucial roles in the activation of innate immunity . toll - like receptors ( tlrs ) ( 11 ) and nod - like receptors ( nlrs ) ( 12 ) are the main prrs that recognize bacterial components . while the importance of microbial sensing and activation by the innate immune system has been well recognized , recent work has also uncovered an important role of bacterial metabolites in the gut immune response . short - chain fatty acids ( scfas ) are the end products of fermentation of dietary fibers by the anaerobic gut microbiota and have been shown to exert multiple beneficial effects on host immune homeostasis ( 13 ) . for example , butyrate - induced signaling inhibits histone deacetylases ( 14 ) , increases the size , and enhances the function of the colonic regulatory t cell pool ( 15 ) . collectively , microbiota - derived molecules , including both bacterial components and metabolites , influence the host immune function . based on the role of gut microbiota in development of the immune system although there is little evidence in this regard so far , several reports have indicated that the microbiota could influence vaccine efficacy and adjuvant activity . vaccines display a protective effect against a broad range of diseases including infection , cancer , and allergy . multiple reports have shown that people in developing countries exhibit reduced responses to oral vaccines . the efficacy of rotavirus ( 16 ) , poliovirus ( 1718 ) , and cholera ( 1920 ) vaccines in developing countries with poor sanitation is lower than that in industrialized nations . nicaraguan children were shown to have blunted antibody responses to oral cholera vaccine as compared to swedish children ( 21 ) . similarly , children in poorer regions of north india displayed reduced mucosal immune responses compared to children in other parts of the country ( 22 ) . furthermore , growing evidence suggests that the gut microbiota , which is known to be different in children from developed and developing countries ( 23 ) , plays an important role in determining vaccine efficacy . for example , an abundance of stool actinobacteria including bifidobacterium was shown to be positively correlated with higher responses to oral and parenteral vaccines and a larger thymus in bangladeshi infants . conversely , a high abundance of clostridiales , enterobacteriales , and pseudomonadales was associated with neutrophilia and lower vaccine responses ( 24 ) . furthermore , small intestinal bacterial overgrowth ( sibo ) , which is defined as excessive bacteria in the small intestine and is common in children in developing countries but rare in industrialized settings , contributed to lower antibodies to cholera toxin after immunization ( 25 ) . since sibo presents malabsorption and systemic symptoms , competition between the host and microbiota for nutrients and defects in the intestinal barrier may cause reduced vaccine responses . indeed , whole blood cells derived from south african infants aged 2 years were shown to be severely hyporesponsive to tlr ligands including lps and secreted lower levels of cytokines and chemokines in response to stimulation of tlrs than the age - matched children from north america , south america , and europe ( 2627 ) . in summary , factors that affect gut microbial communities , such as malnutrition , poor sanitation , increased antigen exposure , antibiotic treatment , nutritional status , and lack of breast milk antibodies , could influence vaccine efficacy . mice treated with clarithromycin or doxycycline showed reduced antibody induction against the hepatitis b virus surface antigen ( hbsag ) vaccine . in contrast , mice treated with the same antibiotics exhibited increased antibody responses to live attenuated salmonella enterica serovar typhi ( ty21a ) vaccine ( 28 ) . macaques , showing a high level of diversity in the intestinal microbial composition , had improved protection upon challenge with virulent shigella dysenteriae after immunization with live attenuated s. dysenteriae vaccine . in addition , oscillospira and streptococcus were found to be positively and negatively correlated , respectively , with vaccine - specific igg and iga induction and pro tective responses ( 29 ) . these findings suggest that intes tinal microbial diversity and particular microbiome compositions can influence antibody responses . it has also been suggested that prebiotics ( compounds that increase the number of certain commensals ) could enhance the efficacy of oral vaccines . prebiotic fructo - oligosaccharide ( fos ) has been shown to improve the efficacy of a vaccine against salmonella infection . treatment with fos prior to vaccination resulted in improved host responses and enhanced protection against infection ( 30 ) . another study showed that a prebiotic mixture containing galacto- and fructo - oligosaccharides ( gos and fos ) enhanced systemic immune responses to influenza vaccine . in addition , the prebiotic mixture increased proportions of certain members of the microbiota , suggesting a role for the specific microbial community in the increased host immune responses ( 31 ) . since bacteria - derived components have potent immunostimulatory capacity , they constitute a major potential source of adjuvants . for example , lipopolysaccharide ( lps ) , peptidoglycan ( pgn ) , cpg dna , and trehalose dimycolate ( tdm ) are known to enhance the immune response against co - administered antigens ( 32 ) . this adjuvant activity is mediated through the activation of tlrs , nlrs , and clrs ( c - type lectin receptors ) that mediate signals responsible for activation of the host innate immune system ( 111233 ) . a systems biology approach to study the innate and adaptive response induced by vaccination of humans with non - adjuvanted influenza subunit vaccine ( trivalent inactivated vaccine , tiv ) revealed that tlr5 levels within three days of receiving the vaccine were positively correlated to the strength of the antibody response against the virus ( 34 ) . tlr5 is a cell - surface receptor specific for flagellin , the monomeric component of bacterial flagellum used for cell motility , and has not been associated with viral recognition . oh et al . found that knockout mice lacking the tlr5 protein produced significantly lower levels of antibodies after receiving tiv than wild - type mice ( 35 ) . because tiv does not interact directly with tlr5 , the authors turned their focus to the microbiome in order to clarify the role of tlr5 signaling in vaccine efficacy . indeed , gf mice and antibiotic - treated mice showed impaired responses to tiv . furthermore , colonization of gf mice with flagellin - expressing , but not flagellin - lacking , bacteria was sufficient to enhance the vaccine efficacy . results of this study indicate that flagellin from the gut microbiota acts as a natural adjuvant to enhance protective immune responses to the tiv vaccine . cholera toxin ( ct ) , derived from vibrio cholerae , is widely used as a mucosal adjuvant in animal immunology . ct is composed of pentameric b subunits that bind to the cell surface gm1 ganglioside receptor , and a monomeric a catalytic subunit that activates the heterotrimeric guanine nucleotide binding protein gs , which in turn stimulates camp production by adenylate cyclase . this mechanism is thought to be responsible for the adjuvant effect of ct ( 3637 ) . although the toxicity of ct prevents its clinical application , elucidation of the mechanisms of its potent adjuvant activity may lead to the development of nontoxic and effective mucosal adjuvants for vaccination . we have shown that the mucosal adjuvant activity of ct relies on nod2 signaling induced by endogenous microbiota ( 38 ) . nasal or oral immunization of gf and antibiotic - treated mice with human serum albumin ( hsa ) + ct exhibited reduced levels of antigen - specific igg1 , recall - stimulated cytokine responses , and reduced follicular helper t ( tfh ) and plasma cells . furthermore , the effect of the endogenous microbiota on the adjuvant activity of ct was impaired in ripk2 ( the adaptor molecule required for nod1 and nod2 signaling)-deficient or nod2 ( recognizes peptidoglycan molecules , muramyl dipeptide ( mdp))-deficient mice , but not in myd88 ( the adaptor molecule required for tlr signaling)-ko or nod1 ( recognizes peptidoglycan molecules , diaminopimelic acid ( dap))-ko mice . gf mice immunized with a combination of hsa + ct and mdp produced high levels of antigen - specific igg1 , suggesting that nod2 signaling promotes the adjuvanticity of ct . furthermore , several members of the mdp - rich endogenous microbiota such as staphylococcus sciuri promoted the adjuvant activity of ct in gf mice in a nod2-dependent manner . this study indicates that the adjuvanticity of ct relies on nod2 signaling triggered by the mdp - rich endogenous microbiota . thus , it can be suggested that environmental factors that affect the composition of the microbiota and stimulatory activity that bacterial components exert on host prrs may be important to determine the capacity of mucosal vaccines to provide protective immunity . it is well known that the efficacy of oral vaccines depends on several factors such as genetic background , prior exposure to antigen via natural infection or vaccination , and nutritional status . current findings suggest that the low responses to oral vaccines could be due to differences in the microbiota composition as a result of genetic , environmental , and nutritional variation . indeed , growing evidence indicates that the microbiota influences vaccine and mucosal adjuvant efficacy ( fig . 1 ) . however , investigations of the relationship between the microbiota and the efficacy of vaccines are at an initial stage , and data accumulated so far are limited . therefore , further understanding of the impact of the microbiota on vaccine effectiveness is necessary . detailed analysis of the role of particular species within communities in vaccine responses will provide novel insights into the development of strategies for improved vaccine efficacy .

a symbiotic relationship between humans and the microbiota is critical for the maintenance of our health , including development of the immune system , enhancement of the epithelial barrier , and acquisition of nutrients . recent research has shown that the microbiota impacts immune cell development and differentiation . these findings suggest that the microbiota may also influence adjuvant and vaccine efficacy . indeed , several factors such as malnutrition and poor sanitation , which affect gut microbiota composition , impair the efficacy of vaccines . although there is little evidence that microbiota alters vaccine efficacy , further understanding of human immune system - microbiota interactions may lead to the effective development of adjuvants and vaccines for the treatment of diseases .

supported nanoparticles are used on a tremendously large scale for catalytic reactions . due to the inherent inhomogeneity of these , often industrial , materials , the basic understanding of their exact atomic structure and relation to their functionality can become very complex . one route to overcome this problem is by design of so - called bottom - up experiments in which well - defined nanoparticles are subjected to controlled environments . such studies can be performed by depositing nanoparticles in ultrahigh vacuum on perfect single crystal substrates , after which their structure is characterized under different thermodynamic conditions and gas environments . in this way , for example , oxidation reduction - induced reversible shape changes in nanoparticles have been discovered . solid oxide fuel cells ( sofcs ) are devices used for energy conversion and are considered as an important future green technology . their function is largely dependent on catalytic processes taking place at their interfaces . two important chemical reactions between the surrounding gas atmosphere and the solid play a decisive role . at the cathode , oxygen is dissociated , and the ions enter the electrolyte . fuel is being oxidized at the anode side , for which the required oxygen reaches the interface through the solid electrolyte . both the cathode and the anode consist of complex materials having a large surface area , such as polycrystalline oxides ( cathode ) or nickel nanoparticles ( anode ) . usually , ni is grown on the electrolyte by wet - chemical methods . here we investigate ni nanoparticles ( nps ) deposited on a polished yttria - stabilized zirconia ( ysz ) substrate as sofc model anode by surface sensitive x - ray diffraction methods . ysz with 9.5 mol % y2o3 content is a widely used sofc electrolyte material because of its high oxygen ion conductivity . nickel films grown on mgo(100 ) have been found to show several preferential orientations , forming a complex epitaxial system and of which the core is stable toward high temperature oxidation . this raises the question how smaller particles in the size regime from 310 nm , as typically encountered on the anodes of sofcs behave when in contact with a solid electrolyte . their sintering behavior in different atmospheres is particularly important for catalytic activity and long - term stability . here we show the results of such a study , whereby the ni nps are first annealed and finally oxidized . we find that ni nanoparticles grow with two very distinct orientations on ysz(111 ) , in contrast to mgo(100 ) . the nio formed during oxidation grows epitaxially with repect to the ni nanoparticles . from the measured particle heights and widths , the energy of adhesion , which is an important quantity with respect to the nanoparticles sintering stability , is determined . the experiments presented in the following were performed at the mpi beamline of the ngstrm - quelle karlsruhe ( anka ) using an x - ray energy of 10 kev . x - ray data consist of x - ray reflectivity , extensive reciprocal space mapping , and crystal truncation rod ( ctr ) data . in addition , ex situ atomic force microscopy ( afm ) measurements were performed in air once the synchrotron experiments had finished . polished single crystals of 9.5 mol % yttria - doped zirconia with orientation were used as substrate having a surface diameter of 10 mm . prior to the ni deposition , the substrates were annealed at 673 k in 10 pa o2 for 120 min , a procedure which was found to result in smooth and well - defined surface structures . the ni nps were grown with a substrate temperature of 623 k at a growth rate of 0.2 nm / min resulting in a nominal 3 nm of deposited material . here , we present results from two such growth runs with different samples , named sample i and sample ii hereafter . for sample i , only the as - prepared nps were structurally characterized , and after the in situ synchrotron experiment , the sample was investigated by atomic force microscopy ( afm ) in air . in a second experiment on sample ii , the ni nps were further treated , and extensive x - ray characterization was carried out . the as - prepared ni nps were first annealed at 973 k for 75 min , then exposed to 10 pa of methane at 573 k for 30 min and finally oxidized at 10 pa of o2 at 573 k for 35 min . after each of these steps , extensive x - ray data were taken , which allow us to determine changes in the orientation , size , and composition of the nps . because the methane exposure did not result in any notable structural changes , these data are not included in the present report . in addition , at each of the sample preparation steps , the ysz surface structure was investigated by measuring several ctrs with a nonzero in - plane momentum transfer . these data do not indicate any considerable changes after ni evaporation and after the oxygen treatment . because these ctrs probe the 3d atomic structure of the ysz(111 ) surface , which is partly covered by the nps , we conclude that each of these processing steps does not result in any significant atomic relaxations of the substrate . afm images were taken ex situ after the first growth run of nickel nanoparticles on ysz(111 ) without further treatments ( sample i ) . particles are clearly observed , and typical heights of 3 nm can be derived . just after growth , with the sample still in the uhv chamber , x - ray reflectivity ( xrr ) measurements were taken ( see figure 2 together with the xrr curves from sample ii described more extensively in this paper ) . the curves of the as - prepared states in both experiments are very similar . analysis of the xrr data is performed by fitting the electron density profile along the surface normal . here we use a so - called slab model , based on the fully dynamical parratt formalism . by comparison of the value obtained for the ni nps to that of a closed bulk ni layer , their coverage can be estimated . at the same time , the average height of the ni nps can be obtained from the xrr measurement . this information is contained mostly in the period of the oscillations of the reflectivity . the result from sample i gives an average height of 3.6 nm , in good agreement with afm performed on the same sample when considering that due to tip convolution effects those values will be systematically lower . in contrast to the height determination procedure described in the section nanoparticle shape and adhesion energy , xrr gives a value averaged over all np orientations . ( left ) afm phase contrast image of the ysz(111 ) surface after nickel evaporation ( sample i ) . ( middle ) height profile from the topographic data along the line as indicated ( left ) . x - ray reflectivity vs the momentum transfer along the surface normal qz(= 4 sin( ) / , with half the scattering angle and the wavelength ) measured after nickel evaporation . shown are the measurements ( symbols ) and fits ( solid lines ) for sample i as - prepared ( gray ) and sample ii as - prepared ( black ) , annealed ( blue ) and oxidized ( red ) nps , whereby the curves are scaled for clarity . the oscillations are a clear indication for the presence of the nps . from fitting an electron density profile to the data , the thickness and coverage of the nps are determined and these results are listed in table 1 . due to coexistence of ni and nio , it is not possible to reliably determine a coverage and merely the total apparent thickness is determined . once the crystalline ni nps were grown , the positions of their bragg peaks with respect to those of the underlying substrate were used to determine their orientation . in the following , several notations will be used interchangeably , depending on the particular frame that is referred to . due to conventions in surface diffraction , use is made of the ysz(111 ) surface unit cell to construct the reciprocal basis vectors of the substrate frame . the direct space axes of this nonprimitive hexagonal cell are a = b = 0.361 nm and c = 0.921 nm . the cell parameters of the conventional cubic lattices of both materials are ani = 0.354 nm and aysz = 0.541 nm . in the remainder of this article , subscripts of ( h , k , l ) coordinates are used to indicate with respect to which basis they are defined : ysz for the ysz(111 ) surface unit cell and ni - bul for the conventional fcc ni lattice and ni-111 for the ni(111 ) hexagonal surface unit cell . table 2 lists different ( h , k , l ) values in the different frames . by mapping out reciprocal space in selected areas , we have found two preferred np orientations and at least one other not yet reported for the epitaxial growth of ni nps . the first major one corresponds to the ni ( 111)-direction parallel to the substrate surface normal . the in - plane directions of the ni lattice were found to align with the substrate surface unit cell directions . this orientational relationship ( or ) is described by yszni111 and [ 110]ysz[110]ni111 and will be denoted or1 in the remainder of this paper . the ( 1,0,l)ni111 bragg peaks are found in a plane ( h,0,l ) at h= 1.44 ( see figure 3 ) , indicating that the ni is completely relaxed and adopts its bulk lattice parameter because this value corresponds exactly to the ratio between the bulk lattice parameters aysz / ani . two peaks , which belong to -oriented particles as described above , at h = 1.44 but at different l - values are seen , corresponding to the ni - bul ( 11 1 ) and ni - bul ( 002 ) reflections . these ni peaks indicate that the ni atoms follow an abc - type stacking along the substrate surface normal . if there were only one unique stacking sequence of the ni atoms , only one of these peaks would be visible , because the 3-fold symmetry axis of the fcc ni along its body diagonal would be preserved . instead , the observation of both peaks indicates that the ni atoms have two different stackings , which results in an apparent 6-fold symmetry axis around the stacking direction , which was also evident from scanning the sample around the surface normal whereby diffraction peaks separated by 60 appeared ( not shown ) . this diffraction feature indicates that the ni nps possess an internal twin structure , that different nps possess different stacking sequences starting from the substrate , or a combination of both of these . from the peak widths along the l direction , it is concluded that internal twinning does not occur frequently because this effect would lead to additional broadening and would result in lower apparent np heights , as explained in more detail in the section nanoparticle shape and adhesion energy . the substrate bragg peak at ( 1,0,2 ) and the corresponding ctr along the l - direction . the ( 1 , 0 , 1)ni111 and ( 1 , 0 , 2)ni111 bragg peaks from ( 111)-orientated ni nps . at ( 1.25 , 0 , 2.53 ) a peak belonging to ( 100)-oriented ni nps and at ( 1.66 , 0 , 1.86 ) a peak orignating from internal twinning as described in the text . the peak at ( 1.02,0,3.28 ) , which is labeled as ( 0 , 0 , 3)ni tilt , originates from the tilted nps as described in the text . table 2 gives an overview of the ( h , k , l ) coordinates expressed in different frames . the hk projection ( inset ) shows the substrate ( black ) and ni ( blue ) reciprocal lattice vectors . another strong ni - bul ( 111 ) peak is observed at ( 1,0,3.28 ) , which originates from np which are 41.33 tilted with respect to the ( 111)-oriented particles . this or2 is described by yszni111r41.33 and [ 120]ysz[120]ni111 , where r indicates a rotation . the full 3d information is confirmed by the observation of a ( 2,2,0)ni bul reflection , belonging to or2 and which is rotated by 34.4 from the substrate surface plane . the angular position of this reflection is obtained by rotating the ( 0,2 , 2)ni bul of or1 , which makes an angle of 30 with the plane of the rsm shown in figure 3 , around the [ 120]ysz direction by 41.33. the atomic - scale arrangement , schematically depicted as a side view along the rotation axis , is shown in figure 4 . the orientation can be understood from a so - called coherent tilt strain - relieving mechanism , which is rather common in thin film heteroepitaxy . for fcc metals , this epitaxial relationship originates from the diffusion of dislocations along ( 111 ) slip planes . the resulting edge dislocations line up laterally at the hetero interface , thereby reducing the strain and tilting the metal lattice . the energetics of this mechanism competes with other types of strain relaxation and can be kinetically hindered by dislocation movement . therefore , depending on the growth technique in combination with temperature , which determines the supersaturation and surface diffusion , different orientations are expected . nanoparticles as investigated here , would actually form the very early growth stage of textured polycrystalline thin films . the np nickel lattice is tilted such that the projection of ( 111 ) planes fits much better to the substrate atomic distance of 0.314 nm along this direction and therefore accommodates strain . the particular arrangement originates from an interfacial strain driven slip mechanism , which is common in fcc metals . by the diffusion of dislocations along the ( 111 ) slip planes toward the interface , the projected d - spacings have a better match with the substrate thereby relieving strain . as a consequence , figure 3 also shows much weaker peaks at ( 1.25,0,2.53 ) and ( 1.66,0,1.86 ) , which are ascribed to ( 100)-oriented nps and from internal twinning around the ni 111-direction pointing toward a side facet , respectively . because these orientations represent only a fraction of all the nps , as judged from the much weaker diffracted bragg intensities , these are left out of further consideration . the or1 found here has been reported before for a variety of different growth methods of ni on ysz(111 ) , like pulsed laser deposition and mbe . because this or results from a simple cube - on - cube stacking and most likely is energetically favorable , it has also been found for the ni - ysz(100 ) interface grown by directional solidification . these studies also report on other ors , but not on the or2 found by our x - ray diffraction study . interestingly , from the geometrical analysis of coincident lattice points , sasaki et al . find that the best match would be formed by yszni bulk and yszni bulk , an or that has experimentally never been verified , also not in this study . this point illustrates that ni - ysz lattice - matching alone is not enough to describe the different ors and that other energy terms such as chemical bonding and dislocation formation and their kinetic interplay during growth play a potentially more important role . moreover , it has been shown previously that the ysz(111 ) possesses a surface morphology with 2d monolayer high islands , which is expected to play a role in the np binding . the average size of the nps can be determined from the widths of their corresponding bragg peaks , under the assumption that the line broadening can be mostly attributed to their finite size and that nonuniform strain plays a minor role . the experimentally observed diffraction peaks are further broadened by the instrument resolution , which in the case here is anisotropic due to the synchrotron radiation characteristics in combination with the beamline focusing optics . from the single - crystal substrate bragg peaks the in- and out - of - plane instrument resolution is determined . because our sample coordinate system is chosen such that the hk - plane lies horizontal and the l - direction is vertical , the instrument resolution can be described by ( h , k,l ) = ( 0.025,0.060 ) , expressed in dimensionless reciprocal lattice units . all the experimentally determined diffraction peaks were well described by gaussians , so that the peak breadths are corrected for the instrumental resolution by = exp2res2 . the average height of the nps is given by the following reciprocal relationship:1where c = 0.890 nm is the direct lattice parameter of the ysz(111 ) surface unit cell and l the peak width ( fwhm ) along the l - direction corrected for the instrument resolution . a similar expression holds for the determination of the average widths ( w ) , for which the peak profile along the h - direction must be considered together with the fact that the h - direction makes an angle of 30 with the real space atomic layers within the hexagonal surface unit cell . the np heights and widths have been determined after each of the treatments , and the resulting values are presented in table 3 . the errors that are listed are calculated from the spread in values obtained from several bragg peaks at different positions in reciprocal space . in a next step , the average sizes are used together with wulff s construction to deduce the nps equilibrium shape when in contact with a foreign substrate . the underlying principle is that the nps are faceted and that those facets with the lowest surface energy will have the largest surface area . this aspect is now combined with the geometrical constraint that the nickel nps are supported by the ysz substrate . it is important to note that the description of the nps shape used in the following is valid for isolated particles ( i.e. , in the absence of particle particle interaction ) . from the afm measurement , shown in figure 1 , it can be concluded that coalescence may have set in for certain particles / areas but probably not for all . a more detailed analysis is hampered by the inherent lateral resolution of the used afm . nevertheless , the sample used for this study is probably not far off a model system with completely isolated nps . for fcc metals like ni , the wulff shape ( i.e. , without support ) is given by a polyhedron with predominantly ( 111)-oriented hexagonal facets together with smaller square ( 100 ) facets . this basic shape is taken as a starting point to construct the shape when in contact with a substrate . using the dimensions and orientation with respect to the substrate , the wulff shape has to be cut at particular positions . for the ( 111)-oriented particles , the wulff shape is shown in the upper part of figure 5a ) . the width of 8 nm and height of 5 nm , as determined previously , require a truncation of the wulff - shape , as shown in the lower part of figure 5a ) . the resulting shape of the ( 111)-oriented nps is then given by a large ( 111)-oriented top facet , six ( 111)-oriented side facets and three ( 100)-oriented side facets . the shape of the tilted nps is determined by rotating the wulff - shape by 41.3 in order to comply with the experimentally determined orientation . then this tilted particle is truncated at the experimentally determined height , see figure 5b ) . ( a ) wulff shape for the unsupported ( 111)-oriented np ( top ) and when supported by the ysz substrate ( bottom ) . the different treatments did not significantly change the nps shapes , although the heights and widths did change , as described in the text and seen in table 3 . the energy of adhesion for relatively round supported nps ( i.e. , which are not very flat and at least half as high as the wulff - constructed free particle ) is given by2with h the height of the np and h * the height of the buried part . it seems a reasonable approximation to relate the experimentally determined heights and widths to the geometric lengths of eq 2 as w = h + h*. taking a value of 111 = 1.89 jm , this gives wadh = 1.4 jm for the ( 111)-oriented ni nps after annealing . ( note : these surface - energy values were checked and recalculated with density functional theory ( dft ) using the latest perdew burke enzerhof ( pbe ) potential , which does not suffer too much from overbinding . ) because the error bar on the widths is relatively large , the criterion for using eq 2 might not hold anymore and they could actually be much flatter , in which case the following relation holds:3 this relation gives , together with 100 = 2.2 j / m ( see parenthetical note above ) , wadh = 2.1 j / m . the adhesion energy value obtained from eq 2 is somehwat smaller than typical values obtained for other materials systems , such as ptrh - al2o3 and pd al2o3 . the value obtained using eq 3 is much closer to those values and is in very good agreement with the value obtained for much larger ( w = 200 nm , h = 150 nm ) ni particles . for the tilted nps , a higher h / w ratio is observed , pointing to a reduced adhesion energy . the occurrence of two distinct np growth directions can be related to a difference in interfacial binding which either could originate from a lateral chemical inhomogeneity of the ysz substrate or a kinetically trapped state . also a size dependence can not be ruled out which is difficult to investigate due to the inherent size distribution of the nps after growth via thermal deposition . at a temperature of 573 k , the nps were exposed to molecular oxygen at a pressure of 10 pa for approximately 35 min , after which the oxygen was pumped off again . figure 6 shows a reciprocal space map after oxidation from the same plane as that shown in figure 3 . clearly observable are two new bragg peaks along the l - direction at h = 1.24 , which originate from nio . from the position of these two peaks with respect to the nearby bragg peaks of the ni -oriented nps the average heights and widths of the -oriented and tilted nps after oxidation are listed in table 3 . additionally , the temporal evolution during oxidation of some structural parameters were followed as well , see figure 7 . the integrated bragg peak intensity , which is proportional to the amount of pure metal , is seen in the decay below half of its initial value , which means that about 65% of the ni contained by the -oriented nps transforms into nio . at the same time , the average height of the -oriented nps changes only by approximately 0.2 nm , which is equal to the estimated error , see table 3 . nevertheless , the data do indicate a trend , whereby the average height of the pure metal ( core ) is reduced . the particular bragg reflection that was followed during oxidation gives an average height somewhat smaller than the value obtained from other bragg reflections and therefore is not identical to the values listed in table 3 . the position of the bragg peak along l can be used to determine changes in the uniform strain of the nps ni ( 111)-planes along the substrate surface normal . by comparing its shift from its initial value , which relates to d0 , the relative strain state d / d0 is calculated . it is seen in figure 7 that as oxidation proceeds , the lattice expands along the surface normal and levels off after an approximately 0.5% increase , a value very close to that found during thin ni film oxidation . we argue that this can be attributed to oxygen incorporation into the ni lattice . from the trend in the intensity curve , it is concluded that the oxidation process had not yet finished , although the lattice did not expand anymore , which indicates that the oxygen dissolution has reached a final state . l plane after oxidation at 573 k and 10 pa of molecular oxygen for 35 min . kinetics of the 111-oriented ni np evolution during oxidation . shown are the average height ( top ) , relative 111 bragg peak intensity ( middle ) and relative strain d / d0 ( bottom ) as a function of oxidation time . in order to rationalize the obtained results , we postulate the following oxidation mechanism . one important ingredient is that small nps oxidize relatively faster than large ones ; their so - called burn - up time is even lower than expected from the oxidation rate of planar surfaces , because the geometry of the electric field around these often more - rounded particles is predicted to accelerate the oxidation process . larger nps will first form an oxide skin around them , and eventually , they also burn up completely , but with a much lower rate . schematically , an intermediate state of this scenario is depicted in figure 8a , which represents the situation after the oxidation step presented here . given a certain particle size distribution , the resulting bragg peak shape figure 8 shows the results for such a calculation , using a hypothetical gaussian width distribution , around an average of 8 nm and a standard deviation of ann = 3 nm . gaussian - shaped diffraction peaks corresponding to each of the np size represented in the distribution are used in the calculation . a cutoff value around 5.5 nm is used , below which the nickel nps are completely oxidized . from the resulting ni and nio therefore , the width of a nio np , which has formed from a metal ni particle , is larger by a factor of 1.19 , which reflects the ratio of their lattice parameters . metal ni . in this particular numerical example , the average ni np width before oxidation is 8 nm , whereas after oxidation , both ni and nio widths are 9 nm . although this numerical simulation certainly does not include all the details of the oxidation process , it does show how the average np sizes can in principle be affected by a preferential oxidation of small particles and how this can lead to average np widths that are larger after oxidation . the simulation presented here does not differentiate between partially and completely oxidized particles , although the experimental results point toward such a situation . this is mostly seen in the large anisotropy between the oxidation of the nps top 111 facet and the average widths . both xrr and high - angle bragg peaks indicate that upon oxidation , the average height of the metal ni nps is slightly reduced , at most by 0.2 nm . it has been observed that during the oxidation of a ni(111 ) single - crystal surface under identical conditions as used in this work , a nio skin of approximately 2 nm forms after approximately 14 h. therefore , it seems reasonable to assume that for the oxidation time investigated here , at most , only one single outermost layer of ni atoms , which constitute the top facets , form an ultrathin nickel oxide . the oxidation process at the other facets , like 100 , seems to proceed much faster and even leads to complete oxidation of nickel nps . ( a ) small nps oxidize comparatively faster and burn up completely , whereas at the same time , an oxide skin has formed on larger nps and the core is still metallic . the evolution of the diffraction peaks is simulated using np width distributions during this partial oxidation process . ( b ) np width distributions before and after oxidation : initial metallic ni nps ( black line ) with an average of 8 nm and a spread of = 3 nm . after oxidation , the width distribution of the metallic core of the partially oxidized particles has become asymmetric and peaks at around 9 nm ( filled white ) . the width distribution of the oxidized metallic ni ( red ) is shifted due to the lattice expansion and is used to calculate the expected nio bragg peaks , see text for more explanation . ( c ) bragg peaks calculated as weighted averages using the gaussian width distributions . shown are the initial ni peak ( black ) , the final ni peak ( red ) , and the newly formed nio peak ( filled red ) . from the diffraction lines , average widths of 8 , 9 , and 9 nm for the initial ni , final ni , and nio particles are found , respectively . the oxidation behavior of the nps belonging to or2 has not been followed in situ , but the overall changes in height and width , as listed in table 3 , are very similar to those of or1 . the intensity loss on the metal ni bragg peaks is about 50% , very similar to that of the peaks belonging to or1 . we therefore conclude that the fact that the particles belonging to or1 have different facets exposed to the gas phase leads to minor changes in oxidation behavior and the stability of the particles is not much different . nickel nps were grown in uhv by pvd using an electron beam evaporator with a nominal average height of approximately 4 nm at substrate temperatures of 623 k. x - ray diffraction revealed that two distinct np orientations form ; there is no random orientational growth which would give rise to powderlike diffraction patterns . the np orientations are or1 : ni ( 111 ) planes parallel to the surface whereby the in - plane directions are also aligned ( niysz and ni[1 10]ysz[1 10 ] ) and or2 : ni 111 planes tilted by 41.33 from the substrate normal in the plane spanned by ysz and ysz[1 10 ] . the average heights and widths of the nps were determined from the x - ray diffraction line breadths . the relatively well - defined height is furthermore extracted from x - ray reflectivity and afm in a separate growth run , which all give systematically comparable values . the average heights and width of nps in both ors increase by approximately 25% , indicating that the particles exhibited already equilibrium shape at the growth temperature . from the height - to - width ( h - to - w ) ratio , together with the ni ( 111 ) and ( 100 ) surface energies , the adhesion energy is determined . depending on the np shape being more round or flat , two different relations hold . because the nps average aspect ratio h / w is close to , either of the relationships might give representative values . this is even more likely when taking into acount the appreciable size distribution , which is more broad in w than in h. the more rounded nps will then have an adhesion energy of 1.4 j / m and the flatter ones 2.2 j / m . this adhesion energy calculation does not hold for the tilted nps forming or2 because of their asymmetric shape . when exposing the nps to methane at a pressure of 10 pa and a temperature of 573 k for 30 min , no changes are observed . a possible adsorption of the hydrocarbons on the nps is thus concluded not to affect the structure of the nps . the most distinct changes are caused by an oxygen treatment at 10 pa and 573 k for 35 min . the intensity of the or1 bragg reflections , which is a measure for the total amount of metal , decreases to less than half its initial value . after the oxidation step , nio bragg reflections are detected , and from their positions in reciprocal space , it is deduced that nio has grown epitaxially , in a cube - on - cube fashion , on the nps from or1 or in contact with the ysz ( 111 ) surface . at the same time , the average height has changed by 0.2 0.3 nm and the average width + 1 3 nm . the average size of the nio is approximately equal to that of the remaining pure metal particles . these results can be rationalized by considering a preferential oxidation mechanism in combination with an anisotropic size distribution , whereby the spread in h is much narrower than that in w. it has been predicted that small , more rounded , nps will oxidize at an enhanced rate compared to larger ones . on the basis of these and our experimental results , we postulate that ni nps smaller than approximately 5.5 nm have burnt up completely and that larger ones have a nio skin formed around them . a simulation , using a hypothetical gaussian width distribution , shows that indeed this scenario can explain the experimentally observed average widths before and after oxidation . this description does not include all the details of the oxidation process , such as exact oxidation rate and nio thickness on the different facets , and even assumes that part of the larger nps does not oxidize at all , which is not very realistic . nevertheless , it shows numerically the effect one important aspect of the mechanism , namely , size - dependence , can have on the final average size . the size - dependent oxidation behavior might also be related to the kink seen after approximately 3 min in the time - dependent intensity reduction of the 111 bragg peak of figure 7 . the average size of the nio particles and/or skin is of the same order of magnitude as the remaining pure ni metal ( cores ) because of two effects : ( 1 ) due to the lattice expansion after oxidation ( i.e. , nio has larger lattice constants than ni ) , a completely oxidized np will be larger than the original one . this effect shifts the average oxidized np size to higher values , thereby compensating for the fact that the smaller metal particles have prefentially oxidized . ( 2 ) the oxide skin on the top facet is formed by a completely structurally correlated nio lattice . therefore , the in - plane nio size will be comparable to the underying metal particle . from ni(111 ) single - crystal oxidation studies , it was found that an approximately 2 nm oxide forms after 14 h at conditions similar to the ones here . extrapolating to the oxidation time used here , it would be expected to have oxidized only a monolayer from the 111 facets , which is about 0.2 nm thin . this value corresponds very well to the reduction in the average height of the ( 111)-oriented nps upon oxidation and would imply that the oxidation proceeds mostly along the ( 100 ) facets . the results obtained here can be discussed in relation to real sofcs , in particular with respect to the so - called redox cycling ( i.e. , the oxidation reduction process of ni ) . during sofc operation , the anode is continuously supplied with fuel , the amount of which is burnt depends on the availability of oxygen . instabilities around the ideal ratio of fuel and oxygen levels can lead to the formation of nio , which is known to lower the sofc performance and lifetime by stress development , lowering of the metal triple phase boundary density , and mechanical failure . our results may add another cause to sofc anode failure , namely , that during the time when there exists a surplus of oxygen , probably the smallest ni nps will completely oxidize and , depending on the reducing properties of the fuel , might not revert to pure metal anymore . after many cycles of such a process , the sofc might gradually loose its efficiency because the smallest particles are most likely to be the most reactive and therefore relatively more important for the functionality . moreover , such a preferential oxidation process , whereby the particle size distribution shifts toward larger values , might result in the sofc operation to drift away from complete reaction leaving unburnt fuel and increasing oxygen levels thereby oxidizing ever more ni . x - ray diffraction revealed two distinct orientations of ni nanoparticles grown in uhv by mbe : -oriented particles and ones with their planes tilted by 41.33 from the surface normal . a combination of diffraction line width analysis , x - ray reflectivity , and afm delivered information on the average np height and width . these sizes were used to determine the energy of adhesion to be in the range of 1.42.2 jm , whereby particles that have a more round shape are more weakly bound than those that have a flatter geometry . annealing the nps in uhv to 923 k led to the observation of sintering : most significantly , their average height increased and the data also indicate an increase in average width . this sinterering process does not lead to a significant shape change from just after growth . exposure to methane at 10 pa at a temperature of 573 k did not show any structural changes . however , oxidation at 10 pa of o2 at the same temperature led to the formation of expitaxial nio on the -oriented particles . an oxidation mechanism is postulated , which is based on a preferentially faster burn up rate for small particles and the formation of an oxide skin around larger ones . such a preferential oxidation mechanism which shifts the np size distribution toward higher values might be one factor responsible for the long - term degradation of sofcs .

nickel nanoparticles supported by the yttria - stabilized zirconia ( 111 ) surface show several preferential epitaxial relationships , as revealed by in situ x - ray diffraction . the two main nanoparticle orientations are found to have their [ 111 ] direction parallel to the substrate surface normal and 41.3 degrees tilted from this direction . the former orientation is described by a cube - on - cube stacking at the oxide metal interface and the latter by a so - called coherent tilt strain - relieving mechanism , which is hitherto unreported for nanoparticles in literature . a modified wulff construction used for the 111-oriented particles results in a value of the adhesion energy ranging from 1.4 to 2.2 jm2 , whereby the lower end corresponds to more rounded particles and the upper to relatively flat geometries . upon oxidation at 103 pa of molecular oxygen and 673 k , a nio shell forms epitaxially on the [ 111]-oriented particles . only a monolayer of metallic nickel of the top ( 111 ) facets oxidizes , whereas the side facets seem to react more severely . an apparent size increase of the remaining metallic ni core is discussed in relation to a size - dependent oxidation mechanism , whereby smaller nanoparticles react at a faster rate . we argue that such a preferential oxidation mechanism , which inactivates the smallest and most reactive metal nanoparticles , might play a role for the long - term degradation of solid oxide fuel cells .

root canal isthmus , a narrow ribbon - shaped communication between two root canals is an important anatomical feature because of the fact that it may contain pulp remnants , necrotic tissues , and micro - organisms and their byproducts . an isthmus is also called a corridor , a lateral interconnection , and a transverse anastomosis . the prevalence of isthmus varies according to the tooth type , root levels , and age . an isthmus might be found in roots with c - shaped canals or in two adjacent canals such as mesial roots of mandibular molars , premolars , and so on . the mesial root of the mandibular first molar exhibits the most number of isthmuses . the majority of isthmuses have been reported in the apical 5 mm of root canals . a study in a chinese population reported that the prevalence of isthmuses decreases with age in molars due to the deposition of secondary dentin . irregularities in root canal system , including isthmuses , are inaccessible spaces for instruments , irrigation solutions , and medicaments , and serve as reservoirs for bacteria , which finally leads to the failure of conventional root canal treatment . in addition , isthmuses overlooked during periapical surgeries might lead to the failure of surgical treatment . ideally , with the present practice of advanced preparation and filling of isthmuses during root - end resection , the success rate of endodontic treatments is expected to increase in most cases . therefore , a thorough knowledge of this anatomic feature in the apical third of root canals in posterior teeth has a great value in increasing the success rate of surgical and nonsurgical endodontic treatments . a large number of studies have been carried out in different parts of the world to determine the prevalence of isthmus in mandibular molars , ranging from 54 to 89% [ table 1 ] . considering ethnical variations and inadequate published data on this anatomical feature , the aim of the present study was to evaluate the prevalence and location of isthmus in the mesial roots of extracted mandibular molars in an iranian population . in this cross - sectional descriptive study , 60 extracted mandibular first and second molars were randomly selected from dental clinics of tehran ( from the north , south , east , and west regions ) . the teeth were rinsed under tap water immediately after extraction and immersed in 10% neutral buffered formalin solution and prepared for two - angle radiographic examination ( straight and 20 mesially ) . the inclusion criteria consisted of mature roots , the presence of two canals in the mesial root , absence of any calcification , internal or external root resorption , and visible cracks . the age , gender , and race of the patients were not considered . all teeth were decoronated and two # 15 k - flexofiles ( dentsply / maillefer , ballaigues , switzerland ) were used to verify the two canals in the mesial roots of the teeth and a periapical x - ray was taken . a low - speed handpiece with a thin metallic disk ( d and z , germany ; length : 0.17 mm , breadth : 2.0 mm ) was used to cut each root at 2 , 4 , and 6 mm distances from the apex perpendicular to the root long axis . each separated root segment , which was 2 mm in thickness , was placed in 5.25% sodium hypochlorite solution for 24 hours to remove any debris or organic material remnants . subsequently , the root samples were immersed in 17% ethylenediaminetetraacetic acid ( edta ; ariadent , asia chemie teb , tehran , iran ) for 30 seconds , then rinsed with distilled water , and dried . finally , the sectioned surfaces of the samples were stained with indian ink and evaluated under a stereomicroscope ( nikon ufx - dx , tokyo , japan ) at a magnification of 30 . photographs were taken using a camera ( nikon fx-35 xx , tokyo , japan ) , and recorded and evaluated by two endodontists . the absence or presence of isthmuses and their types were evaluated and recorded based on the classifications of kim and teixeira at various distances from the apex . the kim classification consists of five types [ figure 1 ] : schematic representation of isthmus classifications described by hsu and kim type i : presence of two canals without a noticeable communication type ii : presence of two canals without a definite communication type iii : similar to type ii but with three canals instead of two canals type iv : extension of the main canal into the isthmus type v : presence of a complete communication or corridor between the two canals the teixeira classification consists of no isthmus , incomplete isthmus , and complete isthmus . a chi - squared test was used to determine the relationship between the prevalence and types of isthmuses with canal location after prevalence and confidence intervals were determined . the results are presented in [ tables 2 and 3 ] and based on the classifications of kim and teixeria . in the 60 mandibular first and second molars evaluated in the present study , isthmus was found in an average of 83% of the mesial roots at 2 , 4 , and 6 mm distances from the apex . the highest prevalence of isthmus was found at a distance of 6 mm from the apex with 92% [ confidence interval ( ci ) : 89.8 - 93.6 ] ; the lowest prevalence was found at a distance of 2 mm from the apex with 70% ( ci : 64.7 - 75.3 ) . the prevalence of isthmus was 88% ( ci : 85.7 - 90.92 ) at a distance of 4 mm from the apex . prevalence of isthmuses , based on the kim classification , at 2 , 4 , and 6 mm distances from the apex in an iranian population prevalence of isthmuses , based on the teixeira classification , at 2 , 4 , and 6 mm distances from the apex in an iranian population based on the kim classification [ figure 2 ] , there was no significant relationship between isthmus type and canal location . the most prevalent isthmus at 2 and 4 mm from the apex was type v but at 6 mm , it was type ii . however , in terms of the teixeira classification [ figure 3 ] , there was a significant relationship between sections at 2 and 6 mm from the apex ( p = 0.039 ) . moreover , the incomplete type was most common in 6 mm ( 67% ) and least in 2 mm ( 18.3% ) . lack of isthmus was most common in 2 mm ( 18% ) and least in 6 mm ( 5% ) . the complete type was most common in 2 mm ( 52% ) and least in 6 mm ( 25% ) . isthmus classifications described by hsu and kim : type i ( a ) , type ii ( b ) , type iii ( c ) , type iv ( d ) , type v ( e ) teixeira classification : no isthmus ( a ) , incomplete isthmus ( b ) , and complete isthmus ( c ) the management of root canal isthmus has been shown to be very essential in nonsurgical and surgical endodontic treatment . complete cleaning , shaping , and obturation of the apical third of root canals are considered as among the most important factors in achieving an excellent prognosis of root canal therapy . an unprepared isthmus in the root canal system , especially in the mandibular and maxillary molars , might contain necrotic debris and tissue remnants , which might serve as a reservoir for bacteria , leading to endodontic failure . therefore , initial anatomical knowledge , recognition , and proper management of an isthmus may be of great value to increase the success rate of surgical and nonsurgical endodontic treatments in posterior teeth . in the present study , isthmuses were found in 83% of the mesial roots of the mandibular first and second molars , which is consistent with the results of the studies by fan et al . in which prevalence rates of 85 , 81 , and 88.5% were reported , respectively [ table 1 ] . teixeira et al . found an incidence of 59% two canals in the mesial root of mandibular molars . the prevalence of isthmus was greatest 3 - 5 mm from the apex . in these cases , 22% were complete and 37% partial in mandibular molars . bidar et al . reported an isthmus incidence of 16% in distal roots with two canals of mandibular molars in a sample of iranian population . this lower rate of isthmus could be explained by different roots ( distal versus mesial ) . however , the authors emphasized that even this percentage would be taken into account during the cleaning and shaping of root canals . furthermore , the highest and lowest prevalence rates of isthmuses in the present study were found at 6 mm and 2 mm distances from the root apex , respectively . therefore , the number of isthmuses increases from 2 to 6 mm distance beyond the apex . previous studies , similar to our study , have shown the highest prevalence of isthmuses at 4 - 6 mm distances from the apex in the mesial roots of mandibular molars . in addition , we found the highest and lowest prevalence rates of complete isthmuses at 2 and 6 mm distances , respectively , indicating a progressive decrease in the number of complete isthmuses from 2 to 6 mm beyond the apex . the prevalence of complete isthmus at 2 mm from the apex , in our study , was higher than that of the findings of gu et al . management of complete isthmus is easier with the use of microsurgical techniques , such as the usage of a dental operating microscope and microsurgical instruments ; however , preparation of incomplete isthmuses is more difficult and requires the accurate use of fine ultrasonic tips . in the present study , a higher rate of incomplete isthmus was found in the 6 mm apical root , indicating a challenging situation during nonsurgical preparation of mandibular molars . additionally , following 3 mm root end resection during periapical surgery , retropreparation and retrofilling to a depth of 3 mm are suggested to clean and fill the 6 mm apically located segment of an isthmus . , the teeth were sectioned and evaluated under a stereomicroscope , similar to the technique used by teixeria et al . and bidar et al . the sectioning , staining , and clearing is a commonly used technique due to its greater accuracy in the detection of isthmus than other techniques . however , microcomputed tomography is a modern technique , which is used at present for the evaluation of the morphology , location , and configuration of isthmus . the technique was first used by mannocci et al . to determine the prevalence of isthmuses in the mesial roots of mandibular first molars . one of the advantages of this technique is a thorough reconstruction of the root canal system without destroying the specimens . if the isthmuses are not cleared of bacteria , there is potential for the treatment to fail , and the presence of unsuspected isthmuses may also affect the quality of the root canal filling . therefore , complete removal of debris and micro - organisms from the apical third of the root canal is an important predicting factor for improving the long - term prognosis of endodontic treatment . a recent study found that the residual bacteria which frequently are entrapped in ramifications , isthmuses , and dentinal tubules makes it necessary to use an antibacterial irrigant and inter appointment medicament to maximize bacterial reduction before filling of the infected teeth . however , the complete eradication of bacteria could not be achieved in apical isthmus after two sessions of endodontic therapy . despite various studies on the evaluation and management of isthmuses and recent advances in nonsurgical endodontic treatment modalities such as modern sonic and ultrasonic irrigation devices , side - vented needle irrigation ( sni ) , and vpro endosafe ( vpro ) , cleaning and shaping of isthmus areas showed that the application of negative pressure techniques for the removal of debris from the isthmus in the mesial root of a mandibular first molar does not lead to the removal of more debris compared to the manual dynamic irrigation technique and none of the techniques completely removes debris from an isthmus . some in vitro studies have shown that none of the isthmuses in the root canals can be completely obturated with root - filling materials during conventional endodontic treatment . it was shown that production of dentinal debris during canal instrumentation and its penetration into the isthmuses of mesial root canals of mandibular molars prevent penetration of sealers and filling materials into the isthmuses despite continuous irrigation during and after instrumentation . therefore , proper management of isthmuses including bacterial reduction and complete filling requires future application of newer technologies and then further studies to verify their efficacies . a recent study by de groot et al . on the cleaning efficacy of laser - activated irrigation of root canals showed that the use of this technique is more efficient in removing debris from the apical third of the root canal compared to passive ultrasonic irrigation and hand irrigation techniques . in addition , the application of er , cr : ysgg laser ( er , cr : ysgg : erbium , chromium - doped : yttrium , scandium , gallium , and garnet ) for the obturation of root canal system resulted in an increased rate of better obturated root canals and isthmuses . therefore , it is postulated that the use of modern technologies such as lasers , modern irrigation devices , and surgical microscopes might result in a more thorough cleaning and obturation of isthmuses during surgical and nonsurgical endodontic treatments . isthmuses are very common in the mesial roots of mandibular permanent molars in the iranian population , with the highest prevalence in those at 6 mm distance from the root apex . therefore , endodontic microscopes and newer technologies should be used for cleaning and obturation of isthmuses to achieve higher success rates in endodontic treatment .

background : management of canal isthmus is considered as an important factor for successful endodontic treatment . accordingly , this study was designed to determine the prevalence , location , and types of isthmus in mesial root canals of extracted mandibular molars in a sample of iranian population.materials and methods : in this cross - sectional descriptive study , 60 extracted molars with two mesial canals were included . the samples were initially decoronated and then , roots were sectioned horizontally at 2 , 4 , and 6 mm levels from the apex via a low - speed handpiece with a thin metallic disk and finally prepared and stained with indian ink . all sections were examined using a stereomicroscope at a magnification of 30 . prevalence , location , and types of isthmus were evaluated based on the classifications by kim and teixeira and all data were statistically analyzed by the chi - squared test . the statistical significance level was established at 0.05.results:eighty-three percent of extracted mandibular molars had an isthmus at the mesial root . this prevalence increased with distance from the apex , that is , 92% at 6 mm from the apex and 70% at 2 mm from the apex . a statistically significant difference was found between the sections at 2 and 6 mm from the apex ( p < 0.05 ) , but no other significant differences between other levels ( p > 0.05).conclusion : isthmus is very common in the mesial roots of the mandibular permanent molars in the iranian population , with the highest prevalence in the 6 mm distance from the root apex . therefore , detection , cleaning , and filling of these apical 6 mm isthmuses are of great benefit in modern endodontics .

sleep can be disturbed by several factors such as illness , stress , and noise . over time these include poor memory , slower reaction time , emotional disturbances , and changes in the immune response ( 1 , 2 ) . however , administration of these drugs is accompanied by side effects including psychomotor impairment , drug dependence , tolerance , amnesia , rebound insomnia , and the potentiating effects of other central depressant drugs ( 3 ) . moreover , some patients with sleep disorders do not respond effectively to current therapeutics . therefore , studies to find new hypnotic agents with fewer side effects and more efficacy have continued . herbal agents have long been a valuable source for developing new therapeutics for disease treatment . ocimum basilicum ( o. basilicum ) is an annual herb belonging to the lamiaceae family and grows mostly in tropical regions such as india , africa , and south asia ( 4 ) . in traditional medicine , o. basilicum is used to treat many disorders such as anxiety , diabetes , cardiovascular diseases , headaches , neurological pain , and seizures ( 5 , 6 ) . it has been demonstrated that the ethyl acetate fraction ( eaf ) of o. basilicum decreases ischemia - induced oxidative stress in the brain and improves short - term memory and motor coordination ( 5 ) . it was reported in some traditional medical texts that o. basilicum leads to sleep and a sedative state . also , some biological activities of o. basilicum were screened by ismail , and it was found that the essential oil of o. basilicum induces anticonvulsant and hypnotic activities ( 7 ) . in folk medicine , maceration is the most widely used form of o. basilicum preparation . however , there is no pharmacological evidence for the sedative - hypnotic effect of o. basilicum macerated extract . the present study was designed to evaluate the sleep - prolonging effects of hydro - alcoholic extract ( hae ) of o. basilicum ) and its fractions . dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st . dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) . the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade . a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) . hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours . the extract was then filtered through a 0.106 mm filter and dried in a water bath . the yield of the dried extract as related to the weight of the dried leaves was 21% . for the preparation of different fractions from hae , 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction , the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) . all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used . then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso . male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food . the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) . first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment , to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) . the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice . after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) . flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) . all materials were injected with a volume of 10 mg / kg ( 11 ) . the onset of sleep is the time that the mice stayed immobile and lost their righting reflex . the time interval between administration of pentobarbital and onset of sleep was considered sleep latency . nine groups , each containing two mice were used for determination of ld50 of hae . groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle . the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded . the mean of these two doses was considered the median lethal dose ( 14 , 15 ) . the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) . pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts . also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials . the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) . then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) . the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 . then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) . briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours . statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) . dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st . dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) . the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade . a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) . hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours . the extract was then filtered through a 0.106 mm filter and dried in a water bath . the yield of the dried extract as related to the weight of the dried leaves was 21% . for the preparation of different fractions from hae , 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction , the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) . all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used . then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso . male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22 1c ) with a 12 hours light / dark cycle and free access to water and food . the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) . first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment , to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) . the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice . after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) . flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) . all materials were injected with a volume of 10 mg / kg ( 11 ) . the onset of sleep is the time that the mice stayed immobile and lost their righting reflex . the time interval between administration of pentobarbital and onset of sleep was considered sleep latency . nine groups , each containing two mice were used for determination of ld50 of hae . groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle . the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded . the mean of these two doses was considered the median lethal dose ( 14 , 15 ) . the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) . pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts . also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials . the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) . then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) . the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 . then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) . briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours . statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) . sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) . as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) . the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively . the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) . the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100 mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) . similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) . the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) . flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity . duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep . the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p < 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) . figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) . the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively . the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae . the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf . values are presented as mean sem ( n = 8) . among the different fractions of hae , however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant . the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively . the mean of these two doses ( 2.4 g / kg ) was considered as ld50 . the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) . it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract , cell viability was 100 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) . regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively . again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) . the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum . sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes ( figure 1 ) . as expected , the reference drug diazepam was able to increase the duration of sleep ( 42 4.6 minutes , p < 0.001 vs. control ) . the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51 3 minutes ( p < 0.001 ) , 79 3 minutes ( p < 0.001 ) , and 46 5 minutes ( p < 0.001 ) , respectively . the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) . the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100 mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) . similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79 3 minutes and 30 2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) . the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) . flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ; p values are presented as mean sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity . duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40 4.5 minutes ( p < 0.001 vs. control ) , 30 2 minutes ( p < 0.001 vs. vehicle ) and 42 2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep . the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p < 0.05 vs. eaf group ; values are presented as mean sem ( n = 8) . figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7 0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5 0.4 minutes ( p < 0.001 ) . the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2 0.2 minutes ( p < 0.001 ) , 4.5 0.5 minutes ( p < 0.01 ) , and 4.4 0.4 minutes ( p < 0.01 ) , respectively . the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae . the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf . values are presented as mean sem ( n = 8) . among the different fractions of hae , however , nbf significantly decreased the latency time to 4.7 0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7 0.4 minutes ) to 5.2 0.2 minutes , this effect was not statistically significant . the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively . the mean of these two doses ( 2.4 g / kg ) was considered as ld50 . the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) . it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract , cell viability was 100 3 , 102 2 , 103 2 , 110 3 , and 113 3% , respectively , as compared to the vehicle ( 100 2.5% ) . regarding l929 cells , the viability was 106 1.5 , 108 2 , 112 2 , 113 1 , and 123 2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively . again , there was no significant difference in the cell viability when compared to the vehicle ( 100 2% ) . the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum . the present study showed that o. basilicum further enhances sleep behavior , confirming that this plant has a hypnotic action as claimed in traditional medicine . to our knowledge , this is the first pharmacological study showing the effects of the macerated extract of this plant on sleep duration and sleep latency . also , this is the first work to determine the ld50 value for hae of o. basilicum and to assess possible cytotoxicity of this extract on neuronal cells . yet , results of this study are preliminary and need to be confirmed by further clinical trials . the hypnotic assessment method was based on prolongation of sleep induced by pentobarbital , which is the most commonly used method for screening sedative - hypnotic agents ( 11 - 13 ) . in agreement with the previously published works and as expected , diazepam significantly increased pentobarbital - induced sleeping time , indicating that our study method was optimized ( 17 , 18 ) . the effect of hae of o. basilicum on sleep latency was lower than that of diazepam . however , the sleep - prolonging effect of hae was comparable to that of diazepam , and , even at a dose of 50 mg / kg , hae had a greater effect . also , the hypnotic effect of hae of o. basilicum is greater when compared with the effect of essential oil of o. basilicum reported by ismail ( 4-fold and 2-fold increase in sleep duration , respectively ) ( 7 ) . according to our results , the hypnotic effect of hae was not dose - dependent in the range of given doses , and the maximum effect occurred with a dose of 50 mg / kg . therefore , we used this dose to investigate the effects of different fractions of hae . the fractions of hae were prepared to obtain better insight into the nature of compounds responsible for the hypnotic effect of o. basilicum : 1 , wf which extracts water - soluble constituents ( e.g. , glycosides , quaternary alkaloids , tannins ) ; 2 , eaf which extracts compounds of intermediate polarity ( e.g. , some flavonoids ) ; and 3 , nbf which extracts low polar agents ( e.g. , sterols , alkanes , and some terpenoids ) ( 19 , 20 ) . although wf , eaf , and nbf at a dose of 50 mg / kg were all able to increase sleep duration , not one of them could enhance sleep duration to the level induced by 50 mg / kg of hae . because hae contains the active constituents of all the above - mentioned fractions , it can be concluded that an additive effect was caused by the interaction between these constituents when hae was administrated . therefore , both polar and non - polar constituents in o. basilicum extract are involved in the sleep - prolonging effect of this plant . among wf , eaf , and nbf fractions , nbf not only induced the maximum prolongation of sleep duration , but also was the only fraction to induce a significant decrease in sleep latency . on the other hand therefore , nbf contains a higher concentration of constituents responsible for the hypnotic effect of o. basilicum . these include terpenoids ( e.g. , linalool , eugenol ) , flavonoids ( e.g. , quercetrin , luteolin ) , alkaloids ( e.g. , rosmarinic acid ) , steroids ( e.g. , beta - sitisterol ) , and saponins ( 21 , 22 ) . it has been shown that o. basilicum contains high amounts of linalool , eugenol , and rosmarinic acid ( 7 ) . linck and coworkers demonstrated that linalool increases barbiturate - induced sleeping time in mice ( 23 ) . similarly , the sleep - prolonging effect of eugenol was reported by sharma et al . rosmarinic acid is found in methanolic extract of o. basilicum and can be isolated from the nbf of plant extracts ( 24 , 25 ) . it plays a major role in the sedative - hypnotic actions of some medicinal plants ( 26 ) . neurons located in the anterior hypothalamus release gamma - aminobutyric acid ( gaba ) to inhibit wake - promoting areas in the hypothalamus and brainstem ( 27 , 28 ) . barbiturates such as pentobarbital act on the gaba receptor s ionophore complex and favor the binding of gaba . benzodiazepines such as diazepam increase the affinity of gaba for its receptor and thereby enhance pentobarbital - induced sleeping time ( 29 ) . consistent with this , we observed that pretreatment of mice with flumazenil decreases the sleep - prolonging effect of diazepam . in agreement with our finding , awad et al . reported that rosmarinic acid , which is found in the extract of o. basilicum , can act as a gaba transaminase inhibitor and therefore increases the brain level of gaba ( 30 ) . therefore , it is rational to suggest that the sleep - prolonging effect of o. basilicum is mediated , at least in part , through the potentiating of the gabaergic system . it is now accepted that some natural compounds interact with the gabaergic system to increase sleep behavior ( 31 , 32 ) . the toxicity assay showed that the ld50 value for hae of o. basilicum is 2.4 g / kg . this dose is far from its hypnotic doses ( 25 - 100 mg / kg ) . also , hae , even at high concentrations , did not decrease the viability of neuronal and fibroblast cells . therefore , it seems that the hypnotic effect of o. basilicum is not accompanied by neurotoxicity . in conclusion , present data demonstrated that the macerated extract of o. basilicum potentiates sleeping behaviors without any cytotoxicity .

backgroundsleep disorders are accompanied by several complications , and currently used soporific drugs can induce unwanted effects such as psychomotor impairment , tolerance , amnesia , and rebound insomnia.objectivesthe present study was carried out to investigate if ocimum basilicum has a sleep - prolonging effect.materials and methodsthis work was an experimental study on 72 mice which were randomly divided into 9 groups : saline ( control ) ; diazepam ( 3 mg / kg , positive control ) ; hydro - alcoholic extract ( hae ) of ocimum basilicum ( 25 , 50 , or 100 mg / kg ) ; ethyl acetate fraction ( eaf , 50 mg / kg ) ; n - butanol fraction ( nbf , 50 mg / kg ) ; water fraction ( wf , 50 mg / kg ) ; and saline containing 10% dmso ( vehicle for eaf and nbf ) . all the test compounds were injected intraperitoneally ( ip ) 30 minutes before pentobarbital administration ( 30 mg / kg ) . duration and latency of pentobarbital - induced sleep were recorded . also , ld50 of hae was determined and the cytotoxicity of hae was tested on neural and fibroblast cells using the mtt assay.resultshae increased the duration of pentobarbital - induced sleep at doses of 25 , 50 , and 100 mg / kg ( p < 0.001 ) . the hypnotic effect of hae was comparable to that induced by diazepam . similarly , wf , eaf , and nbf at 50 mg / kg could increase sleep duration . the sleep latency was decreased by hae ( p < 0.01 - p < 0.001 ) and nbf ( p < 0.001 ) , but not by wf and eaf . the ld50 value for hae was found to be 2.4 g / kg . hae had no effect on the viability of neuronal pc12 cells and l929 fibroblast cells.conclusionsthe present data demonstrated that ocimum basilicum potentiates sleeping behaviors without any cytotoxicity . the main component ( s ) responsible for the hypnotic effects of this plant is most likely a non - polar agent ( s ) which is found in nbf . isolation of the active constituents may yield a novel sedative drug .

a summary of methods is provided below and a detailed description of methods is included in the supplementary information . the toxin nanosponges were prepared by fusing rbc membrane vesicles on preformed poly(lactic - co - glycolic acid ) ( plga ) nanoparticles through an established extrusion process . the size of the nanosponges was obtained from three dynamic light scattering ( dls ) measurements using a malvern zen 3600 zetasizer . the morphology of the nanosponges after absorbing toxins was measured by transmission electron microscopy ( tem ) . for preparation of human rbc nanosponges , the rbcs were collected from whole human blood ( bioreclamation ) and the characterization results were shown in fig . reconstitution of the lyophilized samples was performed by solubilizing the samples in water and the characterization results were included in fig . the in vitro toxin neutralization ability of the nanosponges was examined by mixing 3 g of -toxin with 200 l of 1 mg / ml nanosponges for 30 min , followed by adding into 1.8 ml of 5% purified mouse rbcs . the in vitro toxin absorption capacity of the nanosponges was determined through titrating -toxin to a fixed amount of nanosponges . the interaction of the nanosponges with cells was examined by a scanning fluorescence microscopy by incubating fluorescent nanosponges and rbc membrane vesicles with human umbilical vein endothelial cells ( huvec ) . the in vitro cellular cytotoxicity of nanosponge - sequestered toxins was examined by incubating nanosponges of different concentrations with varied amounts of -toxin , streptolysin - o , and melittin for 30 min , followed by adding to huvecs for 24 hr . the in vivo toxin neutralization ability of the nanosponges was tested through subcutaneous injection of the nanosponge / toxin mixture to the flank region of nude mice , followed by histological analyses . on - site neutralization of -toxin by the nanosponges was conducted by subcutaneously injecting 50 l of 36 g / ml of -toxin solution , immediately followed by a 100 l injection of 2 mg / ml nanosponges . the mice were imaged 3 days later for visualization of skin lesion formation ( fig s11 ) . the in vivo detoxification efficacy was tested through intravenous injection of nanosponges before or after administration of a lethal dose of -toxin to icr mice , followed by monitoring the survival rate of the mice . for the in vivo hepatotoxicity study , one group of mice was sacrificed on day 3 following the injection of the toxin - bound nanosponges and another group was sacrificed on day 7 .

detoxification treatments such as toxin - targeted anti - virulence therapy1 , 2 offer ways to cleanse the body of virulence factors that are caused by bacterial infections , venomous injuries , and biological weaponry . because existing detoxification platforms such as antisera3 , monoclonal antibodies4 , small - molecule inhibitors5 , 6 , and molecularly imprinted polymers7 act by targeting the molecular structures of the toxins , customized treatments are required for different diseases . here we show a biomimetic toxin nanosponge that functions as a toxin decoy in vivo . the nanosponge , which consists of a polymeric nanoparticle core surrounded by red blood cell membranes , absorbs membrane - damaging toxins and diverts them away from their cellular targets . in a mouse model , the nanosponges markedly reduce the toxicity of staphylococcal alpha - hemolysin ( -toxin ) and thus improve the survival rate of toxin - challenged mice . this biologically inspired toxin nanosponge presents a detoxification treatment that can potentially treat a variety of injuries and diseases caused by pore - forming toxins .

in rural australia , undersupply and uneven geographic distribution of the specialist medical workforce has produced an apparent gap between service demand and the availability of radiologists.1 as a result , the responsibility for interpreting radiographic images often falls provisionally to the referring doctor , commonly a general practitioner . radiographic image interpretation involves highlevel skills and complex decision making , and there is a risk that errors may compromise patient care.2 , 3 , 4 although some degree of error in radiographic image interpretation is unavoidable,5 these errors may be reduced by interprofessional collaboration2 , 6 between the referrer and the radiographer . effective communication is integral to interprofessional collaboration and has the potential to positively impact on patient care.7 , 8 , 9 radiographers ' opinions about the presence or absence of pathology on a radiographic image could be valuable in a collaborative approach to diagnosis.3 , 6 , 10 , 11 , 12 however , it is often difficult for radiographers to navigate this interprofessional boundary because communication pathways for sharing radiographic interpretations are unclear.11 in part , this is because radiographic image interpretation was effectively removed from radiographers ' scope of practice in 1925.13 at this time , it was formally decreed that radiographic interpretation was diagnostic work and therefore the responsibility of medically qualified practitioners.13 resultantly , few radiographers are now educationally prepared for image interpretation,14 although they may possess experiential knowledge in recognising abnormalities.15 radiographers have attempted to address the communication gap using radiographer abnormality detection schemes ( rads ) . one welldocumented rads is the red dot system,16 which was designed as a means for radiographers to alert the referring doctor to an abnormality on a patient 's radiographic image . more than 30 years after its introduction , the red dot system is not consistently used across australia.17 communication difficulties which contribute to adverse events in healthcare may arise from the hierarchy of power that exists between different health professionals.7 , 8 , 18 it seems that historical changes to radiography have set in place a restrictive , hierarchical professional structure in which radiographers now practice.19 , 20 a consequence of this structure is that autonomy within radiographer practice is discouraged by paternalism and subordination from medical colleagues.21 this is a characteristic of medical dominance , where the medical profession holds significant power and authority over neighbouring occupations.22 , 23 whenever issues related to interprofessional boundary delineation come into question , such as radiographic image interpretation and communication of results , historical hierarchical relationships may influence and shape the way the interactions take place . awareness of the barriers and enablers of communication in rural radiographic practice will enable a move towards stronger collaborative partnerships between radiographers and their medical colleagues . a twophase doctoral study was undertaken to examine australian rural radiographers ' and their experiences and perceptions of radiographic interpretation and subsequent disclosure . this article reports the research findings of the interpretive component of the study that answers the research question , how do rural radiographers construct ways to negotiate communication and disclosure of their radiographic opinion within their practice world? the study , which employed both descriptive and interpretive research methods had ethics approval from the human research ethics committee ( tasmanian ) network and was completed in 2012 . the inclusion criteria were possession of an australian institute of radiography statement of accreditation , or registration with the medical radiation technologists board of western australia ( prior to national registration ) and providing radiographic services to an area with a population of less than 100,000 . the first phase of the study employed a postal questionnaire to collect descriptive data for development of a demographic profile of the respondent rural radiographers and to examine the context in which this study was situated . prior to dissemination , the questionnaire was piloted , and its content and construct validity were established . it was distributed by third parties to radiographers practising in rural new south wales , western australia and tasmania . the quantitative questionnaire data was analysed using spss ( ms windows , version 15 , chicago , il , usa ) to produce descriptive statistics . the second phase of the study used semistructured interviews to elicit rich , indepth qualitative data . qualitative research methods are well suited to the examination of professional communication24 and allowed an interpretive inquiry focused on understanding meaning , purposes and intentions in how individuals act and interact with others.25 the interview cues were developed initially from the key issues arising from the questionnaire data and later from the analysis of the interview data . the qualitative interview data and openended questionnaire responses were analysed thematically with crosscomparison assisted by nvivo 8 software ( qsr international , melbourne , australia ) to code , organise , explore and analyse the interrelated themes . the data collection , analysis and interpretation occurred as a continuous process allowing emerging themes to influence further data collection . themes were derived that illustrate and describe the dynamics of interprofessional communications between rural radiographers and referring doctors . twentythree of the respondents also volunteered for the indepth , semistructured interviews via the invitation included with the questionnaire . the interviewing ceased once the collective experience of subsequent informants confirmed earlier findings rather than leading to a new insight , indicating that theoretical saturation was reached.26 all interviewees were experienced rural radiographers with an age range of 3662 ( mean 53.4 ) years and 1642 ( mean 32.9 ) years ' radiographic experience . at the time of the interview , all the radiographers were employed in either private practice or public hospitals in communities with a population of less than 50,000 . patient advocacy emerged as an overarching theme and the key driver for radiographers to use various communication pathways with referrers . a number of factors shape professional communication and interaction , including the perceived seriousness of the patient 's condition , delays in diagnosis , the degree of professional confidence and the relationship between the radiographer and the referrer . communication pathways were either direct or indirect , depending upon the various barriers to communication encountered . interceding on behalf of the patient to maximise healthcare outcomes was an overarching feature of rural radiographer 's professional role.rr1 : that 's how i have looked at it , what 's best for the patient rr 9 : going all out and doing what you can to help the management of those patients rr1 : that 's how i have looked at it , what 's best for the patient rr 9 : going all out and doing what you can to help the management of those patients the radiographers clearly regard their role to be more than technicians responsible for image acquisition : patients are central to all of their actions and decision making . it is patient care that incentivises radiographers to ensure timely and appropriate treatment for the patient in the presence of radiographic abnormalities.rr 4 : i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film , particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . rr 9 : i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film , particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . rr 9 : i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see well i think you have to although radiographers are strongly committed to patient welfare , patient advocacy is not easy . direct communication pathways are the most transparent and effective means for achieving the best outcome for the patient . recognising the risk of error in radiographic interpretation , the radiographers used direct communication to ensure that the doctors do not miss significant pathology.rr 1: because they miss so much if i was n't sure i would n't say anything but if there is a definite crack there that i am pretty sure the doctor 's going to miss , it might show in one view , i will say something to them it 's been a few political decisions i 've had to make over the years if i was n't sure i would n't say anything but if there is a definite crack there that i am pretty sure the doctor 's going to miss , it might show in one view , i will say something to them it 's been a few political decisions i 've had to make over the years collaborative practice provides an avenue for the referring doctor to actively seek the radiographer 's opinion.rr 3 : i do get asked for my opinion quite a few times rr 6 : in spite of the fact that we have got teleradiology and pacs [ picture archiving and communication system ] in the first instance it is always your own view that is canvassed . rr 9 : certainly in rural areas not only do you feel that you should , you are actually asked if you can . rr 3 : i do get asked for my opinion quite a few times rr 6 : in spite of the fact that we have got teleradiology and pacs [ picture archiving and communication system ] in the first instance it is always your own view that is canvassed . rr 9 : certainly in rural areas not only do you feel that you should , you are actually asked if you can . in other situations , radiographer 's volunteer an unsolicited opinion to the doctor.rr 2 : i am quite happy to ring them and say look your patient has got a pleural effusion rr4 : i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture rr 2 : i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4 : i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture these collegial , open and direct communications are considered as a collaborative approach to reaching an accurate diagnosis , even if the referrer was inexperienced , as one participant describes.rr 5 : rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them do n't necessarily have the experience rr 5 : rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them do n't necessarily have the experience deciding whether to directly and explicitly communicate any radiographic abnormality to the referrer is influenced by the perceived seriousness of the impending diagnosis and the urgency with which care is required.rr 2 : a bad fracture or something needs to be treated straight away [ whereas ] someone with advanced cancer or whatever , even if i told them at four o'clock in the afternoon the doctor ca n't do anything , can he ? rr 1 : this guy came in and he said my neck 's really sore i looked at the first film , oh , you 've got a fracture here so i went out to him , and it was really unstable , and i said i 've got to ring the doctor but do n't move. within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . rr 2 : a bad fracture or something needs to be treated straight away [ whereas ] someone with advanced cancer or whatever , even if i told them at four o'clock in the afternoon the doctor ca n't do anything , can he ? rr 1 : this guy came in and he said my neck 's really sore i looked at the first film , oh , you 've got a fracture here so i went out to him , and it was really unstable , and i said i 've got to ring the doctor but do n't move. within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . in situations where a patient required urgent attention and interprofessional relationships allow , radiographers assume a proactive role and communicated directly with the referrer . however , sometimes individual traits became barriers to direct communication pathways . for example : rr 3 : it depends a bit on the referrer and their rapport with the radiographer , some will ask our opinions , others rr 6 : there are those [ doctors ] that set themselves apart and are just completely unwilling to accept any advice from anybody rr 3 : it depends a bit on the referrer and their rapport with the radiographer , some will ask our opinions , others rr 6 : there are those [ doctors ] that set themselves apart and are just completely unwilling to accept any advice from anybody other radiographers spoke about the way in which different referrers react to their disclosure of radiographic opinion . for example , one radiographer stated : rr 2 : he just would n't have it . that 's an old fracture , that 's not new , it 's an old fracture. you do n't know what you are talking about , i 'm the doctor. so you have no comeback at that because , yes , they are the doctor . that 's an old fracture , that 's not new , it 's an old fracture. you do n't know what you are talking about , i 'm the doctor. so you have no comeback at that because , yes , they are the doctor . the radiographers revealed a lack of formal training in recognising and describing pathology on radiographic images.rr 6 : well , i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . rr 5 : our interpretations skills came from discussion with colleagues ; we had sessions where we would interpret films , so most of our training was discussion with colleagues , with seniors over pathologies rr 6 : well , i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . rr 5 : our interpretations skills came from discussion with colleagues ; we had sessions where we would interpret films , so most of our training was discussion with colleagues , with seniors over pathologies one consequence of limited knowledge in image interpretation is that radiographers are reticent to offer their radiographic opinion to referrers and this worked to impede communication.rr 7 : i do n't feel comfortable with chests so i rarely say much with a chest . rr 8 : if it 's something that you are unsure of you do n't offer rr 7 : i do n't feel comfortable with chests so i rarely say much with a chest . rr 8 : if it 's something that you are unsure of you do n't offer despite the lack of formal educational preparation for radiographic interpretation , the radiographers described intuitive recognition of radiographic abnormalities.rr 4 : the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . rr 4 : the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . radiographers ' intuitive recognition of radiographic abnormalities could be useful for assisting referrers in reaching a diagnosis but in some instances a lack of a confident radiographic interpretation inhibits their use of direct communication pathways . in order to circumvent the barriers to communication the radiographers used indirect communication pathways to navigate complex interprofessional boundaries . when circumstances do not support direct communication , the radiographers choose either sidestepping direct communication with the referrer or to use a hint and hope approach in order to ensure timely patient care . one way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . in doing so , that particular patient 's images move towards the front of the reporting queue and the radiologist 's report is provided to the referrer more quickly.rr 2 : i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . assigning a radiograph a higher reporting priority , does not necessarily result in faster diagnosis and significant delays can still occur.rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . to minimise the potential negative impact on patient care that these delays may create , some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers , but still provide radiographic interpretation input.rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . laudable when first developed , the red dot system is not universally accepted or even well understood by other health professionals . it is therefore flawed as a communication system , as suggested by this excerpt.rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . in situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other , radiographers sometimes take a more subordinate , but supportive role . they avoid direct language , and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . the radiographers also allow the referring doctor to initially use their own radiographic interpretation skills , but intercede if the abnormality is overlooked.rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . the hint and hope strategy appears to be effective when communicating information about the patients needs , but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . interceding on behalf of the patient to maximise healthcare outcomes was an overarching feature of rural radiographer 's professional role.rr1 : that 's how i have looked at it , what 's best for the patient rr 9 : going all out and doing what you can to help the management of those patients rr1 : that 's how i have looked at it , what 's best for the patient rr 9 : going all out and doing what you can to help the management of those patients the radiographers clearly regard their role to be more than technicians responsible for image acquisition : patients are central to all of their actions and decision making . it is patient care that incentivises radiographers to ensure timely and appropriate treatment for the patient in the presence of radiographic abnormalities.rr 4 : i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film , particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . rr 9 : i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film , particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . rr 9 : i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see well i think you have to although radiographers are strongly committed to patient welfare , patient advocacy is not easy . direct communication pathways are the most transparent and effective means for achieving the best outcome for the patient . recognising the risk of error in radiographic interpretation , the radiographers used direct communication to ensure that the doctors do not miss significant pathology.rr 1: because they miss so much if i was n't sure i would n't say anything but if there is a definite crack there that i am pretty sure the doctor 's going to miss , it might show in one view , i will say something to them it 's been a few political decisions i 've had to make over the years if i was n't sure i would n't say anything but if there is a definite crack there that i am pretty sure the doctor 's going to miss , it might show in one view , i will say something to them it 's been a few political decisions i 've had to make over the years collaborative practice provides an avenue for the referring doctor to actively seek the radiographer 's opinion.rr 3 : i do get asked for my opinion quite a few times rr 6 : in spite of the fact that we have got teleradiology and pacs [ picture archiving and communication system ] in the first instance it is always your own view that is canvassed . rr 9 : certainly in rural areas not only do you feel that you should , you are actually asked if you can . rr 3 : i do get asked for my opinion quite a few times rr 6 : in spite of the fact that we have got teleradiology and pacs [ picture archiving and communication system ] in the first instance it is always your own view that is canvassed . rr 9 : certainly in rural areas not only do you feel that you should , you are actually asked if you can . in other situations , radiographer 's volunteer an unsolicited opinion to the doctor.rr 2 : i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4 : i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture rr 2 : i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4 : i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture these collegial , open and direct communications are considered as a collaborative approach to reaching an accurate diagnosis , even if the referrer was inexperienced , as one participant describes.rr 5 : rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them do n't necessarily have the experience rr 5 : rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them do n't necessarily have the experience deciding whether to directly and explicitly communicate any radiographic abnormality to the referrer is influenced by the perceived seriousness of the impending diagnosis and the urgency with which care is required.rr 2 : a bad fracture or something needs to be treated straight away [ whereas ] someone with advanced cancer or whatever , even if i told them at four o'clock in the afternoon the doctor ca n't do anything , can he ? rr 1 : this guy came in and he said my neck 's really sore i looked at the first film , oh , you 've got a fracture here so i went out to him , and it was really unstable , and i said i 've got to ring the doctor but do n't move. within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . rr 2 : a bad fracture or something needs to be treated straight away [ whereas ] someone with advanced cancer or whatever , even if i told them at four o'clock in the afternoon the doctor ca n't do anything , can he ? rr 1 : this guy came in and he said my neck 's really sore i looked at the first film , oh , you 've got a fracture here so i went out to him , and it was really unstable , and i said i 've got to ring the doctor but do n't move. within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . in situations where a patient required urgent attention and interprofessional relationships allow , radiographers assume a proactive role and communicated directly with the referrer . individual perceptions of role delineation within the health professions influenced radiographer and referrer interaction . for example : rr 3 : it depends a bit on the referrer and their rapport with the radiographer , some will ask our opinions , others they look for other doctors around the hospital to discuss it with . rr 6 : there are those [ doctors ] that set themselves apart and are just completely unwilling to accept any advice from anybody rr 3 : it depends a bit on the referrer and their rapport with the radiographer , some will ask our opinions , others they look for other rr 6 : there are those [ doctors ] that set themselves apart and are just completely unwilling to accept any advice from anybody other radiographers spoke about the way in which different referrers react to their disclosure of radiographic opinion . for example , one radiographer stated : rr 2 : he just would n't have it . that 's an old fracture , that 's not new , it 's an old fracture. you do n't know what you are talking about , i 'm the doctor. so you have no comeback at that because , yes , they are the doctor . that 's an old fracture , that 's not new , it 's an old fracture. you do n't know what you are talking about , i 'm the doctor. so you have no comeback at that because , yes , they are the doctor . the radiographers revealed a lack of formal training in recognising and describing pathology on radiographic images.rr 6 : well , i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . rr 5 : our interpretations skills came from discussion with colleagues ; we had sessions where we would interpret films , so most of our training was discussion with colleagues , with seniors over pathologies rr 6 : well , i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . rr 5 : our interpretations skills came from discussion with colleagues ; we had sessions where we would interpret films , so most of our training was discussion with colleagues , with seniors over pathologies one consequence of limited knowledge in image interpretation is that radiographers are reticent to offer their radiographic opinion to referrers and this worked to impede communication.rr 7 : i do n't feel comfortable with chests so i rarely say much with a chest . rr 8 : if it 's something that you are unsure of you do n't offer rr 7 : i do n't feel comfortable with chests so i rarely say much with a chest . rr 8 : if it 's something that you are unsure of you do n't offer despite the lack of formal educational preparation for radiographic interpretation , the radiographers described intuitive recognition of radiographic abnormalities.rr 4 : the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . rr 4 : the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . radiographers ' intuitive recognition of radiographic abnormalities could be useful for assisting referrers in reaching a diagnosis but in some instances a lack of a confident radiographic interpretation inhibits their use of direct communication pathways . in order to circumvent the barriers to communication sidestepping direct communication with the referrer or to use a hint and hope approach in order to ensure timely patient care . one way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . in doing so , that particular patient 's images move towards the front of the reporting queue and the radiologist 's report is provided to the referrer more quickly.rr 2 : i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . assigning a radiograph a higher reporting priority , does not necessarily result in faster diagnosis and significant delays can still occur.rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . to minimise the potential negative impact on patient care that these delays may create , some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers , but still provide radiographic interpretation input.rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . laudable when first developed , the red dot system is not universally accepted or even well understood by other health professionals . it is therefore flawed as a communication system , as suggested by this excerpt.rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . in situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other , radiographers sometimes take a more subordinate , but supportive role . they avoid direct language , and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . the radiographers also allow the referring doctor to initially use their own radiographic interpretation skills , but intercede if the abnormality is overlooked.rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . the hint and hope strategy appears to be effective when communicating information about the patients needs , but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . one way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . in doing so , that particular patient 's images move towards the front of the reporting queue and the radiologist 's report is provided to the referrer more quickly.rr 2 : i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . i have looked at the films and one of them had a massive pleural effusion on the right side , no air at all i put a priority on it because , i mean the man could n't breathe for a start but also you knew that something was happening in there that needed investigation . assigning a radiograph a higher reporting priority , does not necessarily result in faster diagnosis and significant delays can still occur.rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . rr 2 : the more that you 've been in practice , the more that you know that it 's not a perfect world even if you put a priority on it , some of them get missed and the doctor 's left for the day all those things happen because it 's not a perfect world . to minimise the potential negative impact on patient care that these delays may create , some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers , but still provide radiographic interpretation input.rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . rr 1 : i got to the stage where i used to put red dots on them , if there was something [ abnormal ] . rr 6 : if there is an abnormality in the films then we will red dot them . laudable when first developed , the red dot system is not universally accepted or even well understood by other health professionals . it is therefore flawed as a communication system , as suggested by this excerpt.rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . rr 1 : but other doctors were n't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . in situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other , radiographers sometimes take a more subordinate , but supportive role . they avoid direct language , and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . rr 8 : sort of give the referrer an opportunity by heading them in a direction and quietly saying , now do n't you think that looks a bit extraordinary or something like that . i would say that does n't look right to me , or what do you think about that , does that bulge look normal i would let them make the decision . the radiographers also allow the referring doctor to initially use their own radiographic interpretation skills , but intercede if the abnormality is overlooked.rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . rr 4 : so , you would take your films around and you would go , i think you 'd better look at this , and then you would stand back and let them look at it . and , then if they went round and round whatever what was obviously wrong , i would either say if it was an obvious fracture or something , i would be quite happy to offer an opinion and say , what do you think of that? , and then lead them in to it that way . the hint and hope strategy appears to be effective when communicating information about the patients needs , but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . radiographers can play a valuable role in helping rural doctors to correctly interpret radiographic images . while this role may be familiar to most rural radiographers,10 the communication pathways for interprofessional collaboration are not well defined or standardised . this interpretive inquiry has revealed that the evolution of radiography under the medical dominance model21 , 23 has influenced both radiographers preparation for interprofessional radiographic interpretation and impacted on their communication strategies . radiographic image interpretation is intricately linked to diagnosis , and as a result , radiographers have not historically been formally educated and trained in identifying and describing pathology on radiographs . this leaves a gap in their knowledge , skills and abilities , which leads to a lack of confidence.14 furthermore , unequal power relationships may cause those positioned at the base of the hierarchical pyramid , such as radiographers , to remain silent.22 a flattened hierarchy enables individuals to voice their opinion,8 as is evidenced by radiographers use of direct communication pathways . this study indicates that in some cases , a perceived or evidenced hierarchy remains and , along with a lack of preparation for a role in radiographic interpretation , this has the ability to stifle interprofessional communication . of concern is that as a consequence radiographers tend to enter into a radiographer nurse game,7 , 27 which is an interprofessional communication strategy where doctors are overtly or covertly guided in their clinical decisions by nurses . in the same way that a nurse may make suggestions to guide the doctor 's decision making , the radiographers , within the limitations of their knowledge base , use mechanisms designed to directly or indirectly communicate their radiographic opinion , influencing the subsequent radiographic diagnosis . as a patient advocate workaround strategies to communicate their opinion to the referring doctor and so engage in the radiographer in australia , the educational preparation of radiographers for the higher level competencies required in image interpretation and communication of their radiographer 's opinion is currently ad hoc . this limits radiographers capacity to contribute to patient care and also limits the capacity of the health system to provide higher quality care through interprofessional teamwork . it is essential for safe care that communication is open , direct and transparent7 , so that health professional , such as radiographers , do not feel the need to enter into game playing . improving radiographers ' image interpretation skills , and making the communication pathways between radiographers and referrers more explicit , will positively impact on patient care through the timely collegial sharing of knowledge .

abstractintroductioneffective interprofessional communication is intrinsic to safe health care . despite the identified positive impact of collaborative radiographic interpretation between rural radiographers and referrers , communication difficulties still exist . this article describes the strategies that australian rural radiographers use for communication of their radiographic opinion to the referring doctor.methodsin a twophase interpretive doctoral study completed in 2012 , data were collected from radiographers working in rural new south wales , western australia and tasmania using a paper based questionnaire followed by indepth semistructured interviews . data were analysed thematically in order to identify , analyse and report the emergent themes.resultsthe overarching theme was patient advocacy , where in the interest of patient care radiographers took measures to ensure that a referring doctor did not miss radiographic abnormalities . strong interprofessional relationships enabled direct communication pathways . interprofessional boundaries shaped by historical hierarchical relationships , together with a lack of confidence and educational preparation for radiographic interpretation result in barriers to direct communication pathways . these barriers prompted radiographers to pursue indirect communication pathways , such as sidestepping and hint and hope.conclusiona lack of formal communication pathways and educational preparation for this role has resulted in radiographers playing the radiographer referrer game to overtly or covertly assist referrers in reaching a radiographic diagnosis . the findings from this study may be used to plan interventions for strengthening interprofessional communication pathways and improve quality of healthcare for patients .

the construct of unintended pregnancies ( ups ) is multifactorial and broadly encompasses pregnancies that are either unwanted or mistimed.1 in many instances , ups are likely to end by induced abortion ; worldwide estimates suggest that 50% will be voluntarily terminated.2 in 2008 , it was estimated that 43.8 million abortions occurred worldwide , of which 86% occurred in low-/middle - income countries.3 furthermore , between 2003 and 2008 , the number of induced abortions was found to have decreased in high - income countries but to have increased in low-/middle - income countries . during this same period of time , the proportion of unsafe abortions increased and they were believed to account for 13% of maternal deaths , with the majority of these concentrated in countries with restrictive laws on abortion.4 elective abortion in brazil is considered non - legal , unless the pregnancy resulted from a rape , would cause a life - threatening condition to the mother , or the fetus has anencephaly or any other malformation that is incompatible with the extrauterine life . as a result , data on prevalence and associated costs of abortion are limited and remain a challenge to collect . however , it is recognized worldwide that direct health costs of up and resulting abortions can significantly impact local health services and the families affected.5 health system and societal costs can also arise from ups that do not end in abortion and are carried to parturition . in the united states , the public health costs attributed to unintended births in a single year cost taxpayers $ 11.1 billion ( 2006).6 furthermore , previous studies have reported that ups are more likely to result in preterm births and low - birth - weight babies , which would increase health costs for neonatal care and costs associated with long - term disabilities.7 this is particularly relevant for ups in adolescence , which are more likely to result in low - birth - weight births , which can increase health costs.8 over the past few decades , brazil , like many fast - emerging economies , has experienced a fertility transition noted by a dramatic reduction in the total fertility rate , which currently sits at 1.8 births per woman.9,10 furthermore , during the fertility transition , the rate of contraception has also increased.11 despite increased contraception use , the cumulative rate of spontaneous and induced abortion changed very little between 1996 and 2006 , for which population survey estimates are available.12,13 furthermore , a survey of more than 1,000 women aged 1024 years in a single postnatal unit in brazil reported that more than 50% of all births were unintended , suggesting that possibly significant unmet contraception need still exists.8 similarly , for the whole country , the data from the last demographic and health survey ( dhs ) performed in 2006 indicates that 55% of all births were unintended.14 prevention of up using publicly funded programs in high - income countries has proven to generate significant cost savings for health services and public services.15 previous economic studies evaluating the consequences of up have mostly been performed in high - income countries where publicly funded fertility services and abortion legislation are different from those in low-/middle - income countries.15 the aim of this study was to evaluate the cost consequences of up in brazil , an emerging economy with vastly different fertility planning services than most advanced economies and with restrictive abortion laws . to the best of our knowledge , this is the first published study that describes the economics of up , and it is believed that this work can aid decision makers regarding access to contraceptives . a model was developed to evaluate the humanistic burden and financial impact of ups in brazil . the analysis factored in costs and outcomes for 1 year post - delivery of ups . possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth . all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil . available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries . complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy . data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used . a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) . the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% . the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births . differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy . all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . in brazil the public national health system ( sus ) pays for around 70%75% of all reproductive procedures . however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately . costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system . costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage . the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 . a model was developed to evaluate the humanistic burden and financial impact of ups in brazil . the analysis factored in costs and outcomes for 1 year post - delivery of ups . possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth . all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil . available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries . complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy . data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used . a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) . the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% . the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births . differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy . all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . in brazil the public national health system ( sus ) pays for around 70%75% of all reproductive procedures . however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately . costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system . costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage . the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 . based on the modeled parameters we estimate 1.79 million annual ups and 1.47 million annual planned pregnancies . we estimate annual maternal deaths of 351 , of which 49 ( 14% ) are attributed to abortions and 302 to complications from miscarriages and deliveries . the number of infant deaths within the 12 months following birth was estimated at 32,864 . the number of estimated neonatal admissions for the year 2010 associated with up was 224,631 which included all preterm deliveries and 7.6% of all term deliveries . the disaggregated values for different pregnancy outcomes attributed to up are described in table 2 . costs of abortions are not reimbursed in the public health system and are not included in the analysis . the total costs attributed to up are estimated to be r$4.1 billion annually , of which approximately r$32.9 million ( 0.8% ) was attributed to miscarriage and r$4.07 billion ( 99.2% ) to births and resulting complications . from the direct birth - related costs , antenatal care accounted for approximately r$233 million ( 5.7% ) of birth costs , with labor and delivery costs responsible for r$988 million ( 24.3% ) . the remainder of birth costs were attributed to infant complications , estimated to be r$2.8 billion ( 70.0% ) , with neonatal costs accountable for approximately r$628 million ( 15.4% ) and hospital readmission in the first year costs of r$2.1 billion ( 52.9% ) of all birth costs annually . based on the national cost estimates and the number of annual ups our analysis estimates the costs and outcomes associated with unintended pregnancies to derive a cost per up of r$2,293 . the cost per up factors in a range of health - related costs attributed to those resulting in abortion and resulting live births for those carried to parturition . this figure is broadly aligned with previous analyses in the united states of $ 1,609 per up after factoring in the purchasing power parity between the two countries.35 it is also worth noting that the estimates provided here are likely an underestimate of all costs . for example , the analysis described here focused on the costs that occur within the public health systems . consequently , costs of elective abortions paid for by individuals are not represented in this analysis . furthermore , because elective abortion is illegal in brazil except in extreme and rare cases , hidden costs from elective abortions may filter into the public health system . for instance , costs associated with post - abortive care are likely to be classified as miscarriages when reported to national authorities . additionally , the long - term societal costs that arise from up and reduced educational attainment and lost productivity of young mothers has not been accounted for in our analysis.3639 our analysis highlights that considerable cost savings can be achieved by reducing up , which are thought to represent 55% of all pregnancies in brazil.20 the analysis accounted for both untimed and unwanted births to estimate the up rate and associated costs , in which live births resulting from unwanted pregnancies represented the largest share of health costs , representing 99% of all costs . this is also attributed to the fact that abortion is a relatively inexpensive procedure that is paid for outside of the health service . because a substantial proportion of pregnancies are attributed to mistimed pregnancies , ie , pregnancies that would have occurred at some point in the future , but occurred sooner due to mistimed pregnancy , the actual cost savings to be realized by reducing up is less than described here and would mostly be associated with averting those pregnancies considered to be unwanted . to put the potential cost savings into perspective , an analysis conducted for the state of california , usa estimated that preventing many up in a single year will offer savings of $ 1.1 billion up to 2 years of age of the child , and that every dollar spent on averting up offers $ 2.76 of saving in 2 years and $ 5.33 at 5 years post - delivery.15 a national - level analysis that focused on the federally financed medicaid program in the united states noted that taxpayer savings of $ 4.7$6.2 billion could be achieved by reducing unintended pregnancies.40 based on the cost savings attributed to up , it is anticipated that future health researchers can build on the research described here to better understand the cost savings that may be achieved through improved contraceptive use . the analysis sought to incorporate a broad range of costs associated with up to illustrate that costs extend beyond the point of delivery . for instance , ups often occur in adolescent and young adults and can be predictive of low birth weight , preterm birth , and neonatal admission.8,4143 the precise mechanism by which up influences preterm birth is likely to be multifactorial . however , some studies have observed that women with ups are less likely to receive adequate prenatal care44 and are also more likely to smoke and drink alcohol during pregnancy , which contributes to adverse outcomes.44,45 it is important to highlight these costs because it suggests that ups place unnecessary demand on health services that are often stretched to capacity . since up , for the most part , can be avoided , this illustrates the health service benefits and health service capacity that could be released by reducing up . much of the humanistic burden attributed to up relates to maternal mortality and morbidity of women who pursue unsafe abortions . in our analysis , we estimate 49 annual deaths from abortions representing 13% of the estimated maternal deaths . it has been suggested that access to improved contraceptive services could prevent maternal mortality by 25%35%.46 furthermore , estimates from the world health organization reported that unsafe abortion disables approximately 5 million women each year , suggesting reductions in morbidity could also be achieved through improved contraception use.4 empirical evidence and clinical guidelines agree that the most effective approach to preventing up is through education and contraceptive use , of which long - acting contraceptive ( larc ) methods are the most effective intervention.4751 in particular , among adolescents , prevailing evidence suggests that education and contraception are the main interventions for reducing up.49 in advanced economies , the percentage of women using larc methods has been steadily increasing . a recent study noted that the proportion of women using larc methods has increased from 2.4% of women aged 1544 years since 2002 to 8.5% of women in 2009.52 in the united states , the american college of obstetricians and gynecologists ( acog ) advocates use of intrauterine contraceptives ( both copper - intrauterine device and the levonorgestrel - releasing intrauterine system ) and implants as first - line therapy for adolescent women because they contribute to reduce the number of ups.48 larc has also proven to be cost - saving compared with combined oral contraceptives in the united kingdom . investigators reported that this finding was influenced almost entirely by the lower failure rates associated with larc.50,53 an analysis conducted in the united states also noted that cost savings could be achieved by switching women from oral contraceptives to larcs , and approximately 50% of costs of up were attributed to contraceptive adherence.54 despite the positive benefits of larc , use in brazil lags behind that of other countries . a survey conducted in 2008 in northern brazil reported that only 1% of women attending a post - abortion clinic elected for an intrauterine device , despite 93% of women having knowledge of the product.55 several barriers have been identified , which often limit uptake of larc methods.56 these include health care provider lack of knowledge of risks , myths , misconceptions , and lack of training for insertion of the various devices . also , misinformation and fear of pain at insertion among women can limit demand for larc methods . furthermore , larc can often involve considerable up - front expense , although evidence does suggest that these methods are cost - effective compared with alternative contraceptive methods . because of the expense , reimbursement can influence the rate of uptake . even recognizing the importance of abortion , and especially induced and unsafe abortion , for maternal mortality and morbidity in brazil , there are no reliable and available data confirming a pandemic of such conditions as commonly reported by media and gray literature . data extracted from the 2006 brazilian dhs indicated that only 1.5% self - reported induced abortions among all pregnancies for the whole period of the survey.14,19 a probable contribution for this scenario is the high prevalence of and easy access to contraceptive use among women in reproductive age in the country during the last 2 decades . although all induced abortions are technically classified as unsafe , this is not the impression of the majority of obstetricians working in the field . there is a general belief that complications due to unsafe induced abortions are less and less frequent and that there was recently a marked drop in their occurrence . with no direct information to confirm this , one indirect point of evidence is the decrease in the proportional contribution of abortion as cause of maternal mortality in the country . in fact , official brazilian data confirm that it was 16.4% in 1990 ( representing the third cause of maternal mortality ) , while only 8.9% in 2000 and 9.0% in 2010 ( the fifth cause , behind even the indirect obstetric causes).20 in the meantime , there was a spread of the use of misoprostol as an agent for inducing abortion , not only in health facilities for situations where abortion is allowed , but mainly for situations not legally permitted , wherein the women obtain misoprostol in the black market and self - administer it vaginally . this is believed to be the current most common way of inducing medical abortion and also to be associated with the low rates of severe complications due to abortion , especially infection and hemorrhage , in brazil . there are also at least two possible limitations for the external validity of the current cost estimates . the first refers to the assumption that all miscarriages are really diagnosed and treated with dilation and curettage . however , it is possible that a proportion of individuals will never reach health facilities and receive no intervention . currently , in brazil , the majority of medical abortions are performed by the woman through self - administration of misoprostol acquired off - label or in the black market . with these procedures , the rate of complications due to medical abortions has dropped significantly during the last decade , with some of these cases arriving at hospitals already as complete abortions that need no complementary procedures . although insufficient data are available to support this scenario , this would probably impact strongly on the estimates of post - abortion care , taking into account the correspondent reduced morbidity and mortality . the cost consequences of live births attributed to up in adolescents can extend beyond the observed costs of increased prenatal care , preterm births , and abortions . studies have noted that adolescent women are more likely to dropout from school following pregnancy . furthermore , others have suggested that future generations will follow the reproductive pattern of their parents , which could perpetuate the likelihood of up in the offspring of these mothers.57 as reported , education has been shown to break the association of adolescent fertility across generations , suggesting the longer - term benefits to be achieved by reducing up . our analysis illustrates the cost consequences associated with up in brazil . in the past 2 decades , brazil has made considerable progress toward reducing abortion rates , but , despite these efforts , the humanistic and direct economic costs associated with up remain high.58 to achieve economic savings from reducing the up rate will require education , training of providers , and improved access to effective contraceptive measures . it is believed that the research described herein can inform the burden of up and the benefits of improved fertility planning . the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report . it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries . the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report . it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries .

backgroundunintended pregnancy ( up ) is an unmet medical need with consequences worldwide . we evaluate the costs of up based on pregnancies in brazil from for the year 2010.methodsthe consequences of up were evaluated using decision analysis based on pregnancy rates and outcomes as miscarriage , induced abortion , and live birth , which were factored into the analysis . the model discriminated between maternal and child outcomes and accounted for costs ( in brazilian currency [ real$ , r$ ] ) within the brazilian public health service attributed to preterm birth , neonatal admission , cerebral palsy , and neonatal and maternal mortality . event probabilities were obtained from local resources.resultswe estimate that 1.8 million ups resulted in 159,151 miscarriages , 48,769 induced abortions , 1.58 million live births , and 312 maternal deaths , including ten ( 3% ) attributed to unsafe abortions . the total estimated costs attributed to up are r$4.1 billion annually , including r$32 million ( 0.8% ) and r$4.07 billion ( 99.2% ) attributed to miscarriages and births and complications , respectively . direct birth costs accounted for approximately r$1.22 billion ( 30.0% ) , with labor and delivery responsible for most costs ( r$988 million ; 24.3% ) for the year 2010 . the remainder of costs were for infant complications ( r$2.84 billion ; 72.3% ) with hospital readmission during the first year accounting for approximately r$2.15 billion ( 52.9% ) . based on the national cost , we estimate the cost per up to be r$2,293.conclusiondespite weaknesses in precise estimates in annual pregnancies and induced abortions , our estimates reflect the costs of up for different pregnancy outcomes . the main costs associated with up are in those carried to parturition . the health cost of abortion represents a small proportion of total costs as these are paid for outside of the public health system . consequently , reductions in up will generate not only cost savings , but reductions in woman and child morbidity and mortality .

the treatment of lymphoma depends on the type , location and stage of the disease . the majority ( 50%90% ) of oal is low - grade mucosa associated lymphoid tissue ( malt ) lymphoma . according to the world health organization modification of the real classification , the other 4 main types of oal are follicular cell , mantle cell , diffuse large b cell ( dlbcl ) and lymphoplasmacytic lymphoma . they are all b cell , non - hodgkin s lymphoma , the subtype derived from various stages of b cell maturation . the new classification of lymphoma is dependent on histological , immunophenotypic and molecular genetic analysis . hodgkin s lymphoma does not affect the ocular adnexal structures unless there is widespread systemic involvement or a history of previous systemic hodgkin s lymphoma . according to the ann - arbor staging system lymphoma confined to the orbit is designated as stage i , involvement of adjacent structures ( sinuses , tonsil and nose ) is stage ii . stage iii is abdominal nodal disease below the diaphragm and iv by definition refers to disseminated involvement of one or more extra - nodal sites ( eg , liver , bone ) and e is used when there is a local extra - nodal extent ( eg , ie , iie , iiie and i ve ) . the grading system is similar to nodal lymphomas . broadly speaking , oal with systemic involvement requires systemic treatments whereas low - grade ( emzl or follicular cell ) lymphoma localized to the eye can be very successfully managed with local treatments such as cryotherapy , surgical excision or external beam radiotherapy . when considering local treatment it is important to exclude bilateral ocular involvement . even in the absence of clinical signs staging investigations should include full blood count , hepatic enzymes including ldh , ct abdomen , chest and pelvis , mri or ct of the orbits , and a bone marrow biopsy and aspirate . this is only appropriate for low - grade lymphoma localized to the conjunctiva or orbit with no systemic involvement . surgical excision or cryotherapy is most appropriate for localized stage 1 conjunctival emzl / malt lymphoma.1 this is not curative treatment but can control local disease and relieve local symptoms . tanimoto et al2 reported very good local control of stage 1 malt lymphoma following biopsy or surgical resection alone . patients were followed for a median of 7.1 years , 69% of patients did not require any further treatment . the estimated overall survival rates were 94% at 10 years and 71% at 15 years.2 although , patients should be closely followed for recurrence , histological transformation of the tumor and long - term systemic manifestations of lymphoma . this has been the mainstay of treatment for localized oal for many years with excellent control rates . central nervous system prophylactic radiotherapy is not required as metastatic intraocular or brain involvement is rare.3 the dose of radiation is tapered according to the grade of the lymphoma , 28 to 36 gy for low - grade lymphoma and 30 to 40 gy for high - grade . emzl , lymphoplasmacytic and follicular cell lymphoma are considered low grade , whereas dlbcl and mantle cell are high - grade lymphomas . five - year local control rates for emzl and follicular lymphoma after a dose of more than 30 gy reach 100%.4 however , high rates of delayed systemic recurrence suggest long - term follow up studies are required to assess the true benefit of radiotherapy.5 there is ongoing debate about use of lens shielding radiotherapy to protect the lens from cataract formation . some studies show no effect on local recurrence , whereas in others all recurrences have occurred in patients whose lenses were shielded.6,7 partial orbit volume irradiation leads to unacceptable incidence of intraorbital recurrence and is presently not recommended . ocular complications , including dry eye and cataract are frequently reported in patients that have long - term follow up.8 these localized complications need to be kept in mind by the treating oncologist . ifn - alpha has been used for the treatment of systemic lymphoma for more than 20 years . one study treated 5 patients with intralesional ifn - alpha using an intensive protocol.9 an initial complete response was seen in all patients with stage i oal , one patient with more advanced disease at presentation ( stage iia ) died from systemic lymphoma . long - term follow up studies are required to assess the value of this treatment . rituximab is a monoclonal antibiotic against cd20 positive b cells , which is being considered as an alternative first line treatment for localized cd20 positive oal to avoid the ocular complications of radiotherapy . antibody - induced destruction of cd20 positive b cells can occur by apoptosis , complement mediated cytolysis and antibody - dependent , cell - mediated cytoxicity . rituximab has also been used as monotherapy for localized emzl / malt , in some cases complete regression can be achieved . initial responses are encouraging but the efficacy of rituximab is lower than that reported for gastric malt lymphoma,10 as long - term distal relapse is seen in 50% of cases . very few case series are published on the effect of single agent rituximab on ocular malt / emzl . ferreri and associates achieved regression of malt lymphoma using first line single agent rituximab in all 5 newly diagnosed patients ; however early relapse was seen in 4 of the 5 patients.10 therefore , close follow up intervals are required to detect early relapse when rituximab is used alone . rituximab was not successful for the treatment of local relapse.10 greater success is achieved when rituximab is used in combination with chemotherapy . rituximab and chlorambucil were used as first - line therapy for 9 patients with newly diagnosed emzl . treatment achieved complete remission in 89% with a median follow up of 2 years.11 first - line chemotherapy is often treatment of choice for mantle cell oal , as a high proportion of patients have systemic involvement at presentation . encouragingly , rituximab containing chemotherapy for mantle cell oal had a significantly better 5-year survival than patients receiving chemotherapy alone ( 83% overall 5-year survival verses 8%).12 combination treatment with rituximab and chemotherapy or radioimmunotherapy may achieve a more durable local response and improve patient survival.1113 chemotherapy is reserved for treatment of oal when there is systemic involvement , except for the high - grade dlbcl when systemic chemotherapy is first - line treatment even for stage i disease . more recently chlorambucil and the purine analogs fludarabine , clardarabine and pentostatin have also been used . the extranodal lymphoma study group ( ielsg ) is currently recruiting patients to a clinical trial investigating the role of rituximab alone or in combination with chlorambucil for malt lymphoma ( ielsg # 19).14 novel mechanisms of tumor pathogenesis have related lymphoma to infection leading to new approaches to diagnosis and treatment . chronic antigen stimulation may result in an abnormal lymphoproliferative response that ultimately develops into lymphoma . this association is most profound for gastric malt cell lymphoma , which is related to chronic gastritis from helicobacter pylori infection in more than 90% of cases.15 h. pylori has been isolated from ocular lymphoma specimens . however , h. pylori eradication is not effective treatment for oa malt lymphoma.16 chlamydia psittaci infection has also been linked to oal . ferreri and associates17 identified c. psittaci in 80% of italian oa malt lymphoma specimens . in korea , c. psittaci was isolated in 78% of oa malt lymphoma specimens.18 subsequent reports from south florida , new york state , chicago , netherlands , japan and france failed to identify dna for c. psittaci in any of their oa malt lymphoma specimens.1924 there is wide geographical variation in the prevalence of c. psittaci infection in oa malt lymphoma specimens.25 of the european countries studied , the lowest rate of infection ( 12% ) was present in the uk . results following first line antibiotic treatment are variable.26 abramson and coworkers27 documented a clinical response following antibiotic treatment in 3 patients with oa malt lymphoma . ferreri and associates28 recorded an objective response following oral doxycycline in 4 out of 9 patients with c. psittaci positive oa malt lymphoma . although , grunberger and associates29 were unable to replicate these results . in a multicenter prospective trial , 27 patients with ocular adnexal malt lymphoma were treated with oral doxycycline . c. psittaci dna positive cases had an overall response rate of 64% , the response was of short duration.30 a clinical response was also seen in 38% in c. psittaci dna negative cases.30 lack of response to antibiotic therapy may be a reflection of the biological and genetic heterogeneity of this disease . the extranodal lymphoma study group ( ielsg ) has activated a prospective trial ( ielsg # 27 ) into oal to explore the role of antibiotic treatment and to identify potential infectious agents.14 in summary , advances in our knowledge of the immunophenotypic and genetic alterations in lymphoma cell lines have led to a better understanding of the pathogenesis of the disease . the interplay between a possible infective agent and the immune response has allowed a new wave of treatment possibilities . staging the patient at diagnosis guides subsequent management and local treatments should be reserved for localized disease . however , the practical approach to the patient remains first do no harm and with this in mind ocular oncologists are tempted to administer an initial trial of oral antibiotics for stage 1 malt lymphoma . although , general blanket treatment with antibiotics should be discouraged unless there is proof of an infective agent . we recommend a diagnostic biopsy with immunophenotyping and genetic analysis to determine the grade of the lymphoma combined with pcr analysis to determine the presence of an infective agent . if h. pylori or c. psittaci infection is found a trial of oral doxycycline ( 100 mg twice daily for 3 weeks ) we have also seen some early impressive responses to systemic rituximab ( weekly intravenous infusion over 6 weeks ) . however , close follow up is required as others have reported early local relapse with rituximab alone.10 at present , despite the ocular side effects , localized radiotherapy still remains the standard first - line treatment for stage i low - grade oal .

most lymphomas that involve the ocular adnexal structure are low grade , b cell , non - hodgkin s lymphomas . the treatment depends upon the grade and stage of the disease . high grade lymhoma requires treatment with systemic chemotherapy whereas the localized low grade ( extranodal marginal zone lymphoma ) can be successfully managed with local radiotherapy . chlamydia psittaci infection is associated with low grade ocular lymphoma ; however there is wide geographic variation in the strength of this association . blanket antibiotic therapy is not advised unless there is proof of an infective agent . the monoclonal antibody , rituximab , may be successful for cd20 positive lymphoma , although it is likely that rituximab will have better long - term results when used in combination with systemic chemotherapy .

inactivation of the mismatch repair ( mmr ) genes mlh1 , msh2 , msh6 and pms2 causes lynch syndrome ( ls ) , an autosomal dominant predisposition for colorectal and endometrial cancer . inactivation of the mismatch repair pathway can also occur sporadically , through somatic mlh1 hypermethylation or by acquired biallelic somatic inactivation ( variant affecting function or loss of heterozygosity ( loh ) ) of the mmr genes . inaccurate dna repair leads to a high frequency of somatic variants , with loss of mmr leading to hypermutated ' tumors with 10100 variants / mb . ls tumors are characterized by microsatellite instability ( msi ) and immunohistochemical loss of expression of mmr proteins . however , germline variants affecting function can not be detected in up to 59% of patients displaying msi and/or loss of mmr , referred to as suspected ls ' ( sls ) . recently , germline and somatic variants in the exonuclease domains ( edms ) of dna polymerase ( pole ) and polymerase d ( pold1 ) were described . these pole / pold1 variants affect proofreading function and lead to an ultramutated phenotype with a variant incidence exceeding 100 variants / mb . germline pole - edm variants can result in a ls phenotype and microsatellite instable colorectal cancers ( crcs ) . the exact role of somatic pole / pold1 variants in tumors with high microsatellite instability ( msi - h ) remains unclear . the aim of our study was to identify the underlying genetic cause of disease in a cohort of 64 sls cases selected on the basis of msi , loss of mmr , young onset and often a family history for ls by screening the mmr , pole and pold1 genes in both leukocyte and tumor dna . this study included 64 patients with lynch - associated tumors recruited in four academic centers in the netherlands between 1997 and 2014 : leiden university medical centre ( n=37 ) , maastricht university medical centre ( n=11 ) , erasmus medical centre ( n=9 ) and university medical centre utrecht ( n=7 ) . demographic and clinical data , as well as informed consent , were obtained at the time of diagnosis . this study was approved by the local medical ethical committee of the lumc ( p01 - 019e ) . patients were selected based on loss of mmr ( as indicated by immunohistochemical staining ) and/or msi . unexplained tumors with low msi or tumors with inconclusive ihc results were also included in this study ( see supplementary table 1 and supplementary methods ) . fifty - eight ( 91% ) patients fulfilled bethesda criteria , and families of 24 ( 38% ) patients also fulfilled amsterdam ii criteria . patients were previously screened in a diagnostic setting for germline mmr variants . whereas 57 patients showed no disease - causing germline variants , 7 patients were found to have a germline variant of unknown significance ( vus ) . of the total cohort , 75% of patients presented with crc ( n=48 ) , 14% with endometrial cancer ( ec , n=9 ) and 11% with another ls - associated tumor two patients were excluded from the analysis because of poor dna quality . of the remaining 62 tumors , tumor and leukocyte dna was sequenced for variants in the exonic regions of mlh1 , msh2 , msh6 , pms2 , pole and pold1 using the ion pgm system ( life technologies , carlsbad , ca , usa ) . raw data analysis , alignments and variant calling was carried out using the default parameters in torrent suite v4.0 ( thermo fisher scientific , waltham , ma , usa ; see supplementary methods ) . the full data set was filtered and prioritized by variant frequency ( > 10% ) and coverage ( > 50 ) . the in silico prediction programs were used to predict effect on function ( see supplementary methods ) . all variants ( likely ) affecting function , including two variants with a 9% variant frequency , were validated with sanger sequencing . for all pms2 variants , pms2-specific primers were created to confirm that the variant is present in pms2 and not in a pms2 pseudogene . loh was determined for every heterozygous snp by comparing the ratio of allele a with allele b in leukocyte and tumor dna samples . furthermore , for every heterozygous snp , the allelic imbalance factor ( aif ) was calculated and fisher 's exact test was performed to determine whether the difference between normal and tumor is significant . if all heterozygous snps of one gene showed loh with an aif > 2 and fisher 's exact p - value < 0.05 , loh was called ( supplementary table 1 ) . seven patients with a germline mmr vus ( class 3 ) were included in this study ( see supplementary table 1 ) . in all cases , the variant was detected with ngs in leukocyte and tumor dna . during the course of the study , three of these germline variants were reclassified as class 4 or 5 ( ( probably ) affects function ) by the international society for gastrointestinal hereditary tumors incorporated ( insight ) . six cases with somatic mlh1 hypermethylation fulfilling revised bethesda criteria ( three from families fulfilling amsterdam ii criteria ) were sequenced for underlying hereditary defects explaining the family history , but no germline variants were found . one of these mlh1 hypermethylated tumors carried a somatic mlh1 variant likely to affect function and one displayed mlh1 loh ( supplementary table 1 ) . one ( n=27 , 44% ) or two ( n=13 , 21% ) somatic aberrations ( variant or loh ) in a mmr gene were found in a total of 62 tumors ( see supplementary table 1 ) . of the 13 tumors with two somatic aberrations , 12 had variants in mlh1 or msh2 and were msi - h . although the majority ( 83% ) of tumors showed < 10 somatic variants in the genomic region analyzed , 10 cases displayed a larger number of somatic variants , ranging from 16 to 375 somatic variants within the sequenced area of 31 kb . out of 10 tumors , 9 showed a pole or pold1 variant that ( probably ) affects function ( table 1 ) . of the highly mutated tumors , two carried novel germline heterozygous pole / pold1 variants that are predicted to affect proofreading ( supplementary table 2 ) . of these two germline cases , tumor sls-67 was also found to carry two somatic mlh1 variants , explaining the tumor phenotype ( loss of mlh1 and pms2 expression and msi - h ) . the second tumor , sls-16 , was msi - l , showed positive mlh1 and msh2 staining ( msh6 and pms2 were not tested ) and had no somatic mmr variants . seven of the highly mutated tumors showed somatic pole / pold1 variants likely to affect function . six tumors carried a somatic pole / pold1-edm hot spot variant ( pole : c.857c > g , c.856c > t , c.1231g > t , c.1366g > c , c.1367c > t or c.1376c > t and pold1 c.1433g > a ) that has previously been described to impair proofreading . in the seventh tumor ( sls-105 ) , a novel pole c.846_847delinstt variant was detected . this variant lies close to a known pole hot spot site ( pole c.857 ) and is predicted to affect function by two out of three prediction programs ( supplementary table 2 ) . four pole / pold1-edm mutated tumors displayed additional somatic nonsense pole variants outside the exonuclease domain ( see supplementary table 3 ) . eight of the nine ultramutated tumors with a pole / pold1-edm variant found in our study showed msi ( 3 msi - h and 5 msi - l ) . in six of these ultramutated tumors , ihc detected loss of at least one of the mmr proteins and all six tumors displayed somatic variants in the affected mmr gene likely to affect function . tumor sls-19 with two pole variants was found to have two somatic mlh1 aberrations , as well as two somatic pms2 aberrations , and ihc showed solitary loss of pms2 expression . reanalysis of staining also showed ambiguous mlh1 staining ( cytoplasmic enhancement and vague , focal nuclear staining ) . tumor sls-09 displayed a nonsense msh6 variant and missense msh2 variant predicted to affect function , and ihc showed loss of msh6 expression and weak positive msh2 expression . in three pole / pold1-edm mutated tumors with positive or inconclusive mmr expression and msi - l phenotype , no somatic mmr variants ( likely ) to affect function were found . however , in one of those three tumors ( sls-05 ) , solitary mlh1 loh and pms2 loh without variants was found ( see table 1 ) . in three non - ultramutated tumors , a pole / pold1-edm variant was found ( sls-80 , sls-87 and sls-101 , see table 1 ) . these variants have not been described before , but are predicted to affect function ( see supplementary table 2 ) . two variants co - occur with a germline msh6 vus and a somatic msh6 variant ( sls-80 and sls-87 ) , whereas one ( sls-101 ) co - occurs with a somatic pms2 variant . four additional non - ultramutated tumors showed pole ( sls-18 , sls-21 ) or pold1 loh ( sls-12 , sls-49 ) in all heterozygous snps ( supplementary table 1 ) , without germline or somatic pole variants . pole - edm variants are reported to be the mutagenic factor driving ultramutation in tumors . the same report also noted that the increased mutational load seen in pole mutated tumors exceeds that expected because of loss of exonuclease activity . the number of variants detected in the sequenced area in the present study implicates an ultramutated phenotype , with > 100 variants / mb in all pole / pold1-edm mutated tumors in this cohort . as only a limited region ( 31 kb ) was sequenced , we can only extrapolate the total number of variants per mb . in our cohort , the pole / pold1 mutated mmr - deficient tumors display two deficient pathways increasing the mutational load . comparing frequencies of the different variants found in these tumors , it might be concluded that faulty proofreading may be the initiating event in some of these tumors , possibly resulting in loss of mmr and thereby in msi . interestingly , four tumors show pole / pold1 loh without germline or somatic pole / pold1 variants . these tumors however do not show the typical ultramutated phenotype , whereas single variants without loh do show that . this phenomenon of loh without variants affecting the exonuclease domain has not yet been described . furthermore , three tumors show somatic pole / pold1 variants , without the ultramutated phenotype . all three variants are missense , but are predicted to affect function ( supplementary table 2 ) . as these variants are not found in ultramutated tumors , evidence of deleterious functional effect is lacking . in conclusion , targeted next - generation sequencing of 62 sls cases led to the detection of 9 highly mutated tumors with a germline ( n=2 ) or somatic ( n=7 ) pole / pold1-edm variant . even though pole germline variants have previously been shown to co - occur with somatic mmr variants , in this study we found germline and somatic pole / pold1 variants in a cohort selected for sls characteristics . importantly , although current literature mainly addresses pole / pold1 variants in mss tumors , somatic pole / pold1 variants in sls patients are likely to be overlooked . however , a very recent recommendation for genetic testing and surveillance states that mmr deficiency should not be an exclusion criterion for genetic testing of germline pole / pold1 variants . our results further emphasize the importance of pole / pold1 germline and somatic screening in unexplained msi - h and mmr - deficient tumors .

many suspected lynch syndrome ( sls ) patients who lack mismatch repair ( mmr ) germline gene variants and mlh1 or msh2 hypermethylation are currently explained by somatic mmr gene variants or , occasionally , by germline pole variants . to further investigate unexplained sls patients , we analyzed leukocyte and tumor dna of 62 sls patients using gene panel sequencing including the pole , pold1 and mmr genes . forty tumors showed either one , two or more somatic mmr variants predicted to affect function . nine sls tumors showed a likely ultramutated phenotype and were found to carry germline ( n=2 ) or somatic variants ( n=7 ) in the pole / pold1 exonuclease domain ( edm ) . six of these pole / pold1-edm mutated tumors also carried somatic mmr variants . our findings suggest that faulty proofreading may result in loss of mmr and thereby in microsatellite instability .

noise - induced hidden hearing loss ( nihhl ) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift ( pts ) . this is different from the conventional definition of noise - induced hearing loss ( nihl ) , which is based on changes in auditory sensitivity or threshold shift . therefore , noise exposure recommendations are based on the likelihood that a particular dose of exposure will result in a pts . physiologically , variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells ( ohcs ) in the cochlea , which provide mechanical amplification of soft sounds [ 2 , 3 ] . noise exposures that result in only a temporary threshold shift ( tts ) have a reversible impact on ohc function , which is manifested by the recovery of otoacoustic emissions ( oae ) [ 46 ] and cochlear microphonics ( cm ) [ 711 ] . the functional changes in these measures parallel the recovery of hearing thresholds , as well as the repair of structures such as stereocilia and the tectorial membrane [ 7 , 12 ] . by contrast , noise exposure at higher levels and/or for longer durations can cause permanent damage to , or even the death of , ohcs and , hence , lead to pts . therefore , the ohcs and the structures surrounding them , including the tectorial membrane and the supporting cells , are considered to be the major loci of cochlear damage that result in noise - induced threshold shifts [ 13 , 14 ] . although some early reports claimed that reversible noise - induced ihc pathologies were responsible for tts [ 15 , 16 ] , ihcs are relatively insensitive to noise - induced cell death . however , it has long been recognized that the synapse between ihcs and primary spiral ganglion neurons ( sgns ) can be damaged by noise [ 1719 ] . these early studies showed that this manifests mainly as damage to the postsynaptic terminals ; however , there is clear evidence from more recent studies that noise induces damage to both pre- and postsynaptic structures . more importantly , disruption of the synapses can be permanent , resulting in degenerative death of sgns . the finding that damage to ribbon synapses can occur without pts is significant because of the potential impact of such damage on hearing function . because the physiological damage is not accompanied by a permanent shift in hearing threshold , it would likely be missed by a standard ( i.e. , threshold - based ) hearing assessment and has thus been referred to as nihhl . nihhl first manifests as reduced output of the auditory nerve at high sound levels , without affecting the hearing threshold . this reduction has been found in both animals [ 6 , 2023 ] and human subjects with a history of noise exposure but with normal audiograms . since the thresholds of the auditory nerve remain unchanged , the function relating compound action potentials ( cap ) amplitude with sound levels in nihhl animal is different from that in animals with threshold changes . schematic curves of cap input / output functions are presented in figure 1 for a comparison across normal control and those with different pathologies . theoretically , if the damage is restricted to ohcs , the major change in cap input / output ( i / o ) curve is restricted around threshold and the amplitude reaches the control value at high sound levels . in the case of nihhl , cap reduction is mainly at high sound level , with no difference at low sound level , suggesting a suprathreshold deficit . when the damage occurs at both ohcs and the ihc - sgn synapses , the reduction of cap amplitude is seen across all sound levels . as nihhl is initiated at the synapse between the ihcs and sgns , which silences the auditory nerve fibers ( anfs ) that extend from them , the corresponding disorder is categorized as a cochlear neuropathy ( i.e. , cochlear synaptopathy ) [ 25 , 26 ] . presumably , the reduction in the amplitude of the auditory nerve response without threshold elevation is due to selective loss of anfs that have high thresholds , which is supported by single - unit recording studies [ 20 , 27 ] . given the important features of those low - spontaneous - rate anfs in auditory coding , the neuropathy or synaptopathy in hidden hearing loss is not simply a reduction in the number of functional anfs . furthermore , the synaptopathy in nihhl is likely to be related to the synaptic repair after initial damage by noise , rather than a simple initial loss . in addition , the functional deficits seen in nihhl may also involve the contribution from central auditory plasticity [ 26 , 2832 ] . in this review , we summarize the available data for noise - induced damage and repair around ihc sgn synapses and discuss the evidence for the contributions of cochlear malfunction and central plasticity to nihhl . accumulated evidence has shown that the synapses between ihcs and type - i sgns are sensitive to noise and the damage to this synapse is likely to be the bases for nihhl . the synapse is characterized by presynaptic dense bodies termed ribbons [ 3335 ] , which are spherical or ellipsoidal in shape , 100200 nm in diameter , and surrounded by synaptic vesicles . the ribbons are built up from ribeye protein subunits [ 37 , 38 ] and anchored to the active zone of the presynaptic membrane via bassoon proteins [ 3941 ] . the functional role of ribbons has been recognized as tethering and conveying synaptic vesicles to the active zones [ 42 , 43 ] , where the release of neurotransmitters at these synapses is modulated by a specific l - type calcium ion channel ( i.e. , cav1.3 ) [ 44 , 45 ] . noise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses [ 6 , 22 , 23 , 4648 ] . the damaged synapses exhibit various degrees of swelling of the terminals , resulting in disruption of the synaptic connections between ihcs and sgns [ 20 , 46 , 48 ] . immunohistological staining has revealed similar losses for ribbons and terminals [ 6 , 22 , 23 , 49 ] . the mechanism for the damage to the postsynaptic terminal is glutamate - mediated excitotoxicity ( reviewed in ) . one possible mechanism of ribbon loss is the loss of cell - cell contact that is required for the maintenance of the pre- and postsynaptic complexes [ 5053 ] . our electron microscopy evaluations did not reveal any residual presynaptic complexes without ribbon and postsynaptic terminals . therefore , it is likely that the entire presynaptic structure breaks down when the postsynaptic terminal is damaged . a brick assembly model , in which a ribbon is built up from multiple ribeye subunits , has been proposed for ribbon construction in retina photoreceptor cells . moreover , the ribbons in retina sensorial cells can be partially broken down by light , but they rapidly reassemble in the dark , probably serving as a mechanism of adaption to bright light [ 5458 ] . in the retina , the ribbon size appears to be a determining factor for the quantity of neurotransmitter released . however , the dynamic disassembling / reassembling process has not been identified in the cochlea , and changes in the ribbon size and the relationship with the release of neurotransmitters have not been investigated in the cochlea . additionally , disassembly and reassembly , as well as ribbon size , are modulated by ca signaling involving cav - channels , presynaptic ca levels and storage , and guanylate cyclase - activating protein-2 ( gcap2 ; see the review by schmitz ) . interestingly , optical stimulation of photoreceptors causes hyperpolarization of the presynaptic membrane and a decrease in [ ca]i , as opposed to depolarization and the large increase in [ ca]i in ihcs in response to sound . the decrease in [ ca]i in photoreceptor cells is followed by a conformational change of gcap2 , which results in the disassembly of the ribbons . in the cochlea , it is not known whether there is a gcap - mediated pathway that controls ribbon size . as the membrane potential of ihcs is depolarized with increasing sound levels , resulting in an influx of ca , the role of ca in ribbon assembly is unlikely to be the same as it is in the retina . the first quantitative study of noise - induced ribbon synapse damage in cba mice reported that the number of ribbon synapses was reduced to 40% compared with the control 1 day after brief noise exposure that did not lead to pts . the synapse count recovered to 50% within 1 week , but no further recovery was observed , and this 50% loss of synapses was considered permanent . sgn death observed 2 years after the noise was found to match the 50% permanent loss of synapses . however , a study on guinea pigs carried out by the same research group found a similar loss of ribbon synapses 2 weeks after exposure to noise that did not cause pts , but this study found a much smaller final loss of sgns . this suggests that some sgns , which had originally lost their synapses with ihcs , survived and reestablished synapses with ihcs . our studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to pts [ 22 , 23 ] . although this recovery was not complete , approximately 50% of the initial loss of paired ribbon and postsynaptic density ( psd ) puncta in the basal half of the cochleae was seen 1 day after noise , and the loss was recovered to < 20% within 1 month . comparing the aforementioned data from mice and guinea pigs , it appears that there may be some species difference in the ability to regenerate synapses following noise - induced hearing damage . however , a recent study of c57 mice reported that the loss of ribbon synapses induced by non - pts - inducing noise was largely reversible . this discrepancy in synapse regeneration following noise exposure requires further investigation . in a recent review , it was argued that the recovery of ctbp2/psd counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up / downregulation of the synaptic protein rather than regeneration of synaptic connections . however , there are several lines of evidence for the possibility of synapse repair following noise - induced damage . first , it has been reported that plastic changes occur in the presynaptic component , including the existence of multiple presynaptic ribbons around an active zone and the changes in the size and location of ribbons following noise exposure . second , the change in the amplitude of the compound action potential ( cap ) corresponded to the changes in ribbon / psd counts : a large initial reduction in cap amplitude and synapse counts were followed by a significant recovery after the noise exposure . third , changes in many single - anf coding activities were not seen at the time that the synapses were damaged but rather manifested later ( see section 3 ) with the recovery in both the cap and synapse number , suggesting that those changes occurred in the anfs that connect ihcs via repaired / reestablished synapses . further work is required to determine the mechanisms and factors that influence the repair of both pre- and postsynaptic components . ribbon synapses exhibit spatial differences around ihcs ; that is , the synapses at the modiolar side of an ihc have relatively small ribbons but larger postsynaptic terminals , whereas those at the pillar side have relatively large ribbons but smaller terminals . liberman et al . reported that anfs are functionally categorized by their spontaneous rate ( sr ) , which is inversely related to the fiber 's threshold and dynamic range [ 6365 ] . it is widely accepted that low - sr anfs exhibit synapses with ihcs on their modiolar side , whereas high - sr units exhibit synapses on the pillar side ( this is based on data obtained using intracellular tracer injections ) . the low - sr units are considered critical for hearing in noisy environments due to their larger dynamic range , higher thresholds , and the ability to follow the quick change of the amplitude of acoustic signals . by contrast , high - sr units are responsible for the sensitivity to quiet sounds and are saturated by high - level background noise [ 26 , 63 , 64 , 67 , 68 ] . in nihhl , low - sr anfs appear to be more vulnerable to noise than high - sr units . selective loss of low - sr anfs has been found following exposure to noise that did not lead to pts . presumably , this selective loss of low - sr units should produce coding deficits , which can be predicted based on the unique features of those units . however , no coding deficits were examined and reported in this study . on the other hand , we reported a time delay in the development of coding deficits by single anfs in guinea pigs following a similar noise exposure that did not cause pts ; these deficits were attributed to intensity coding and temporal coding as summarized in sections 3.1 and 3.2 . intensity coding in the cochlea is defined as the ability of anfs to encode the sound intensity or the change of sound intensity . this ability is determined primarily by the spike rate ( or the change of spike rate ) of individual anf in response to sound intensity change and the number of functional anfs . therefore , the intensity coding deficits can be evaluated in both evoked field potential and single - unit recordings . deficits in intensity coding were first suggested by a reduction in wave i of the auditory brainstem response ( abr ) [ 6 , 49 ] , as well as a reduction in the amplitude of the cap , as this is likely due to the loss of functional anfs following synapse disruption . the fact that the reduction is more significant at higher sound levels has been considered evidence for selective damage to low - sr fibers , which have higher thresholds [ 25 , 26 , 69 ] . the deterioration in intensity coding following no - pts noise exposure was manifested as a reduction in the driven spike rates ( peak , sustained , and total rates ) of anf units that were tested only at one sound level . such changes are significant only in low - sr anf units and are seen at a later time rather than immediately following exposure . this time delay in the development of coding deficits suggests that ( 1 ) the reduction in driven spike rates occurs in the anfs to which the synaptic connections to the ihcs are reestablished following the initial disruption and ( 2 ) the repaired synapses are functionally abnormal , with less efficient neurotransmitter release . temporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals . in human subjects , the process involves both bottom - up and top - down mechanisms ; but in animal models , only bottom - up mechanisms are tested ( see reviewed by ) . many different tests have been used to detect the bottom - up mechanisms of temporal coding , some of them based on the peristimulatory changes of firing rate showing latency and adaptation . as reviewed above , the major function of presynaptic ribbons in ihcs is to facilitate the synaptic transmission . indeed , such deficits were manifested as an increase in response latency of anfs in animals with nihhl . this was first demonstrated as a significant delay in cap peak latency and then further supported by the delayed latency of peak in psth ( or peak latency ) of anfs in our single - unit study . in another very recent report , such delay was reported in abr as the marker of cochlear synaptopathy . we also found a reduction in the ratio of peak to sustained rates in animals that were exposed to noise . this ratio is considered an index of the ability of a neuron to encode dynamic signal changes ( see review by ) . using a paired - click paradigm , we found that the anf response of noise - exposed animals to the second click recovered more slowly from the masking effect of the first click . these results reveal poorer coding to the transient features of acoustic signals by anfs , which were examined in previous studies to show the deterioration in of temporal coding in animals with bassoon mutation [ 39 , 41 ] . whereas an increase in peak latency was seen shortly after exposure to noise , changes in the peak rate and the peak / sustained spike ratio , as well as a slower recovery of the spike rate to the second click , were not seen until later , suggesting an association between the deficits and the synapse repair . a temporal deficit in phase - locking responses has been proposed based on selective loss of low - sr units and the functional features of this group of anfs [ 25 , 26 , 69 ] , but it has not been tested at the single - unit level . so far , there appear to be two models for the development of coding deficits in nihhl . one model suggests that the coding deficit or synaptopathy is simply due to the loss of low - sr anfs . since those units have unique functions in signal coding , the loss of those functions is predicted as the consequences . that is , the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption . we found that the noise - induced synaptic damage in guinea pigs under nihhl is largely repairable , leaving only a small amount of synapses not being reestablished . therefore , the coding deficits or synaptopathy can not be simply attributed to the loss of sr units . since the coding deficits are seen at the time when the synapse counts are largely recovered , we believe that the coding deficits likely occur in the repaired synapses ( most of them innervating low - sr anfs ) . studies are needed to verify which model is more likely the case in human subjects . it has long been recognized that subjects with normal audiograms may have perceptual difficulties , and this is especially true in the elderly . age - related hearing loss with threshold elevation is termed peripheral presbycusis , whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis . for example , temporal processing deficits and difficulties of hearing in noisy environments are two major problems experienced by the elderly . these problems were recognized long before the discovery of cochlear damage associated with nihhl and were considered to be the result of central auditory processing disorders [ 7175 ] . it was generally accepted that any perceptual deficits observed without changes to hearing thresholds and cognitive functioning can be attributed to central dysfunctions . based on recent progress in functional deficits in cochlear coding , such separation between peripheral and central presbycusis is likely to be incorrect . the so - called central presbycusis may , at least in part , result from disorders in the auditory periphery . the coding deficits related to the loss of low - sr anfs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified . data on changes in the sr distributions of anfs suggest the reestablishment of synapses following an initial disruption that was selective to low - sr units . although our data revealed abnormalities in some aspects of coding in the auditory nerve , further work is required to investigate coding deficits in nihhl . such studies can not be replaced by speculation based on the selective loss of low - sr fibers ; for example , one can not be certain how the auditory nerve changes its response to amplitude modulation until it is measured at the single - unit level . two possibilities must be considered : ( 1 ) the surviving anfs may change their function and ( 2 ) the initially lost low - sr fibers may be repaired but with changed function . it should be noted that there is now a tendency in the literature to consider nihhl to be a purely peripheral issue , a result of the overcorrection of the central presbycusis . however , despite the strong evidence for a peripheral contribution , the central contribution to the problems seen in nihhl should not be neglected . in other words , it may be more constructive to assume that there are both peripheral and central contributions to nihhl . it is well known that hearing loss ( with elevated threshold ) can induce central changes , which can result in deteriorations in signal processing . studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare . one such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in nihhl ( i.e. , reduced auditory nerve input to the brain ( measured as a smaller abr wave i ) ) but normal hearing threshold . in an earlier study in rats , tinnitus was found 6 months after exposure to noise that caused minimal loss of hair cells and pts but significant loss of anfs . one of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition , resulting in hyperactivity and/or hyperresponsiveness in the central auditory system ( see reviews in [ 7780 ] ) . the types of hearing loss producing such central enhancement include cochlear ablation , drug- and noise - induced damage . while direct effect of drugs and noise on central neurons needs to be differentiated , a similarity across those hearing loss models is the reduction of cochlea output to the auditory brain , which may be the main initial factor causing the imbalance between excitation and inhibition . in this sense , while most of studies in central plasticity using nihl model correlated the central enhancement with the amount of threshold shifts [ 29 , 8185 ] , at least one study has reported central enhancement in mice exposed briefly to noise at a moderate level that did not cause pts , presumably producing only nihhl . unfortunately , the reduction in auditory input from the cochlea was not quantified in this study . taken together , available data suggest that cochlear damage , with or without threshold elevation , can lead to central plasticity by reducing input from the auditory nerve . further work is required to establish the central contribution to coding / perception difficulties in nihhl , and previous studies on central processing disorders in subjects with nihl should be reevaluated to differentiate the central contributions from the peripheral ones . in a brief summary , we use figure 2 to summarize the available data for the mechanisms of perception difficulty experienced by subjects with history of noise exposure but normal or near normal thresholds . in this schematic diagram , we include the two potential models of noise - induced synaptopathy in cochleae . in model 1 , the coding deficits are speculated based on the role of low - sr anfs in signal coding . both models result in a reduction in the cochlear output to the auditory brain , which in turn will result in plastic reorganization of the brain . auditory signal processing disorders experienced by subjects with long - term nihhl should include what are inherited from the coding deficits developed in the auditory peripheral and those associated with the plastic changes of auditory brain . although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required , evidence suggesting the occurrence of nihhl in human subjects is being accumulated . this is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds . since the deficits are demonstrated at suprathreshold levels , it is clear that normal hearing thresholds do not guarantee normal hearing functions , especially in subjects with history of noise exposure [ 24 , 86 ] . the second line of evidence is the reduction in the output of the auditory nerve in subjects with a history of exposure to noise . interestingly , the combination of a reduction in wave i and an increase in wave v / i ratio may be considered evidence of increased central gain and is likely responsible for the generation of tinnitus in hidden hearing loss [ 32 , 87 , 88 ] . the third line of evidence comes from the age - related sgn degeneration seen in the examination of human temporal bones . unfortunately , there is , as yet , no clear human evidence that degeneration of sgns is expedited by exposure to noise that does not cause threshold elevation . the clinical implications of nihhl are manifested by the fact that noise exposure causing nihhl occurs frequently in daily life and impacts much more general population . such noise exposure has been generally considered to be safe according to current safety standards for exposure to noise . the evidence from the studies reviewed here indicates that the resulting damage to the ribbon synapses from noise that did not induce pts can be repaired even though the repair is incomplete . more importantly , the signal coding deficits are developed in association with the synapse repair . since the damage and repair occur repeatedly , the damage on signal coding can be accumulated during aging and likely contributes to the perceptual difficulties experienced by the elderly . this impact of noise exposure on signal coding is obviously different from the contribution made by the hearing loss defined by threshold shifts . in future , the coding deficits and related synaptic repair in nihhl should be further investigated in a laboratory setting . since the ribbon synapse is the first gating point for temporal processing in auditory pathway , the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging . whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift . moreover , the coding function of anfs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent . we are currently collecting data using electron microscopy , as well as conducting an analysis on the potential changes of the molecular structures of ribbons and psds , in an attempt to elucidate the morphological / molecular mechanisms responsible for functional changes of repaired ribbon synapses . it would also be interesting to understand the reasons for the extreme sensitivity of low - sr synapses to noise , as well as elucidate possible methods to prevent damage . laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea , as well as reveal the factors that influence repair in order to promote it . to translate the knowledge to clinic , investigation is needed to establish good measures for detecting nihhl in human subjects . although abr wave i is useful for evaluating synaptopathy caused by noise that does not induce pts , its reliability and sensitivity are questionable in human subjects where the abr amplitude is small , and other methods should be explored . a very recent report suggests the use of abr latency as the marker of nihhl . the study tested human subjects with normal hearing thresholds and reported a big variation in the threshold of envelope interaural timing difference , which was negatively correlated with the shift of abr wave v latency by background noise : the higher the threshold ( poorer sensitivity ) , the smaller the shift . the observation of the latency shift with masking is supported by the fact that the low - sr anfs have longer latency than high - sr fibers and are resistant to background noise [ 91 , 92 ] . it is not clear why the study did not report the change in wave v amplitude by masking . theoretically , the masking should produce greater reduction in wave v amplitude in subjects with selective loss of low - sr units . moreover , no information about the history of noise exposure was reported and it is not clear whether the poorer performance in temporal cue detection was due to noise - induced synaptopathy or other reasons . to date , the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low - sr anfs , the subcortical steady state responses ( sssr ) [ 93 , 94 ] . based on the animal studies , this test should be carried out using amplitude - modulated signals at relatively high intensity and a shallow modulation depth . the input intensity of the driving signal should fall within the saturation range of the high - sr fibers . high frequency carrier waves with a high intensity and with shallow amplitude modulation are especially useful for evaluating the function of low - sr fibers . this is supported by modeling the loss of low - sr fibers . to differentiate the sssr contribution from the auditory nerve from that of central neurons a recent mouse study found that the modulation frequency close to 1 khz was optimum with a high frequency carrier without concern of modulation depth . however , a recent human study reported a successful detection of the low - sr unit loss using off - frequency maskers and a shallow modulation depth .

recent studies on animal models have shown that noise exposure that does not lead to permanent threshold shift ( pts ) can cause considerable damage around the synapses between inner hair cells ( ihcs ) and type - i afferent auditory nerve fibers ( anfs ) . disruption of these synapses not only disables the innervated anfs but also results in the slow degeneration of spiral ganglion neurons if the synapses are not reestablished . such a loss of anfs should result in signal coding deficits , which are exacerbated by the bias of the damage toward synapses connecting low - spontaneous - rate ( sr ) anfs , which are known to be vital for signal coding in noisy background . as there is no pts , these functional deficits can not be detected using routine audiological evaluations and may be unknown to subjects who have them . such functional deficits in hearing without changes in sensitivity are generally called noise - induced hidden hearing loss ( nihhl ) . here , we provide a brief review to address several critical issues related to nihhl : ( 1 ) the mechanism of noise induced synaptic damage , ( 2 ) reversibility of the synaptic damage , ( 3 ) the functional deficits as the nature of nihhl in animal studies , ( 4 ) evidence of nihhl in human subjects , and ( 5 ) peripheral and central contribution of nihhl .

the extracellular matrix ( ecm ) is a protein rich entity which supports tissue structure , cell adhesion , cell - cell communication and differentiation 1 . numerous studies have found that ecm proteins are aberrantly expressed in carcinomas and actively participate in tumor progression . in particular , fibronectin is receiving increasing interest as a result of its participation in multiple stages of tumor progression . under homeostatic conditions , fibronectin has been shown to play a role in cell growth , differentiation , migration , and is additionally involved in processes such as wound healing and blood coagulation 2 . with regard to cancer , fibronectin is not only increased in tumors where its altered expression has been shown to promote tumor growth 3 - 5 , migration 6 , 7 and invasion 8 , 9 but fibronectin has also been reported to limit tumor cell responsiveness to therapy 10 - 12 ( figure 1 ) . for instance , the introduction of fibronectin to differentiated mammary epithelial cell acini cultured atop the basement membrane extract matrigel caused the cells to re - enter the cell cycle thus reversing their growth arrest and acinar morphology 13 . work in our lab has shown that loss of caveolin-1 , the main structural protein of caveolae , results in increased expression of fibronectin , tenascin - c and collagen in murine mammary glands ( thompson et al , unpublished data ) . the altered ecm is accompanied by changes in mammary gland architecture characterized by increased numbers of ducts that possess a larger circumference and area when compared to wild type controls , a result which may be in part related to increased fibronectin expression observed in the stroma ( thompson et al , unpublished data ) . the purpose of this review is to illustrate the mechanisms in which aberrant fibronectin expression influences tumorigenesis and therapy resistance . in particular , this review will focus on the interactions between cell receptor ligands and fibronectin and how this interaction influences downstream signaling events that facilitate tumor progression . further , this review will highlight potential strategies whereby fibronectin deposition , engagement with cell surface receptors and activation of cell signaling pathways may be exploited to halt tumor progression . in this manner , it is with anticipation that a new generation of novel therapeutics may be developed to better combat fibronectin 's participation in tumorigenesis . fibronectin has been shown to play a central role in processes associated with tumor progression . in particular , 51 integrin and fibronectin have not only been shown to be upregulated in tumors , but have also been reported to participate in tumor cell proliferation . for example , nam et al found that fibronectin and the extra domain ( ed)-a splice variant of fibronectin were associated with higher 51-integrin expression in malignant as opposed to normal breast epithelial cells cultured atop matrigel 5 . interestingly , the authors also reported that total fibronectin , ed - a fibronectin and 51-integrin was markedly upregulated in the malignant breast cancer lines but not in non - malignant breast epithelial cells 5 . similarly , mierke et al also determined that 51 integrin was upregulated in mda - mb-231 breast cancer cells and further demonstrated that fibronectin augmented the invasiveness of 51 expressing breast cancer cells cultured atop collagen hydrogels 3 . to investigate a mechanism responsible for 51-fibronectin in tumor growth , mitra et al examined the activation of the receptor tyrosine kinase c - met in human heya8 and skov3ip ovarian cancer cell lines following 51 engagement with fibronectin 4 . results showed that binding of cellular 51 to fibronectin resulted in increased activation of the c - met / fak / src signaling pathways in ovarian cancer cells 4 . blocking ovarian cancer cell 51 interaction with fibronectin reduced c - met mediated focal adhesion kinase ( fak ) and src phosphorylation in vitro and in vivo and additionally reduced tumor weight and proliferation in xenograft tumors 4 , suggesting that 51-fibronectin interactions regulate cell signaling pathways important for ovarian cancer growth . in an effort to further examine the effects of integrins on ovarian tumor cell proliferation , kenny et al examined adhesion and proliferation of human ovarian cancer cells following culture atop a de - cellularized ecm derived from human omental tumors 8 . when cultured in the presence of anti- 51 , v3 and rgd peptides , the authors observed decreased adhesion and proliferation of ovarian cancer cells cultured atop the de - cellularized ecm supporting a role for fibronectin - integrin engagement in ovarian cell proliferation 8 . a defining feature of tumors is the ability of cancer cells to evade apoptosis . in an effort to delineate a role for fibronectin - integrin interactions on tumor cell resistance to apoptosis , han et al used wortmannin to block pi3-k in human bronchial epithelial beas-2b and 16-hbe cell lines following 51 cellular engagement with fibronectin 14 . the authors found that wortmannin caused an increase in dna fragmentation indicative of apoptosis 14 . in the absence of wortmannin , fibronectin stimulated 51-mediated phosphorylation of pi3-k and downstream signaling of pi3-k / akt , which in turn , down - regulated p21 and the tumor suppressor gene p53 and upregulated cyclin d1 in the bronchial epithelial cells 14 . in addition to integrins , fibronectin binding to cell surface receptors such as syndecans has been reported to regulate tumor cell ecm attachment and growth . this is evidenced by work from huang et al who examined the effect of tenascin - c on tumor cell adhesion to fibronectin 15 . using mda - mb-435 breast cancer and t98 g gliobastoma cell lines , the authors found that tenascin - c bound to the fniii13 of the hepii site on fibronectin blocked syndecan-4 mediated tumor cell binding to fibronectin 15 . overexpressing syndecan-4 reversed the effects of tenascin - c induced inhibition of tumor cell adhesion to fibronectin , suggesting that tenascin - c bound to the fniii13 site on fibronectin disrupts syndecan-4-fibronectin signaling 15 . similarly , syndecan 1 and 4 engagement with fibronectin was reported to promote mda - mb-231 breast cancer cell adhesion and migration on collagen and proliferation on fibronectin matrices , an observation which was reversed upon incubation with anti - syndecan 1 and 4 antibodies 16 . aside from direct cellular interactions with fibronectin on tumor progression , others have reported a role for fibronectin in tumor immunosuppression . for example , sengupta et al examined the role of sirna against fibronectin in glioma tumor growth and immune cell activation 17 . using gl261 glioma tumor cells , the authors observed delayed tumor cell proliferation and growth in mice injected with fibronectin silenced gl261 cells 17 . this observation was in part , attributed to a reduction in the number of t - regulatory cells , which inhibit the activity of t helper cells . while the full length form of fibronectin plays an important role in tumorigenesis , isoforms of fibronectin , such as the ed - a and ed - b variants , have been reported to regulate tumor growth . for instance , rybak et al demonstrated markedly higher expression of ed - a fibronectin in the vasculature of murine liver metastases from f9 teratocarcinoma cells with minimal expression observed in non - diseased organs , pointing to a role for ed - a fibronectin in angiogenesis 18 . indeed , a later study demonstrated that ed - a overexpressing colorectal cancer cells exhibited upregulated expression of the pro - angiogenic cytokine vascular endothelial growth factor - c ( vegf - c ) and promoted tumor growth and lymphangiogenesis in mice 19 . to determine a mechanism responsible for these observations , the authors report that ed - a upregulation of vegf - c is accomplished via ed - a mediated activation of the pi3k / akt pathway . dose dependent reductions in vegf - c were observed following cancer cell treatment with an inhibitor of pi3k 19 . aside from a role in angiogenesis , ed - a fibronectin was reported to be upregulated in patient colorectal tumors , especially those that were of advanced stage 20 . investigating cd133/cd44 colorectal tumor cells , a subset of cells believed to be responsible for colon cancer initiation , the authors found that these cells expressed higher levels of the ed - a receptor 91 and further demonstrated that ed - a interaction with 91 was required for tumor cell proliferation 20 . to elucidate a mechanism responsible for their observations , ou et al found that ed - a sustains wnt/-catenin signaling , necessary for the progression of colorectal cancer cells , via integrin mediated activation of a fak / erk pathway 20 . in a more recent study , ed - a fibronectin was shown to increase the population of myeloid derived cells , known to impair immune responses to cancer cells , from osteoblasts 21 . to determine whether the absence of fibronectin impaired myeloid cell numbers and thus tumor formation , the authors subcutaneously injected melanoma cells into either a fibronectin knock out model ( cko ) in which fibronectin expression in differentiating osteoblasts was conditionally deleted or a wild type animal ( wt ) and evaluated for tumor formation 21 . tumor growth and the presence of myeloid cells in tumors were reduced in cko animals but not in wt animals 21 . although the authors do n't attribute these findings to a specific reduction in the ed - a fibronectin isoform , they go on to demonstrate that culture of myeloid cells from cko animals with ed - a fibronectin resulted in a reduction of melanoma apoptosis , suggesting that this pro - tumor effect results from ed - a activation of myeloid cells 21 . ed - b fibronectin has also been reported to play a role in angiogenesis where it has been shown to promote vegf expression , tube formation and proliferation of human vascular endothelial cells 22 . specifically , the authors found that ed - b fibronectin was upregulated in vascular endothelial cells in response to high glucose , tgf-1 and endothelin-1 , a finding which was reversed following administration of inhibitors to tgf-1 and endothelin-1 22 . these findings would suggest that growth factor signaling is an important regulator of ed - b fibronectin mediated vascular morphogenesis 22 . despite a role in angiogenesis , ed - b fibronectin expression has been documented to be more highly expressed in the stromal and epithelial compartments of colorectal and breast carcinomas , but not in endothelial cells 23 , suggesting that additional work is needed in order to fully elucidate the role of ed - b fibronectin in tumorigenesis . regardless of its role in tumorigenesis , ed - b fibronectin has more recently been exploited as a molecular marker for prostate cancer . here , han et al synthesized a small peptide to target upregulated ed - b expression in tumor xenografts harboring human prostate carcinomas , demonstrating that the peptide bound to ed - b fibronectin produced by the tumor cells 24 . it 's conceivable that this strategy may be utilized for both imaging and diagnostic purposes as well as delivery of therapeutics . overall , these studies suggest that fibronectin , via engagement with several integrin receptors and interference with immune function , favors tumor cell survival and proliferation . additionally , multiple isoforms of fibronectin also participate in tumorigenesis , highlighting the importance of understanding how the different splice variants of fibronectin not only interact with cell surface receptors , but how this interaction activates downstream signaling pathways important for tumor growth . while fibronectin appears to play a key role in multiple facets of tumor progression , it has also been shown to participate in processes associated with tumor migration , invasion and metastasis . to examine the role of fibronectin on tumor cell migration , lou et al overexpressed sox2 to determine its role in ovarian tumor cell metastasis 6 . here , the overexpression of sox2 upregulated fibronectin gene expression resulting in increased migration and invasion of the a2780 human ovarian cancer cell line in transwell chambers 6 . downregulation of fibronectin using sirna resulted in a reversal of cell migration and invasion despite high expression of sox2 indicating that sox2 signals via fibronectin during tumor cell metastasis 6 . to further explore the role of fibronectin on tumor cell migration , wei et al sought to demonstrate whether nicotine enhanced the migration of sw480 colorectal cancer cells through nicotinic - acetylcholine receptor ( 7-nachr ) mediated induction of cox-2 7 , reported to regulate emt 25 . the authors found that inhibiting 7-nachr and cox-2 not only limited fibronectin expression , but also increased e - cadherin expression , reducing sw480 cell migration 7 . in the absence of cox-2 inhibition , nicotine enhanced colon cancer cell migration via 7-nachr and cox-2 mediated up - regulation of fibronectin expression indicating that inhibition of cox-2 and 7-nachr reduced migration of colorectal cancer cells as a result of a reduction in fibronectin expression 7 . integrin engagement has been reported to play a crucial role in fibronectin mediated tumor cell invasion . a recent study reported an indispensable role for fibronectin and the 51 integrin on the invasive capabilities of heya8 and skov3ip1 human ovarian cancer cell lines 8 . here , the authors first showed that 3d co - culture of ovarian cancer cells with mesothelial cells supported fibronectin expression from mesothelial cells via activation of a tgf-r1/rac1/smad3 signaling pathway , a phenomenon which was important for cancer cell adhesion , proliferation and invasion as silencing of fibronectin reduced these cellular responses 8 . in an attempt to further elucidate a role for fibronectin in tumor cell progression , the authors administered antibodies against 5 and 1 integrins to 3d co - cultures of ovarian cancer cells and mesothelial cells and discovered that tumor cell invasion and proliferation was reduced up to 40% 8 . in - vivo , the authors found that treatment of mice with antibodies against 5 and 1 reduced the number of metastases and tumor weight from orthotopically injected ovarian cancer cell lines 8 . these results suggest that ovarian cancer cells stimulate fibronectin expression from mesothelial cells via a tgf- pathway and that tumor cells adhere , proliferate and invade in response to 5 and 1 integrin engagement with fibronectin . in a similar study , it was shown that inhibition of 5 and 1 integrins significantly reduced ovarian tumor cell adhesion to a 3d model consisting of primary human mesothelial cells and fibroblasts and additionally limited the number of metastases in ovarian cancer xenografts 26 . downregulation of e - cadherin on ovarian cancer cells was specifically shown to augment 5 integrin expression via activation of fak and erk1 26 . further , sirna of e - cadherin was shown to increase cancer cell adhesion to fibronectin and invasion in a matrigel matrix 26 , suggesting that ovarian cancer cell adhesion to fibronectin and invasion in a 3d matrix are regulated by decreased e - cadherin mediated 5 integrin expression . in addition to ovarian cancer , work by others have shown that mda - mb-231 breast cancer , t24 bladder carcinoma , and 786-o renal adenocarcinoma cells expressing 51 integrins exhibited increased invasion in 3d collagen matrices which had been polymerized with fibronectin , further highlighting that integrin engagement with fibronectin supports tumor cell invasion in 3d 3 . interestingly , the addition of soluble fibronectin at the start of the assay reduced tumor cell invasion in collagen matrices , suggesting that differences in the polymerization status of fibronectin may differentially regulate tumor cell behavior 3 . a role for v3 interaction with fibronectin for example , 3d matrices of clotted plasma , reported to play an important role in tumor metastasis 27 , or fibrin , the principal component of clots , were found to induce invadopodia , cellular protrusions which participate in the early stages of cell migration and invasion , formation from several tumor cell lines 9 . furthermore , it was found that primary metastatic kidney tumor cells exhibited an increased expression of invadopodia formation in addition to fibronectin and v3 integrin which together upregulated the expression of the epithelial - mesenchymal ( emt ) transcription factor slug 9 . these results would suggest that v3 and fibronectin promote tumor cell invadopodia formation via activation of the emt transcription factor slug . in order to metastasize to distant sites , tumor cells make use of altered cell signaling pathways and matrix metalloproteinases ( mmps ) , which degrade and remodel the ecm allowing tumor cell migration . with regard to a role for fibronectin in altered cell signaling , balanis et al showed that adhesion of mda - mb-231 breast cancer cells to fibronectin elicited robust stat3 activation , a result which was dependent on cellular expression of fak and the tyrosine kinase pyk2 28 . the authors further demonstrated that administration of fibronectin to mda - mb-231 cells cultured atop 3d hydrogels resulted in cellular outgrowth and a phenotype previously linked with enhanced metastatic properties 28 . inhibition of 1 integrin or the use of a small inhibitor against stat3 reversed these findings , suggesting that in 3d , mda - mb-231 breast cancer cells utilize fibronectin-1 integrin mediated stat3 signaling during invasion 28 . to illustrate a cooperative role of fibronetin and mmps in tumor invasion , meng et al sought to determine whether a549 lung cancer cell invasion occurred as a result of fibronectin - induced fak activation of mmp9 29 . the authors found that fibronectin stimulated fak phosphorylation and src recruitment , resulting in activation of downstream targets , erk1/2 and pi3k / akt in a549 cells , promoting invasion and migration of the cancer cells in transwell chambers 29 . inhibition of erk1/2 and pi3k resulted in a reduction of migrating and invasive a549 cells as a result of downregulation of mmp9 , implicating a role for fibronectin - fak - mmp9 in lung cancer invasion 29 . together , these findings not only suggest an important role for integrin engagement with fibronectin , but cooperation between fibronectin and altered cell signaling and mmp expression in processes associated with tumor invasion and metastasis . chemotherapeutic agents have undoubtedly contributed to improved survival rates for individuals with cancer . despite these successes , tumor cell resistance to therapies and relapse while a number of factors have been implicated in chemotherapy resistance , fibronectin has received interest . in a study by pontiggia et al the efficacy of tamoxifen , an estrogen receptor antagonist , was used to examine how the microenvironment limits tumor cell sensitivity to tamoxifen 12 . here , the authors co - incubated human and mouse breast tumor cells with fibronectin and measured tumor cell sensitivity to tamoxifen 12 . the addition of fibronectin promoted tamoxifen resistance via fibronectin - mediated activation of 1-integrin which in turn , stimulated the pi3k / akt and mapk / erk 1/2 signaling pathways in both tumor cell lines 12 . disrupting the interaction between the 1-integrin and fibronectin reversed the conferred resistance , leading to drug - induced apoptosis of the tumor cells 12 . similarly , yuan et al found that 1 integrin and fibronectin were markedly increased in tamoxifen resistant as opposed to tamoxifen sensitive mcf7 breast cancer cells 30 . the overexpression of 1 was associated with increased expression of the epidermal growth factor receptor ( egfr)/erk signaling pathway . inhibition of 1 improved tamoxifen sensitivity of resistant mcf7 cells and further reduced cellular migration following culture in the presence of conditioned media from carcinoma associated fibroblasts ( cafs ) 30 . interestingly , culture of tamoxifen resistant mcf7 cells in caf conditioned media resulted in enhanced cell migration due to caf produced fibronectin , a result which was diminished following cellular treatment with the 1 integrin inhibitor 30 . in addition to tamoxifen , cetuximab , an antibody that inhibits cell proliferation when bound to the egfr , has been reported to have limited efficacy as a result of tumor cell resistance 10 . for example , a549 human lung adenocarcinoma cells and h1299 human non - small cell lung carcinoma cells were reported to have diminished cell cytotoxicity to cetuximab and radiation following culture atop fibronectin coated surfaces 10 . the observed reduction of cytotoxicity was a result of tumor cell 51 integrin engagement with fibronectin as silencing these integrins restored tumor cell sensitivity to cetuximab 10 . interestingly , cetuximab was also shown to promote increased fibronectin expression from both tumor cell lines , a finding which was reported to result from activation of the p38-mapk - atf2 signaling pathway 10 . sirna silencing of fibronectin improved cytotoxicity of cetuximab in h1299 and a549 tumor cell lines , demonstrating that excess fibronectin in combination with 51 integrin engagement with fibronectin facilitates cetuximab resistance 10 . in a similar study , changes in tumor cell apoptosis were analyzed following etoposide treatment of h69 small cell lung cancer cells which had been cultured in fibronectin coated dishes 31 . the results showed that tumor cell 2 , 3 , 6 , and 1 integrin interaction with fibronectin inhibited chemotherapy induced apoptosis , evidenced by decreased caspase 3-activity 31 . using breast cancer as a model , spangenberg et al reported that inducible expression of the erbb2 proto - oncogene in mcf7 breast cancer cells resulted in upregulated gene expression of 5 and 1 integrins 32 . the authors found that mcf7 cells exhibited increased resistance to cisplatin and 5-fluorouracil as a result of erbb2 induced 51 integrin expression and exhibited a 2 fold reduction in responsiveness to these agents when the cells were cultured on fibronectin . to overcome chemotherapy resistance from integrin - fibronectin interactions , nam et al showed that disrupting the peptide bond between fibronectin and 51-integrin in combination with radiation therapy promoted apoptosis and reduced the expression of 51 in cultures of malignant human breast cancer cells cultured atop a 3d matrigel 5 . the authors found that the pro - survival effect of malignant tumor cell 51 interaction with fibronectin in a 3d environment was a result of 51 mediated activation of akt signaling as akt kinase and akt phosphorylation were downregulated following 51 inhibition 5 . similarly , it was found that primary glioblastomas resistant to the anti - angiogenic agent bevacizumab exhibited a higher expression of 1 integrins and fak , a phenomenon the authors attributed to high intra - tumoral hypoxia following anti - angiogenic therapy 33 . to determine whether inhibition of 1 integrin improved therapeutic responses , the authors treated subcutaneously grown u87 mg glioblastomas with alternating doses of a 1 inhibitor and low dose ( 1mg / kg ) bevacizumab 33 . the results showed a significant improvement in tumor regression in the dual treated animals 33 . these results were comparable to a high dose regimen ( 10mg / kg ) of bevacizumab alone , suggesting that the addition of a 1 integrin inhibitor improves the therapeutic efficiency of bevacizumab 33 . given these and other similar studies , it 's apparent that targeting the cellular interactions with fibronectin and/or fibronectin itself may be a desirable strategy to improve patient outcomes . while the aforementioned studies have provided encouraging evidence to address therapeutic targets against integrin engagement with fibronectin , further research is necessary in order to find novel strategies for inhibiting tumor growth via targeting fibronectin biosynthesis and/or fibronectin regulated cell signaling . matrix rigidity is a common feature of tumors and has been shown to support tumor growth , invasion and metastasis 34 . in response to matrix rigidity , focal adhesions , fak , a structural and signaling component of focal adhesions , has not only been reported to be upregulated in response to matrix rigidity 35 , but has additionally been shown to regulate fibronectin - directed matrix fibrillar organization 36 . ilic et al reported that fibronectin fibrils were more sparse and thinner in fak null embryos compared to wildtype littermates and fibroblasts , a result which was demonstrated to be independent of fibronectin gene or protein expression and synthesis , suggesting that fak activity is necessary for fibronectin matrix organization 36 . interestingly , in a separate study , fibronectin expression was shown to drive fak signaling 29 , suggesting that both fak and fibronectin may interact in a positive feedback loop . given the role of fak in fibronectin expression , it 's conceivable that targeting fak expression may be a mechanism to reduce fibronectin expression and thus tumor progression . meng et al showed that fibronectin - induced fak tumor cell migration and invasion was decreased following treatment of a549 lung tumor cells with the src inhibitor pp2 29 . specifically , the authors reported that inhibition of src resulted in decreased fak phosphorylation suggesting that a reduction of fak activity may be a useful strategy to reduce fibronectin mediated effects on tumor cell migration and invasion 29 . in addition to indirect targets of fak , it may be possible to directly reduce fak activity via use of a novel atp competitive kinase inhibitor , pf-04554878 , which was recently shown to promote apoptosis and reduce proliferation of pancreatic neuroendocrine tumor cells 37 . aside from targeting faks , it may be possible to directly reduce matrix stiffness in tumors , thereby preventing fak - induced fibronectin expression . levental et al demonstrated that expression of lysyl oxidase ( lox ) , an enzyme which cross - links collagen fibers 38 , not only increased fak expression , but augmented matrix rigidity and breast tumor progression , a phenomenon which was reversed upon treatment with an inhibitor of lox 39 . in support of a role for lox in fibronectin expression , adam et al showed that atrial samples from patients with atrial fibrillation exhibited increased expression of lox and fibronectin 40 . administration of a small molecule inhibitor of lox to neonatal cardiac fibroblasts attenuated lox - induced fibronectin expression via downregulation of angiotensin ii and connective tissue growth factor 40 . although this work was conducted in cardiac fibroblasts , it 's foreseeable that inhibiting lox may be a means to reduce tumoral fibronectin expression . overall , it is likely that targeting the molecular machinery which is responsible for supporting matrix rigidity will reduce both fibrosis and fibronectin expression and in effect decrease tumor progression . while indirect targets against fibronectin should undoubtedly yield more favorable outcomes for cancer patients to this end , tomasini - johansson et al developed a small peptide inhibitor of fibronectin , pur4b , which was shown to limit fibronectin polymerization in the matrix 41 . work by chiang et al has shown that administration of pur4b in animals which had received a partial ligation of the external or internal carotid arteries resulted in reduction of intima , media and adventitial thickness of the vessels and further decreased the accumulation of fibronectin and collagen 42 . furthermore , hielscher et al demonstrated that treatment of co - cultures of fibroblasts and breast cancer cells with pur4b not only reduced fibronectin deposition and organization in the ecm , but also inhibited the deposition of other matrix proteins 43 . additionally , inhibition of fibronectin matrix assembly also attenuated vascular morphogenesis of endothelial cells cultured atop the fibronectin - deficient matrix 43 . these studies illustrate that fibronectin not only serves as a substrate regulating the assembly of other ecm proteins but is additionally important for supporting angiogenesis . as such , direct inhibition of fibronectin polymerization via a small peptide may impair the assembly of other matrix proteins and capillaries , thus depriving tumors of a supportive scaffold and the oxygen and nutrients necessary for continued growth . indeed , the use of such anti - cancer peptides have not only been used to directly treat cancer but have also been used as cytotoxic drug carriers 44 . as such , it 's possible that delivery of a peptide inhibitor of fibronectin polymerization may be utilized in conjunction with a chemotherapy regimen to improve outcomes for cancer patients . a benefit from use of an inhibitor of fibronectin polymerization is that the off target effects of the peptide should be minimal as fibronectin is n't actively produced in tissues with the exception of wounds and during development . as such , one may need to complex the inhibitor in a delivery vector to improve both tumor targeting and sustained delivery of the peptide at the tumor site . rna interference is rapidly becoming a powerful tool to silence the expression of genes and has been extensively investigated for its utility as a form of cancer therapy . previous studies have shown that silencing fibronectin reduced tumor cell proliferation and growth 17 and tumor cell migration 6 . as such , silencing fibronectin gene expression via rna silencing may be an effective strategy to enhance therapeutic responses . despite its obvious advantages , the major drawback associated with use of gene based therapies is the delivery of these to the tumor . traditionally , sirna have been delivered locally where they 've had much success with regard to treating ocular diseases 45 . for delivery of fibronectin sirna to tumors , it will be important to utilize vectors which not only target the tumor , but penetrate the tumor cell membrane to deliver the packaged sirna particles . cell - penetrating peptides ( cpp ) , which consist of short peptide sequences of 10 - 30 amino acids , have been shown to cross the cellular plasma membrane 46 . derived from viral proteins or mammalian proteins with translocation capabilities , cpps have been used to deliver sirna to in - vitro cell cultures 47 and in transvascular delivery of sirna to mice 48 . although additional work will be necessary , the use of these vectors may be an avenue by which to deliver fibronectin sirna to tumors . another means by which to target fibronectin expression could come from use of vaccinia viruses , double stranded dna viruses used to treat cancer . use of vaccinia viruses has received increased interest as strategies to engineer more potent oncolytic forms of vaccinia viruses has not only resulted in greater tumor specificity but have been shown to be an efficient means by which to lyse tumor cells 49 . recently , a genetically engineered vaccinia virus was reported to target both malignant ovarian cancer cells in addition to the fibroblasts in the tumor stroma 50 . given that the vaccinia virus was able to infect fibroblasts , it 's possible that utilizing a similar strategy may halt the production of fibronectin from cancer associated fibroblasts as fibroblasts are a principle cell type responsible for ecm production . together , the aforementioned studies have provided a clearer perspective on the mechanisms whereby fibronectin exerts its tumorigenic effects in cancer . these studies suggest that fibronectin is not only aberrantly expressed in tumors , but also promotes tumor progression , metastasis and therapy resistance via activation of a number of downstream signaling pathways . with advances in this knowledge , it 's foreseeable that employing strategies to inhibit tumor cell association with fibronectin , fibronectin - mediated cell signaling and/or fibronectin biosynthesis in the ecm will not only result in unprecedented discoveries about the mechanisms fibronectin exerts on tumor cell behavior , but will additionally result in development of novel therapeutic approaches to treat cancer .

fibronectin is a matrix glycoprotein which has not only been found to be over - expressed in several cancers , but has been shown to participate in several steps of tumorigenesis . the purpose of this review is to illustrate how aberrant fibronectin expression influences tumor growth , invasion , metastasis and therapy resistance . in particular , this review will focus on the interactions between cell receptor ligands and fibronectin and how this interaction influences downstream signaling events that aid tumor progression . this review will further discuss the possible implications of therapeutic drugs directed against fibronectin and/or cellular interactions with fibronectin and will additionally discuss novel approaches by which to limit intra- and extra - tumoral fibronectin expression and the cellular events which lead to aberrant fibronectin expression . it is anticipated that these studies will set a basis for future research that will not only aid understanding of fibronectin and its prognostic significance , but will further elucidate novel targets for therapeutics .

they originate either in bone ( osseous plasmacytoma ) or in soft tissues ( extramedullary plasmacytoma ) . it can occur as the sole manifestation of plasma cell neoplasm , as a metastasis from another extramedullary plasmacytoma or as a consequence of multiple myeloma . it has no characteristics of multiple myeloma , but the development of multiple myeloma has been observed in 836% of patients months or years later . these plasma cell tumors can occur anywhere and have to be differentiated from other neoplasms , infectious processes and chloromas . the head and neck region has been reported as a frequent site for amyloid deposits . one of the most common forms of amyloid proteins are amyloid light chain ( al - type ) , which is derived from plasma cells , contains immunoglobulin light chains and is associated with plasma cell dyscrasias . fine needle aspiration smears of a 50-year - old female patient with cervical lymphadenopathy were sent to the department of pathology without any clinical details . cytology smears ( giemsa stain ) showed sparse cellular aspirate with amorphous granular basophilic material , predominantly scattered lymphocytes , plasma cells and occasional epithelioid cells [ figure 1 ] . this amorphous material was interpreted as caseous necrosis and the possibility of tuberculous lymphadenitis was suggested . subsequently , patient 's clinical findings were provided with the request for review of slides as the patient had not responded to antituberculous treatment . in the details provided , a nasal growth was also mentioned to be present in the lateral wall of the nasal cavity , which was globular , firm and pink in colour and measured 3 2 2 cm . lymph node aspirate showing amorphous granular basophilic material which mimicked caseous necrosis ( giemsa , 400 ) repeat aspiration from the cervical lymph node , imprint smears and biopsy from the growth were performed . repeat aspiration and imprint smears revealed high cellularity with amorphous basophilic material , large number of plasma cells with varying degree of maturity , myeloma cells and occasional scattered epithelioid cells . an occasional foreign body type , multinucleated giant cell with intracytoplasmic amorphous basophilic material was also seen . plasma cells had prominent eccentric nuclei , coarse chromatin , perinuclear halo and basophilic cytoplasm and myeloma cells had fine chromatin , prominent nucleoli and little or no halo . presence of numerous plasma cells and myeloma cells raised the possibility of plasmacytoma [ figure 2 ] . the amorphous extracellular material and similar intracytoplasmic material within giant cells raised the possibility of amyloid within the cells . subsequent congo red staining of the cytology and histopathology slides , showed the amorphous material to be orange - red in color . it gave an apple - green birefringence when viewed under polarized light , confirming it to be amyloid . thus , the diagnosis of plasmacytoma with amyloidosis was suggested and confirmed by a histopathology study of the biopsy tissue . there was no clinical , radiological or laboratory evidence of plasma cell dyscrasias or systemic amyloidosis . repeat lymph node aspirate showing plasma cells ( uninucleate and binucleate ) , myeloma cells , scattered lymphocytes and amorphous basophilic material ( giemsa , 400 ) extramedullary plasmacytoma is a soft tissue neoplastic lesion that is made up of monoclonal plasma cells ( plasma cell dyscrasias ) . it can be primary , without evidence of disease in other foci , or part of a systemic process during the course of multiple myeloma . thus , these patients should be carefully evaluated for the presence of disseminated disease / multiple myeloma before arriving at a diagnosis of extramedullary plasmacytoma . studies should include complete blood cell count , serum calcium levels , radiologic skeletal survey , magnetic resonance imaging ( mri ) of the spine , pelvis , humeri and femurs , immunoelectrophoretic examination of serum and urine , 2 microglobulin assay and bone marrow biopsies . diagnosis should be made only if all studies for disseminated disease are negative , as was seen in our case . these tumors can occur anywhere in the body , but nearly 8090% of all extramedullary plasmacytoma occur in the head and neck region . most of these cases arise in the aerodigestive tract , probably because of the abundance of lymphatic tissue in this area . in one study the frequently affected sites in decreasing order of frequency included nasal cavity or paranasal sinuses , nasopharynx , oropharynx and larynx . extramedullary plasmacytoma occurs in patients between 50 and 60 years of age , and is more common in men ( 4:1 ) . plasmacytomas arising in the nasal cavity or nasopharynx usually present as a soft bleeding mass or as a polypoid mass , covered mostly by an intact overlying mucosa . cervical lymphadenopathy and nasal discharge may be the associated presenting symptoms in an occasional patient . microscopic examination of cytological smears show numerous plasma cells , including binucleate , multinucleate and pleomorphic forms . on histopathological examination , plasmacytomas are very vascular tumors with minimal stromal component and sheets of plasma cells with varying degrees of differentiation and immature plasma cells . differential diagnosis of plasmacytoma includes plasma cell granuloma , plasmacytoid lymphoma and large cell lymphoma of immunoblastic type . immunocytochemical study with antibody to kappa and lambda light chains is an important approach to differentiate these conditions . kappa or lambda light chain - restricted population confirms the diagnosis of plasmacytoma and suggests a pathogenetic relationship between plasmacytoma and amyloid deposition . sakai et al . diagnosed extramedullary plasmacytoma of the tonsil by demonstrating diffuse immunoreactivity for lambda light chains within plasmacytoid cells . immunoblastic lymphomas usually involve lymph nodes and show cytoplasmic igm heavy chain ( igg and iga heavy chains in plasmacytomas ) and express pan b cell surface antigens . eilam et al . reported a case of plasmacytoma of the nasal cavity that involved the palate , ethmoidal and maxillary sinuses and contained deposits of amyloid . reported a case of extramedullary plasmacytoma of the parotid gland with extensive amyloid deposition masking the cytologic and histopathologic picture . amyloid refers to the extracellular fibrillar proteinaceous substance deposited in various tissues and organs of the body in a wide variety of clinical settings clinically , the symptoms depend on the magnitude of the deposits and on the particular sites or the organs affected . regarding its imaging appearance , affected bony conchae and sinus walls show a fluffy appearance adjacent to amyloid deposit on computed tomography scan ; calcification has been mentioned as a nonspecific finding . foreign body giant cell reaction may be evoked about the amyloid deposits , as was also seen in our case . it can be seen on mri as a peripheral enhancement in the region of amyloid deposits with contrast material administration . differential diagnosis of this material on lymph node cytology smears include caseous necrosis , tumor necrosis and colloid . completely amorphous granular material without identifiable cell remnants suggest caseous necrosis , seen predominantly in tuberculosis caused by mycobacteria and other bacterial diseases and fungal infections . the type of granulomatous reaction , clinical details and bacteriological data are necessary to differentiate these conditions . neoplastic conditions , specially the lymphomas ( non - hodgkin 's and hodgkin 's ) and metastatic carcinomas , show tumor necrosis and therefore should be excluded . tumor necrosis appears as numerous cell shadows with pyknotic nuclei it may be homogenous or granular , resembling caseous necrosis . colloid appears as a dense , blob - like material with sharp outlines on cytology smears from the thyroid . rectal / bone marrow biopsy and fine needle aspiration of abdominal subcutaneous fat is suggested to rule out systemic amyloidosis . the diagnosis of amyloid is based almost entirely on its staining characteristics . with light microscope and standard tissue stains differentiation between amyloid and other amorphous eosinphilic substances , such as hyaline material , collagen and fibrin , is impossible . thioflavin t and other fluorescent dyes offer greater sensitivity in detection of amyloid but both hyaline and fibrin give positive results . thus , the gold standard for diagnosis of amyloid is congo red staining , which stains the tissue pink or red in color by light microscopy and gives green birefringence by polarized microscopy due to a -pleated sheet conformation of amyloid fibrils .

amyloid material on lymph node cytology smears can mimic caseous necrosis . we report one such case where a 50-year - old lady presented with a nasal mass and cervical lymphadenopathy . fine needle aspiration cytology smears of the cervical lymph node were interpreted as tuberculous lymphadenitis based on the presence of an occasional epithelioid cell and caseous material . the patient did not respond to antituberculous therapy and was revaluated . repeat aspiration from the lymph node showed numerous plasma cells and myeloma cells in addition to the amorphous material which was confirmed to be amyloid on staining with congo red . a diagnosis of plasmacytoma with amyloidosis was rendered . imprint smears from nasal mass , detailed hematology workup and subsequent histology confirmed the diagnosis .

alzheimer 's disease ( ad ) is a neurodegenerative disorder which causes a progressive and irreversible loss of cognition and physical function . the number of ad patients will increase sharply in the near future , and it has been estimated that over 80 million individuals will be diagnosed with this condition by the year 2040 . to date , no cure is available to modify the progression of ad , although available pharmacological treatments have been proven to positively control cognitive and functional symptoms of the disease and improve quality of life of patients and their families for the time they are responsive to treatment [ 3 . ] most often , care for ad patients is provided by informal caregivers including relatives and friends . caregivers are individuals who are called to assist patients in their activities of daily living , to administer medications to them and to deal with the patients progressive personality change and physical , cognitive and emotional deterioration . such aspects make providing care to people with ad a burdensome experience which affects the physical and psychological state of caregivers and their quality of life . managing and administering medications to ad patients may substantially contribute to the caregivers workload and distress . compliance to treatments and efficacy of medications may depend on how well caregivers are able to manage drug administrations . the caregivers satisfaction with treatments is crucial for a good compliance to treatments , and it has been associated with an increased likelihood of positive clinical outcomes , decreased need of sedatives and anxiolytics , and delayed patient institutionalization . currently , there are four medications approved for the treatment of ad by regulatory authorities . three are acetylcholinesterase inhibitors ( acheis ) including donepezil , rivastigmine and galantamine ; the fourth is memantine , an n - methyl - d - aspartic acid receptor antagonist . since the rivastigmine patch has been marketed , experimental data from randomized clinical trials have suggested that the transdermal application would be preferred by caregivers of ad patients compared to oral medications . to date , no information is available regarding patient compliance to treatment and caregiver satisfaction with respect to different formulations of ad medications in a real clinical setting . the present study aimed at comparing compliance to treatment of community - dwelling patients with mild - to - moderate ad treated with transdermal application compared to oral formulation drugs and caregiver satisfaction in a real clinical setting . the secondary purpose of the study was to provide a full description of sociodemographic and clinical characteristics of mild - to - moderate ad patients and to draw a sociodemographic profile of their caregivers . a sub - study was conducted to perform the linguistic and psychometric validation of the caregiver medication interview ( cmi ) , which is the instrument adopted in the current investigation to assess patient compliance to treatment and caregiver satisfaction . the axept study consisted of a main study , which was conducted to primarily compare patient compliance to treatment and caregiver satisfaction with respect to oral and transdermal formulations of anti - ad medications , and a sub - study , which was conducted to validate the cmi instrument . the main study had an observational , cross - sectional , multicenter design . in the sub - study , a sample of participants was observed longitudinally and assessed with the cmi at the baseline visit and 1 week later ( follow - up visit ) to measure the psychometric properties of this instrument . the study was conducted in 45 memory clinics uniformly distributed throughout italy between september 8 , 2010 and january 31 , 2011 . patients with ad were considered eligible for participation if : ( a ) they had a mini - mental state examination ( mmse ) score between 18 and 26 at the screening visit ; ( b ) they were 18 years old or more ; ( c ) they signed an informed consent , and ( d ) they were treated either with acheis or memantine in monotherapy regimens , and such treatment was initiated 46 months prior the baseline visit as either the first anti - ad treatment ever received or a new drug option after switching from an old treatment . all the above - mentioned inclusion criteria had to be met for patients to be enrolled in the study . patients were excluded from the study if they were treated with a combination of acheis or with acheis plus memantine . caregivers had to be present at the baseline visit and had to provide care to the patients at least 4 h per day for at least 4 months prior to the baseline visit . they had to speak and understand the italian language and to be able to answer a questionnaire including a numeric rating scale . participants in the main study were assessed at one point in time during the baseline visit . participant assessment was conducted by site physicians who received specific training on the study procedures during a centralized investigator meeting . information was collected by direct interviews with patients and caregivers and extracted from medical charts . the caregivers were asked about the daily amount of time spent providing care to patients . information on the patients weight , height , blood pressure , heart rate , disease duration , swallowing difficulties , medical history and concomitant gastrointestinal or dermatological symptoms or diseases was also collected . the activities of daily living scale ( adls ) , scoring from 0 ( total dependence ) to 6 ( total independence ) , was used to evaluate the patients functional status . data on the patients current anti - ad pharmacological treatment were recorded , including the drug ingredient , dose , formulation , start date of treatment and eventual reason for switching to a different medication . the patients use of concomitant medications with particular respect to psychotropic drugs was also assessed . the cmi is an ad hoc questionnaire developed for the purpose of the axept study . it was initially developed in the english version by the axept researchers as an instrument which could have been used internationally . it is available in two forms , one designed to be used in case of oral treatment and one designed to be used in case of transdermal applications . it consists of three questions directed to the caregiver and exploring the ease of administration , the global compliance and the satisfaction relative to the treatment the patient is receiving . for questions on compliance and satisfaction , the caregiver has to indicate , on a numeric rating scale , a score between 0 and 10 , with 0 being the lowest degree and 10 the highest degree of compliance and satisfaction . data collected from direct interviews with caregivers and from medical charts during the baseline visit were used to assess the characteristics of mild - to - moderate ad patients and their caregivers . according to the methods proposed by divine et al . for estimating the sample size for the wilcoxon - mann - whitney test , it has been calculated that a sample of 730 participants was required to observe a p(x > y ) equal to 0.57 with a power of 0.90 and a 0.05 type i error . given that x and y are random observations from the two distributions being compared ( transdermal and oral formulation ) , p(x > y ) represents the probability that x is greater than y. since previous data were missing , it has been conservatively assumed that p(x > y ) equals 0.57 , under the null hypothesis that p(x > y ) equals 0.50 . a zero variance associated with ties and an allocation ratio of 3 to 4 between the transdermal and the oral formulation according to data on prescription patterns in italy have also been assumed . characteristics of the study participants according to the type of drug formulation received were assessed using descriptive statistics . compliance to treatment and caregiver satisfaction were described according to the type of drug formulation ( oral or transdermal ) . descriptive statistics included mean , standard deviation , median , mode , quantiles and interquartile range . comparison between patient compliance and caregiver satisfaction according to the type of drug formulation was conducted using the wilcoxon - mann - whitney test . statistical analysis was performed using sas v 9.1.2 . for the purpose of the current study , the english version of the cmi was translated into the italian language , and a linguistic and psychometric validation of the italian version was performed . the linguistic validation process included the following steps : forward translation of the english cmi into italian : two forward translations were produced by two independent professional translators who were native italian speakers ; reconciliation : creation of a preliminary combined version ( reconciled version ) on which both translators and investigators agreed ; backward translation of the italian reconciled version into english : the preliminary italian version was translated back into english by a professional translator who was a native english speaker and had no access to the original version of the instrument ; comparison between the backward translated version with the original english version ; mistranslations and inaccuracies were detected and the preliminary italian version was revised accordingly in a consensus meeting including the backward translator and the investigators ; comprehension test : the italian revised version of the cmi was administered to a sample of 18 caregivers of patients who were potential study participants according to the inclusion criteria ; these caregivers were identified from the three expected highest recruiting sites ; this was a qualitative examination performed to determine possible problems in the respondents understanding of the instructions ; creation of a final italian version by the investigators and implementation of this instrument in the study . the psychometric evaluation of the translated cmi was then conducted in a sub - sample of 188 caregivers participating in the study and selected by the six expected highest recruiting sites . the cmi was administered to the sub - study caregivers during the baseline evaluation and 1 week later . the reliability of the instrument was evaluated by measuring its internal consistency , expressed by cronbach 's , and its test - retest reliability , expressed by the intraclass correlation coefficient ( icc ) and the spearman correlation coefficient ( ) . no gold standard among the instruments which measure compliance to treatment and caregivers satisfaction is available in the literature . therefore , the convergent validity was evaluated comparing the cmi scores with the responses obtained from the same caregivers who were administered with a visual analogue scale ( vas ) . for the purpose of this study , a vas including a 10-cm line with one extreme indicating the lowest and the other extreme indicating the highest compliance and satisfaction was used . the icc and spearman 's were measured to estimate the convergent validity of the italian version of the cmi compared with the vas . the axept study consisted of a main study , which was conducted to primarily compare patient compliance to treatment and caregiver satisfaction with respect to oral and transdermal formulations of anti - ad medications , and a sub - study , which was conducted to validate the cmi instrument . the main study had an observational , cross - sectional , multicenter design . in the sub - study , a sample of participants was observed longitudinally and assessed with the cmi at the baseline visit and 1 week later ( follow - up visit ) to measure the psychometric properties of this instrument . the study was conducted in 45 memory clinics uniformly distributed throughout italy between september 8 , 2010 and january 31 , 2011 . patients with ad were considered eligible for participation if : ( a ) they had a mini - mental state examination ( mmse ) score between 18 and 26 at the screening visit ; ( b ) they were 18 years old or more ; ( c ) they signed an informed consent , and ( d ) they were treated either with acheis or memantine in monotherapy regimens , and such treatment was initiated 46 months prior the baseline visit as either the first anti - ad treatment ever received or a new drug option after switching from an old treatment . all the above - mentioned inclusion criteria had to be met for patients to be enrolled in the study . patients were excluded from the study if they were treated with a combination of acheis or with acheis plus memantine . caregivers had to be present at the baseline visit and had to provide care to the patients at least 4 h per day for at least 4 months prior to the baseline visit . they had to speak and understand the italian language and to be able to answer a questionnaire including a numeric rating scale . participants in the main study were assessed at one point in time during the baseline visit . participant assessment was conducted by site physicians who received specific training on the study procedures during a centralized investigator meeting . information was collected by direct interviews with patients and caregivers and extracted from medical charts . the caregivers were asked about the daily amount of time spent providing care to patients . information on the patients weight , height , blood pressure , heart rate , disease duration , swallowing difficulties , medical history and concomitant gastrointestinal or dermatological symptoms or diseases was also collected . the activities of daily living scale ( adls ) , scoring from 0 ( total dependence ) to 6 ( total independence ) , was used to evaluate the patients functional status . data on the patients current anti - ad pharmacological treatment were recorded , including the drug ingredient , dose , formulation , start date of treatment and eventual reason for switching to a different medication . the patients use of concomitant medications with particular respect to psychotropic drugs was also assessed . the cmi is an ad hoc questionnaire developed for the purpose of the axept study . it was initially developed in the english version by the axept researchers as an instrument which could have been used internationally . it is available in two forms , one designed to be used in case of oral treatment and one designed to be used in case of transdermal applications . it consists of three questions directed to the caregiver and exploring the ease of administration , the global compliance and the satisfaction relative to the treatment the patient is receiving . for questions on compliance and satisfaction , the caregiver has to indicate , on a numeric rating scale , a score between 0 and 10 , with 0 being the lowest degree and 10 the highest degree of compliance and satisfaction . data collected from direct interviews with caregivers and from medical charts during the baseline visit were used to assess the characteristics of mild - to - moderate ad patients and their caregivers . according to the methods proposed by divine et al . for estimating the sample size for the wilcoxon - mann - whitney test , it has been calculated that a sample of 730 participants was required to observe a p(x > y ) equal to 0.57 with a power of 0.90 and a 0.05 type i error . given that x and y are random observations from the two distributions being compared ( transdermal and oral formulation ) , p(x > y ) represents the probability that x is greater than y. since previous data were missing , it has been conservatively assumed that p(x > y ) equals 0.57 , under the null hypothesis that p(x > y ) equals 0.50 . a zero variance associated with ties and an allocation ratio of 3 to 4 between the transdermal and the oral formulation according to data on prescription patterns in italy have also been assumed . characteristics of the study participants according to the type of drug formulation received were assessed using descriptive statistics . compliance to treatment and caregiver satisfaction were described according to the type of drug formulation ( oral or transdermal ) . descriptive statistics included mean , standard deviation , median , mode , quantiles and interquartile range . comparison between patient compliance and caregiver satisfaction according to the type of drug formulation was conducted using the wilcoxon - mann - whitney test . for the purpose of the current study , the english version of the cmi was translated into the italian language , and a linguistic and psychometric validation of the italian version was performed . the linguistic validation process included the following steps : forward translation of the english cmi into italian : two forward translations were produced by two independent professional translators who were native italian speakers ; reconciliation : creation of a preliminary combined version ( reconciled version ) on which both translators and investigators agreed ; backward translation of the italian reconciled version into english : the preliminary italian version was translated back into english by a professional translator who was a native english speaker and had no access to the original version of the instrument ; comparison between the backward translated version with the original english version ; mistranslations and inaccuracies were detected and the preliminary italian version was revised accordingly in a consensus meeting including the backward translator and the investigators ; comprehension test : the italian revised version of the cmi was administered to a sample of 18 caregivers of patients who were potential study participants according to the inclusion criteria ; these caregivers were identified from the three expected highest recruiting sites ; this was a qualitative examination performed to determine possible problems in the respondents understanding of the instructions ; creation of a final italian version by the investigators and implementation of this instrument in the study . the psychometric evaluation of the translated cmi was then conducted in a sub - sample of 188 caregivers participating in the study and selected by the six expected highest recruiting sites . the cmi was administered to the sub - study caregivers during the baseline evaluation and 1 week later . the reliability of the instrument was evaluated by measuring its internal consistency , expressed by cronbach 's , and its test - retest reliability , expressed by the intraclass correlation coefficient ( icc ) and the spearman correlation coefficient ( ) . no gold standard among the instruments which measure compliance to treatment and caregivers satisfaction is available in the literature . therefore , the convergent validity was evaluated comparing the cmi scores with the responses obtained from the same caregivers who were administered with a visual analogue scale ( vas ) . for the purpose of this study , a vas including a 10-cm line with one extreme indicating the lowest and the other extreme indicating the highest compliance and satisfaction was used . the icc and spearman 's were measured to estimate the convergent validity of the italian version of the cmi compared with the vas . patients and caregivers consecutively visiting the participating memory clinics since the beginning of the study were screened for enrolment . the duration of the screening period was 5 months , and a competitive enrolment was allowed . a total of 896 patients and their caregivers were screened ; 41 of them were excluded because they did not meet the inclusion criteria . sociodemographic , cognitive , functional and clinical characteristics of the study patients by type of drug formulation received are reported in table 1 . the mean age of patients was 77 years in both groups , and over 60% were female . both groups showed a mild - to - moderate cognitive impairment ( mean mmse score 21.0 ) and a good level of preserved functional abilities ( mean adl score 5.0 ) . cardiovascular diseases were highly prevalent in both groups , with nearly 50% of patients affected by hypertension and 20% diagnosed with any cardiac disease . patients on oral medications were more likely to be diagnosed with psychiatric illnesses such as major depression , bipolar disorder and schizophrenia than patients on patch ( 14.6 vs. 7.9% ) . however , the patients compliance to treatment and the caregivers satisfaction by type of drug formulation did not change after stratifying the sample on the presence of psychiatric illnesses . moreover , correlation analyses showed that psychiatric illnesses were not associated with the type of drug formulation and with compliance and satisfaction . the prevalence of gastrointestinal diseases was slightly higher among patients treated with oral medications compared to those treated with the rivastigmine patch . dermatological conditions were rarely reported , with a slightly increased prevalence in the group receiving oral medications . other conditions including chronic obstructive pulmonary disease , asthma , cancer , swallowing problems and renal or liver failure were infrequent and equally distributed in the two groups . the estimated mean time from the onset of symptoms to ad diagnosis was nearly 2 years in both groups . over 17% of patients in both groups had switched from a previous anti - ad medication to the current one received at the study time . the most frequently reported medication changes were from either donepezil or oral rivastigmine to patch ( 40% ) and from either patch or oral rivastigmine or donepezil to memantine ( 34% ) . the most frequently reported reason for switching was low tolerability of the previous treatment . with respect to other psychotropic medications , 30% of patients on oral medications and 26% of patients on patch were taking antidepressants mostly belonging to the class of selective serotonin re - uptake inhibitors . antipsychotic prescription was not frequent in the study population , with more atypical than conventional medications used in both groups . a low prevalence of use was also reported for benzodiazepines and anticonvulsants in both groups . characteristics of the caregivers in the study according to the type of drug formulation their patients were receiving are reported in table 2 . the mean age of the caregivers was 58 years , and over 70% were female . over 95% of the caregivers were relatives , and more than half of them were living with the patient . the mean time spent providing care reported by caregivers in both groups was around 12 h per day . the rivastigmine patch was the most frequently prescribed anti - ad drug in the study population . over 46% ( n = 396 ) of the study patients were on rivastigmine patch , while 54% ( n = 461 ) were receiving any type of oral anti - ad drugs . with respect to oral drugs , 25% of patients were treated with donepezil and 21% were receiving memantine . the median and the mode in addition to the range of the daily drug dose are reported in table 3 . nearly 80% of caregivers of patients on patch were not concerned about patient adherence to anti - ad medical treatment compared to 64% of caregivers of patients on oral medications ( fig . did not report any difficulties in remembering to administer treatment compared to 73% of caregivers of patients on oral medications ( fig . both compliance to treatment and satisfaction were significantly higher in the group treated with the rivastigmine patch compared to the group on oral medications ( table 4 ) . nearly 90% of caregivers of patients on transdermal application versus 78% of caregivers of patients on oral medications reported a score between 9 and 10 at the cmi question on compliance . over 60% of caregivers of patients treated with the patch compared to 46% of caregivers of patients on oral medications reported a score between 9 and 10 at the cmi question on satisfaction . the translated version of the cmi showed a high level of internal consistency ( cronbach 's = 0.74 ) and a high test - retest reliability ( icc = 0.96 and = 0.94 for items assessing compliance ; icc = 0.98 and = 0.97 for items assessing satisfaction ) . convergent validity of the instrument compared with the vas was also high ( icc = 0.79 and = 0.72 for items assessing compliance ; icc = 0.92 and = 0.92 for items assessing satisfaction ) . findings from the axept study indicate that the use of a transdermal application may improve patient compliance to anti - ad treatment and caregiver satisfaction . these results are in line with previous experimental data which highlighted the benefits of a transdermal application for patients and caregivers . specifically , more than 70% of the 1,059 caregivers of ad patients included in the ideal trial preferred the patch over capsules for drug delivery . caregivers reported that their preference for the patch was based on the ease of use and following the schedule , which was eventually related to spare of time and reduced workload . the only previous large observational study investigating caregiver satisfaction with drug treatment in ad suggested that donepezil orally disintegrating tablets may have been preferred by caregivers ; however , this study did not include patients on rivastigmine patch . such results appear to support the current hypothesis that new effective drug formulations may be preferred by caregivers of ad patients due to the ease and convenience of administration . it has been shown that the use of the rivastigmine patch is associated with reduced side effects and access to optimal dosages with possible improved efficacy due to constant drug delivery , steady plasmatic drug levels and prolonged cholinesterase inhibition . in general , the patch represents a user - friendly mode of drug delivery in geriatric patients who are likely to present with multimorbidity and complex polypharmacy . the potential for overdose due to erroneous multiple administrations may be reduced by using a transdermal application . finally , the patch provides caregivers with a visual reminder that the medication has been correctly administered , thus helping improve patient compliance to treatment . all these advantages may relieve the strain among caregivers in managing drug administration while improving treatment compliance and effectiveness . recently , the who has recommended studies focusing on the role of the caregiver and particularly investigating the level of caregiver satisfaction in relation to daily workload and drug administration . also , the fda has stated that effective health outcomes in ad should be identified taking into account the needs of both patients and caregivers . in this respect , the caregivers perspective may provide unique insight into clinical research because of their own experience with this condition . cognitive and functional impairment , behavioral symptoms and susceptibility to side effects make compliance to drug regimens in ad patients particularly challenging . it has been estimated that over 70% of ad patients require assistance in managing and taking medications . the development and implementation of strategies to help caregivers administrate medications to their patients may indeed reduce the strain related to such tasks and increase their level of satisfaction with the care delivered . a reduced level of caregiver burden and distress has also been associated with a decreased risk of developing behavioral symptoms in ad patients and with an overall improved quality of life for their families . such positive effects result in a more prolonged in - home care provision for patients , with an overall reduction of costs of institutionalization . the study population included patients with multimorbidity who were relatively preserved in their functional abilities , as it may have been expected in consideration of their mild - to - moderate level of cognitive impairment . cardiovascular diseases and metabolic conditions such as diabetes and dyslipidemia were highly prevalent in our study patients , and this is in accordance with a growing body of literature which indicates that ad patients are likely to have an even worse cardiovascular and metabolic profile compared to elderly individuals who are not cognitively impaired . the management of comorbidities represents an additional contribution to the overall burden of care , requiring caregivers to monitor medical symptoms and signs and administer complex drug therapy regimens . in our study patients , the time interval between first appearance of symptoms and ad diagnosis was nearly 2 years . this estimate is in line with previous european data indicating that for a clinical diagnosis of ad nearly 3 years are required in the uk , 2 years in france and spain and less than 1 year in germany . the role of families and general practitioners has been recognized as crucial to the process of early identification of ad symptoms , and public health interventions to target possible barriers to an early diagnosis have been proposed . an early diagnosis allows to provide medical treatments which are most effective in the early stages of the disease , to implement adequate multidisciplinary interventions and to offer tailored socioeconomic support and services to patients and their families . the sociodemographic profile of caregivers of community - dwelling patients with mild - to - moderate ad has been described in this study . caregivers are likely to be women of middle or old age , mostly daughters or spouses , living with the patients . they spend half a day , 7 days per week in providing assistance to the patients . this is in line with previous epidemiological data indicating that family caregivers are those who provide most of the care and social support to the ad patients , thus being daily confronted with the psychological and socioeconomic burden of the disease . the needs of these people are among the most urgent public health issues of our society and need to be addressed by tailored interventions aimed at providing social , financial and psychological support . the cross - sectional study design does not make possible to establish any temporal relationship between the exposure to a particular drug formulation and the level of compliance to treatment and caregiver satisfaction reported . data on concomitant medications other than psychotropic drugs were not collected . also , no information was gathered regarding the presence and severity of behavioral and psychological symptoms of dementia . the instrument used to assess the patient compliance and caregiver satisfaction is based on a report of the caregivers perspectives , which may not necessarily correspond to the patients opinions . although all caregivers in the study reported to administer medications to their patients , it is not possible to exclude that some patients may have self - administered treatments . finally , results have been derived from a sample of community - dwelling mild - to - moderate ad patients and their caregivers in italy and they may not be extendable to other populations . the current study has provided evidence regarding patient compliance to and caregiver satisfaction with ad pharmacological treatments derived from the observation of a real clinical setting . caregivers of patients treated with a transdermal application appeared to be more satisfied and reported higher levels of compliance to treatment than caregivers of patients receiving anti - ad oral medications . these findings indicate that new convenient modes of drug administration may be appreciated by caregivers and may contribute to reduce their daily workload , thus producing beneficial effects for the patients and their families . clinicians should consider such evidence together with efficacy and safety data and the patients individual clinical profile when choosing the best medical treatment option for persons with ad . the following questions ask about your opinion about the capsule medication used by the patient with alzheimer 's dementia for whom you provide care . please answer all of the questions as honestly as you can and without help from anyone . taking your medication in the dose and at the times prescribed can be very important to the action of the medication . during the past 1 week of therapy with the oral medication for alzheimer 's dementia , to the best of your knowledge : the following questions ask about your opinion about the treatment received by the patient with alzheimer 's dementia for whom you provide care . please answer all of the questions as honestly as you can and without help from anyone . taking your medication in the dose and at the times prescribed can be very important to the action of the medication . during the past 1 week of therapy with the patch medication for alzheimer 's dementia , to the best of your knowledge : roberto bernabei from universit cattolica del sacro cuore , roma ; daniela gragnaniello from universit sant'anna , ferrara ; emilio luda di cortemiglia from ospedale di rivoli , rivoli ; paolo maria rossini from universit cattolica del sacro cuore , roma ; emanuele cassetta from ospedale fatebenefratelli , roma ; alfonso iudice from ospedale santa chiara , pisa ; luca cipriani from istituto nazionale riposo e cura per anziani ( inrca ) , roma ; alberto pilotto from casa sollievo della sofferenza , san giovanni rotondo ; luigi di cioccio from ospedale santa scolastica , cassino ; claudia bauco from servizio geriatrico integrato dottore angelico , aquino ; maria lia lunardelli from policlinico s. orsola malpighi , bologna ; niccol marchionni from universit degli studi di firenze , firenze ; vito ferrara from ospedale regionale f. miulli , acquaviva delle fonti ; mario barbagallo , from universit di palermo , palermo ; maurizio russotto from ospedali riuniti villa sofia cervello , palermo ; salvatore ferrara from azienda sanitaria provinciale di siracusa , siracusa ; massimo franceschi from multimedica holding , castellanza ; giancarlo comi from fondazione san raffele del monte tabor , milano ; carlo valente from azienda ospedaliera usl 4 , prato ; antonio lacetera from ospedale santa croce , fano ; evelina bianchi from ospedale civile san bortolo , vicenza ; domenico consoli from ospedale g. jazzolino , vibo valentia ; flavio devetag from ospedale santa maria del prato , feltre ; piero secreto from casa di cura beata vergine della consolata fatebenefratelli , san maurizio canavese ; marinella turla from ospedale vallecamonica , esine ; gianpaolo ben from ospedale civile san martino , belluno ; francesco saverio caserta from assistenza anziani asl napoli 1 , napoli ; fabio di stefano from asl vco omegna , omegna ; massimo zanasi from ospedali riuniti , foggia ; ciro mundi from ospedali riuniti , foggia ; mauro minervini from opera don uva , bisceglie ; sandra fanfoni from ospedale nuovo regina margherita , roma ; carlo serrati from ospedale san martino , genova ; stefania boschi from policlinico le scotte , siena ; cristina paci from ospedale civile madonna del soccorso , san benedetto del tronto ; leonardo cocito from universit degli studi di genova , genova ; carlo de lena from universit degli studi di roma la sapienza , roma ; lucilla parnetti from universit di perugia , perugia ; lino pasqui from ospedale civile di monselice , monselice ; maria giovanna marrosu from policlinico universitario di monserrato , monserrato ; edoardo dal monte from ospedale civile salute degli infermi , faenza ; antonio tetto from ospedale san leopoldo mandic , merate ; giovanni giannelli from centro esperto asl cesena , cesena ; stefano viola from ospedale civile san pio da pietralcina , vasto ; carlo sabba from ospedale policlinico consorziale , bari , italy .

background / aimsaccording to experimental data , a transdermal application is preferred by caregivers of alzheimer 's disease ( ad ) patients compared with oral medications . the axept study compared compliance to treatment among community - dwelling patients with mild - to - moderate ad treated with transdermal application compared to oral medications and caregiver satisfaction in a real clinical setting.methodsdata from 45 memory clinics in italy were collected between september 8 , 2010 and january 31 , 2011 . compliance to treatment and caregiver satisfaction were measured using the caregiver medication interview.resultsa total of 855 ad patients and their caregivers participated in the study . nearly 80% of caregivers of patients on patch were not concerned about adherence to treatment compared with 64% of caregivers of patients on oral drugs . among caregivers of patients on patch , 94% did not report any difficulties in remembering to administer treatment compared with 73% of caregivers of patients on oral medications . the highest level of compliance and satisfaction was reported by caregivers of patients on transdermal application.conclusioncaregivers of patients treated with a transdermal application appeared to be more satisfied and reported a higher level of compliance than caregivers of patients receiving anti - ad oral medications .

thirty - three eyes of 33 patients who underwent implantation of pc iols into the ac between may 2006 and july 2008 were evaluated retrospectively . fourteen eyes had inadequate support due to posterior capsular rupture during phacoemulsification , and 19 eyes were previously aphakic . the procedures were performed under topical or subconjunctival anesthesia . if the remaining capsular support was considered to be insufficient for iol implantation during phacoemulsification , an anterior vitrectomy was performed and acetylcholine was injected into the ac for miosis . the vacuum level of the vitrectomy was set to 200 mmhg and the frequency to 50 cuts / min . two iridectomies were performed on the midperipheral iris with a vitrectomy cutter at the 7 and 1 oclock positions . the iols were implanted in the ac with the haptics passing through the iridectomies to the pc . the first five patients received polymethylmethacrylate ( pmma ) iols ( aurolab , madurai , tamil nadu , india ) , while single piece foldable iols ( ocuva , vsy , istanbul , turkey ) were implanted in the remaining 28 patients . ( a ) intraoperative posterior capsule rupture and desantralization of the sulcus - fixated iol . ( d - f ) using the same probe at 50 - 100 mmhg vacuum , two iridectomies were created at the 1 and 7 oclock positions . ( g and h ) iol haptics were implanted into the iridectomies using a dialer with the aid of ocular viscoelastic substance . ( i - o ) suturation of the inferior and superior iol haptics to the iris using mccunnel 's technique the procedures were performed under topical or subconjunctival anesthesia . if the remaining capsular support was considered to be insufficient for iol implantation during phacoemulsification , an anterior vitrectomy was performed and acetylcholine was injected into the ac for miosis . the vacuum level of the vitrectomy was set to 200 mmhg and the frequency to 50 cuts / min . two iridectomies were performed on the midperipheral iris with a vitrectomy cutter at the 7 and 1 oclock positions . the iols were implanted in the ac with the haptics passing through the iridectomies to the pc . the first five patients received polymethylmethacrylate ( pmma ) iols ( aurolab , madurai , tamil nadu , india ) , while single piece foldable iols ( ocuva , vsy , istanbul , turkey ) were implanted in the remaining 28 patients . ( a ) intraoperative posterior capsule rupture and desantralization of the sulcus - fixated iol . ( d - f ) using the same probe at 50 - 100 mmhg vacuum , two iridectomies were created at the 1 and 7 oclock positions . ( g and h ) iol haptics were implanted into the iridectomies using a dialer with the aid of ocular viscoelastic substance . ( i - o ) suturation of the inferior and superior iol haptics to the iris using mccunnel 's technique the mean age of the 16 men and 17 women was 56.3 12.2 years . fourteen eyes had inadequate posterior capsular support due to posterior capsular rupture during phacoemulsification and underwent primary iol implantation . nineteen eyes that had previously been left aphakic for various reasons underwent secondary iol implantations . mild corneal edema , which resorbed in 5 - 7 days , was detected in eight of 14 patients with primary iol implants . pupillary block developed in two of the first five patients who had received a pmma iol , and neodymium - doped yttrium aluminum garnet ( nd : yag ) laser iridotomies were performed to relieve the block in these eyes . one of the patients with a pmma iol presented with dislocation of the haptic into the ac . the haptic was resutured to the iris with a 10/0 nylon suture for this patient . none of the patients had longlasting corneal edema , iris atrophy , uveitis , corneal contact with iol , or retinal detachment during the follow - up period . pigment dispersion was seen in four patients ; however , we did not document any pigmentary glaucoma . while mild guttata was seen in five eyes during the follow - up period , none of the eyes had corneal decompensation . a marked increase in visual acuity was observed in the patients with posterior capsular rupture during surgery compared to the preoperative levels ( 20/50 versus 20/25 ) , while visual acuity increased moderately from 20/40 to 20/30 in those who were previously aphakic and underwent secondary iol insertion . posterior capsular rupture is also a well - known intraoperative complication of cataract surgery . in the presence of a posterior capsular tear , an iol can be placed in the sulcus if the capsular rim is available , or in the bag if the tear is small . if the remaining capsule does not offer sufficient support for the iol implantation , the surgeon may choose one of following options : ac iol , iris - claw iol , iris - sutured iol , pc iris - sutured iol , sutureless iol with iris anchors , or scleral - fixated lol . ac iols and scleral - fixated iols are generally recommended when capsular support is insufficient or absent . complications associated with ac iols , including postoperative inflammation , pupillary transformation , glaucoma , and the loss of corneal endothelial cells have been reported . even though there is no contact between scleral - fixated iols and the fine structures of the ac angle and corneal endothelium , these iols also have some drawbacks , which limit their usage . recently , totan and karadag reported sutureless scleral fixation of a three - piece foldable iol using 25-gauge transconjunctival sutureless vitrectomy trocars in patients with insufficient posterior capsule support . iris - claw iols may be a good alternative , however higher costs limit their extensive usage . by implanting a pc iol in the ac through two iridectomies , the optics and haptics of the iol are placed in positions that are far from the corneal endothelium and the ac angle . we observed that patients had transparent corneas , centralized iols , and iol haptics that fitted into the iridectomies throughout the follow - up period . the number of patients with complications was very small , and the complications were negligible and not vision - threatening . we also found that primary iol implantations gave better postoperative visual results compared to secondary implantations . the main limitation of the study is the lack of availability of data associated with corneal endothelium pre- and postoperatively . we created the iridectomies at one and seven oclock positions 180 apart from the midperipheral iris . , iol dislocation may be due to large iridectomies , loose sutures or suture release , or the inappropriate positions of opposed iridectomies . however , iol dislocation in this technique can easily be overcome by resuturing the haptic into position . the surgical correction of an iol dislocation in our technique is less traumatic and complicated than that for sclera - fixated or posterior iris - fixated iols . this study suggests that this technique is a practical alternative that leads to favorable visual outcomes and minimized risk of complications in eyes without adequate capsular support . however , a long - term study on a large population is required to confirm these findings .

we aimed to evaluate the implantation of a posterior chamber intraocular lens ( iol ) in the anterior chamber ( ac ) with the haptics passing through two iridectomies to the posterior chamber . a total of 33 eyes of 33 patients with inadequate posterior capsular support due to either previous aphakia or posterior capsular rupture during cataract extraction were included in the study . a double iridectomy was performed on all patients using a vitrectomy probe on the midperiphery of the iris . iols were implanted in the ac , and the haptics were passed through the iridectomies to the posterior chamber . the mean follow - up time was 25.3 months . ac hemorrhage occurred in five patients during the iridectomy procedure . corneal edema was detected in eight of 14 patients with primary iol insertions . haptic dislocation was detected in only one patient . this technique may be a good alternative to scleral - fixated iol implantation in eyes with aphakia .

malaria remains one of world s major infectious diseases and an impediment to economic development . one third of the world s population are at risk of infection , around 250 million people develop clinical infections annually , and at least half a million die each year ; most are children aged under five years . the disease is caused by infection of circulating red blood cells by one of five species of the protozoan parasite , plasmodium ; p. falciparum causes most morbidity and mortality . the majority of antiplasmodial drugs are now severely compromised due to acquired resistance by the parasite , and the development of efficacious vaccines remains a challenge . novel approaches to treatment and a greater understanding of disease pathogenesis and the host response to infection are desperately needed . outcome to malaria infection is determined largely by the increasing parasite mass and the response of the host to the infection . importantly , less than 1% of infections progress to life - threatening stages , underscoring the efficacy of host protective mechanisms . the innate protective response limits parasite growth early in an infection in a non antigen - specific manner . it also allows time for the subsequent development of an adaptive response , which is capable of clearing the infection and protecting against clinically symptomatic malaria . it is also antigen - specific , and several years and many exposures are needed to build an effective immunity against the multitudinous array of parasite antigens in any given endemic region . innate immune mechanisms are therefore crucial in all malarial infections to buffer against the early growth of blood - stage parasites . we recently reported that platelets are an important component of the host innate immune response against malaria infection . in addition to their well - defined role in hemostasis , platelets are increasingly implicated in immunological processes , including direct pathogen - killing functions ( reviewed by yeaman and colleagues , ref . they express receptors that bind host immune response modulators ( e.g. , antibodies and cytokines ) and toll - like receptors that bind microbial products . they also express the cd154 co - stimulatory molecule and influence the development of adaptive immune responses . importantly , their location in the circulation makes them ideal sentinels against any nascent infection . platelets respond to a variety of microbial cells by releasing immunomodulatory molecules and by directly killing microbial pathogens . malaria infections are commonly accompanied by a thrombocytopenia or loss of platelets , the severity of which closely mirrors the increasing parasite mass . it is now clear from our study and others that platelets protect the host during erythrocytic infection . mice with pre - existing platelet deficiencies are more susceptible to infection and exhibit higher loads of viable parasites . treatment of normal mice with aspirin , a platelet activation and aggregation inhibitor , also reduces survival to infection . pre - treatment of these platelets with inhibitors ( including aspirin ) blocks the parasite killing effect . platelets bind preferentially to p. falciparum ie , mainly through interactions between the platelet - expressed scavenger receptor protein , cd36 and the p. falciparum erythrocyte membrane protein ( pfemp1 ) , produced by the parasite and trafficked to the erythrocyte surface . we believe that platelets are active early in infection to slow the initial growth of malaria parasites in the bloodstream , providing greater opportunity for other defense mechanisms to control the infection and ensure survival . more recent studies by our group and others have provided additional mechanistic and molecular insight to how platelets kill the intraerythrocytic parasite . central to these findings is a platelet derived cxc - type chemokine called platelet factor 4 ( pf4/cxcl4 ) , which is released by activated platelets and kills the parasite . approximately 25% of the protein released by platelets comprises of pf4 , and concentrations surrounding activated platelets reach high micromolar ranges . the parasite - killing activity of platelets appears to be entirely due to pf4 . neutralizing anti - pf4 antibodies completely block the activity of human platelet lysate and pf4-deficient platelets from mice fail to kill parasites . love and colleagues elegantly demonstrated that upon entering the cell , the protein relocates to the parasite digestive vacuole ( dv , site of hemoglobin digestion ) , resulting in specific lysis of the organelle and death of the parasite . therefore pf4 is a unique example of a host - derived molecule with direct plasmocidal activity . pf4 belongs to growing list of chemokine molecules called kinocidins , which have a remarkable capacity to function as both chemotactic and antimicrobial molecules . the different functions are localized to structurally distinct domains , and the latter at least is separable . chemotactic activity resides in the n - terminal portion of the protein where the classical cxc chemokine motif exists . this domain interacts with chemokine receptors on various target cells , leading to receptor - mediated signaling events . the c - terminal domain , rich in positive charged amino acids , mediates the antimicrobial effects of pf4 and other kinocidins . peptide scanning analyses have mapped the bactericidal and fungicidal activity of the rabbit pf4 ortholog , platelet microbiocidal protein 1 to the carboxyl terminal 15 residues , 5 of which are lysines . notably , love and colleagues showed that a peptide encompassing the carboxyl terminal 12 residues of human pf4 ( c12 ) had plasmocidal activity . this function was abrogated when three out of the four lysine residues were substituted to alanines . the charged , amphipathic nature of kinocidin c - terminal domains gives them propensity to interact with cell membranes . in bacteria , this has been variously shown to result in disruption of membrane potential , cell permeabilization and inhibition of protein and dna synthesis . consistent with these former effects , pf4 and c12 treatment of p. falciparum results in lysis of the dv . however , exactly how and why the dv is specifically targeted by pf4 is unclear , especially given that the molecule must cross three separate membranes in order to reach the dv ( red cell plasma membrane ( pm ) , parasitophorous vacuole membrane and parasite pm ) . pf4 binds to duffy , a red cell antigen receptor for chemokines ( darc / fy ) and our results show that platelet and pf4-mediated killing of intraerythrocytic parasites requires darc . the likely mechanism involves the binding of pf4 to darc on the infected cell surface , following by internalization of the pf4-darc complex . binding occurs via a domain on darc known to bind a number of other chemokines . also , other chemokines with a high affinity for darc out - compete the pf4 parasite binding and killing of parasites . the molecular details of how the complex may translocate into the parasite ( as well as the existence of the complex within the parasite ) remain to be demonstrated . but interestingly the uptake of host membrane proteins by intraerythrocytic plasmodium , including darc , has been documented . uptake of darc appears to be an active process and requires the tubovesicular membrane ( tvm ) network that derives from the parasitophorous vacuole membrane and connects the parasite with the erythrocyte pm . the tvm develops within the first 24 h following merozoite invasion and host proteins accumulate in conjunction with parasite maturation . uptake is also selective for certain host proteins , especially those that reside in cholesterol - rich detergent - resistant host membranes ; more abundant cytoskeletal molecules such as glycophorin - a , band-3 and glut1 are excluded . interestingly , establishment of the tvm and maximal accumulation of darc coincide with the parasite stage most sensitive to pf4 ( trophozoites ) . the intracellular fate ( and function ) of these imported host proteins remains to be determined . in the case of darc , details of parasite intracellular localization are not known . as well as establishing if pf4-directed lysis of the dv requires darc , it will be of interest to determine if darc translocates to the parasite dv membrane , and thereby provide a plausible mechanism for the delivery of pf4 from the activated platelet to this organelle . the protective role played by platelets in human malarial infections has yet to be directly answered , but there are a number of lines of evidence that support the affirmative . depletion of platelets ( or inhibition of platelet function ) in murine models of malaria where outcome is determined by bloodstream parasitemia ( as opposed to the inflammatory response - based cerebral malaria syndrome ) results in reduced survival . although a similar platelet depletion study is difficult to perform in humans , we do know that thrombocytopenia , which is a common clinical accompaniment of all malarial infections , has been correlated with a poor outcome in falciparum malaria . platelets have also been implicated as susceptibility factors in the development of cerebral malaria ( cm ) , a complex collection of syndromes specific to p. falciparum infections and a major cause of death . central to the pathophysiology of cm is the accumulation or sequestration of ie in the cerebral microvasculature , causing the obstruction of blood flow , leukocyte accumulation , localized intravascular inflammation , activation and damage of the endothelium and disruption of the blood - brain barrier . platelets are often found at sites of ie sequestration in both human cm and mouse cm models where they are believed to mediate ie binding in the microvasculature , and release molecules that affect endothelial cell viability and promote leukocyte adhesion . the parasite - killing activity of platelets may also contribute to the pathophysiology of cm . platelet - directed killing may moderate the local inflammatory responses to live parasites , or dead parasite toxins and exudate could contribute to the inflammatory and cell - damaging milieu . the effect of non - steroidal anti - inflammatory drugs , especially aspirin , may add an additional level of complexity in assigning the importance of platelets in human malaria . we showed previously that aspirin prevented platelet - mediated killing of cultured p. falciparum and reduced survival in p. chabaudi - infected mice . aspirin is cheap and readily available , and its usage as an antipyretic remains widespread ( although contraindications such as dengue fever and reyes syndrome have led to revisions on usage guidelines in many westernized countries ) . a recent cochrane review found that the available data are insufficient to conclude if aspirin ( as well as other antipyretics ) are beneficial or detrimental in malaria . therefore studies to access the impact of aspirin on malaria are highly warranted . the polymorphism responsible for the duffy - negative blood group , a t to c substitution in a gata1 promoter binding site , prevents erythrocytic expression of the duffy gene . the c allele is very common in african populations ; frequencies are close to 100% in most of central and western africa . our findings on the requirement for darc in killing of parasites by platelets have led us to speculate that platelet - mediated protection may be inadequate in duffy - negative ( darc deficient ) individuals . duffy - negativity provides protection against p. vivax infection by virtue of the requirement of the receptor for merozoite invasion ( although the simplicity of this assumption has been recently challenged ) , and p. vivax is rarely seen in africa . however since erythrocytic expression of duffy is also necessary for the platelet - mediated killing of p. falciparum parasites , we might expect to see increased severity or a poorer outcome to falciparum malaria in duffy - negative populations . at a broad level , this appears to be the case . using the best available global estimates , rates of infection and death due to p. falciparum are much higher in africa compared with other parts of world ( asia and south america ) , where duffy - negativity is rare . but many other factors also contribute to this difference , such as the type of parasite and its level of virulence , vector control measures , rates of host immunity , access to healthcare and other socioeconomic factors . in africa as much as half of malaria - associated deaths are attributable to cm . the failure of platelets to kill sequestered parasites growing in duffy - negative red cells may counter intuitively exacerbate this complication by allowing viable parasites to remain within the cerebral vasculature . importantly , studies are needed to test the relationship between duffy - antigen status and platelet protection where , ideally , p. falciparum disease severity , incidence and outcome would be compared within duffy - negative and positive individuals living in the same area and affected by the same parasite strains and other environmental factors . if a lack of platelet protection is indeed detrimental in duffy - negative individuals , it is challenging to reconcile evolutionarily why the duffy - negative polymorphism should remain common in africa . however the events that led to , and maintained , the duffy - negative allele in africans are likely to be complicated . it is widely held that the polymorphism arose through positive selection at a distant time in human evolutionary history to protect against , what was presumably then , a lethal form of p. vivax . compared with p. vivax , the advent of p. falciparum infection in humans appears to have been a more recent event . therefore fixation of the duffy - negative allele may have preceded p. falciparum in humans . there may also be other positive selective pressures for the polymorphism that we do not know about . the protective power of the platelet in malaria infection is only beginning to be appreciated . while convincing evidence of their importance in clinical infection is still needed , advances in understanding the molecular detail of parasite killing process have led to some very interesting findings . useful novel antimalarial molecules based on the pf4 platelet effector molecule are an exciting prospect . the findings may also help to explain why p. falciparum is a particular problem in africa . we must continue to direct our efforts to control this scourge of humankind , especially in this part of the world .

platelets , as well as regulating blood hemostasis , are an important component of the body s defense against invading microbial pathogens . we previously reported that platelets protect during malaria infection by binding plasmodium - infected erythrocytes ( ie ) and killing the parasite within . more recent studies have now revealed the platelet plasmocidal factor , platelet factor 4 ( pf4 ) and the red cell - expressed duffy - antigen molecule as the central players in the parasite killing activity of platelets .

diabetes is the most frequent cause of end - stage renal disease in industrialised countries [ 1 , 2 ] . clinically , diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate . this severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [ 3 , 4 ] . at present the most important identified risk factors are diabetes duration , arterial blood pressure , and glycaemic regulation . as evident from an increasing incidence of affected patients , however , there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy . clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events . the inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines , for example , il1 the impact of complement activation on the diabetic kidney may well , in part , be mediated through induction of cytokine response and inflammation [ 911 ] . several studies have linked diabetic late - complications to the complement system of the innate immune system [ 12 , 13 ] . the complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity . the activation of the complement system results in the release of multiple inflammatory signaling molecules . ultimately complement activation leads to the formation of pore - forming membrane attack complexes ( macs ) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress . however , in mammalian cells , it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation . furthermore , macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [ 16 , 17 ] . the latter effects of mac may explain the link between complement and diabetic kidney damage . three activations pathways exist : the classical , the alternative , and the lectin pathway . the present paper focuses on the lectin pathway , in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system , that is , mannan - binding lectin ( mbl ) , h - ficolin , l - ficolin , m - ficolin , and collectin - k1 . the three ficolins utilize a fibrinogen - like domain that binds , for example , n - acetylglucosamine , n - acetylgalactosamine , and n - acetyl - neuraminic acid , whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . a very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes . the observational design of the study , however , limits its ability to study a cause - effect relationship . when ficolins bind they all initiate activation of associated serine proteases ( mbl associated serine proteases , masps ) , which subsequently cleave the complement factors , c2 and c4 , leading to further complement activation [ 20 , 21 ] . eventually , complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . the balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . in diabetes , inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [ 2325 ] . it is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [ 24 , 25 ] . most significantly , an association is seen between diabetic nephropathy and the lectin pathway [ 2628 ] . we have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [ 29 , 30 ] . we speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes . this study aimed to investigate the impact of ficolin b ( the orthologue to human m - ficolin ) on the development of diabetic nephropathy in a mouse model of type 1 diabetes . we used 11-week - old , female ficolin b knockout mice and age - matched , female c57bl/6j bomtac wild - type mice ( taconic , ry , denmark ) . the knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background ( own breeding ) . in each cage there were three to eight mice and they had free access to tap water and standard chow ( altromin number 1324 ; lage , germany ) . the environment was stable with a 12-hour light - dark cycle , temperature at 21 1c , and humidity of 55 5% . the ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group ; thus four groups were made : ( 1 ) diabetic knockout mice ( n = 6 ) , ( 2 ) nondiabetic knockout mice ( n = 7 ) , ( 3 ) diabetic wild - type mice ( n = 11 ) , and ( 4 ) nondiabetic wild - type mice ( n = 11 ) . diabetes was induced by intraperitoneal injections of streptozotocin ( stz ) dissolved in a cold 10 mm citrate buffer ( doses of 55 mg / kg body weight , sigma aldrich , st louis , mo , usa ) on five consecutive days . the 18-week experiment was initiated when the mice were classified as diabetic ( blood glucose > 15 mm ) . animals with more than 15% sustained weight loss , signs of illness , or persistent ketonuria were excluded from the study . blood glucose was measured from tail vein by contour ( bayer diabetes care , kgs . lyngby , denmark ) . with combur test d strip ( roche diagnostics gmbh , mannheim , germany ) two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels . furthermore , two mice from the diabetic knockout group were excluded because of weight loss > 15% of body weight . the excluded mice were not included in the number of animals per group indicated above . spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals . the blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes ( sarstedt , nmbrecht , germany ) . the animals were anesthetized by an intraperitoneal dose of ketamine at 0.5 mg / g body weight and xylazine at 0.2 mg / g body weight ( ketaminol 4 vet and narcoxyl vet , resp . , urinary albumin excretion was determined by mouse albumin elisa quantification kit ( bethyl laboratories , inc . , urine creatinine was measured by isocratic high - performance liquid chromatography ( hplc ) on a zorbax scx300 column ( agilent , usa ) using a slight modification of a method first reported by yuen et al . . in brief , 5 l urine was added to 100 l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine . after 15 min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25 l 5 mm sodium acetate , ph 4.1 . duplicate samples ( 10 ul each ) were fractionated on a 50 mm 2.1 mm zorbax scx300 column with an in - front scx guard column . isocratic hplc was performed at a flow rate of 1 ml / min , and uv absorbance was monitored at 225 nm . a standard curve was created by including a 2-fold dilution series of creatinine anhydrous ( sigma aldrich ) . this study was designed with two independent factors ; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance . the main focus of interest was the interaction between the diabetic factor and the knockout factor ; that is , does ficolin b modify the effects of diabetes on the effect parameters ? if no interaction was found , the independent effects of diabetes and ficolin b on the kidney were estimated . for pairwise comparison , normal distributed data was tested with student 's t - test , whereas otherwise the wilcoxon mann - whitney rank sum test was used . data are given as mean ( 95% confidence interval ( ci ) ) unless else is stated . at baseline , the knockout mice on average weighed 20.0 g , which was slightly less than the wild type mice , 20.8 g ( p = 0.04 ) . no difference was found between the two diabetic groups or between the two nondiabetic groups ( table 1 ) . after 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status ( p < 0.001 ) . the nondiabetic mice weighed 3.2 g ( ci : 2.0 g4.3 g ) more than the diabetic mice . furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type ( p < 0.05 ) . as presented in table 1 , blood glucose , estimated as area under the curve ( auc ) , did not differ between the two diabetic groups ( p = 0.69 ) or between the two nondiabetic groups ( p = 0.13 ) . the kidney weight was equally increased in diabetic wild - type mice , 24% ( ci : 13%36% ) , and in the diabetic knockout mice , 29% ( ci : 12%47% ) , compared to the respective control groups ( figure 2(a ) ) . no interaction between knockout and diabetes was found ( p = 0.60 ) , indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy . the considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight . ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction ( p = 0.11 ) . furthermore , no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type , 1.95 mg / g , and the diabetic knockout , 2.89 mg / g ( p = 0.09 ) . the albumin - to - creatinine ration ( acr ) was higher among the diabetic wild - type mice , 76 mg / g ( ci : 50103 mg / g ) , compared to the nondiabetic wild - type mice , 44 mg / g ( ci : 2563 mg / g ) , p = 0.07 . similarly , the acr of diabetic knockout mice was 96 mg / g ( ci : 71122 mg / g ) compared to the nondiabetic knockout group , 34 mg / g ( ci : 2344 mg / g ) , p < 0.001 . as depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout , p = 0.21 . in the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for , or a crucial contributory factor in , the pathophysiology of diabetic nephropathy . in our study , the kidney weight and to some extent the acr were altered by diabetes as expected . the diabetes - induced increase in kidney weight , measured by comparing the diabetic mice with the nondiabetic mice , was not statistically different between the wild - type and ficolin b knockout mice . the diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice . taking the lower body weight of the knockout mice into account , the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice . similarly , the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age , body weight , and genetical background was a strength to the study , as the diabetes factor and the knockout factor were the only modulators of the outcome . most importantly both diabetic groups did reach and sustain blood glucose levels of above 15 mm . at study end , the body weight differed among groups , which impeded the analyses of the diabetic kidney damage , because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus . our study provides important new information on the association between the lectin pathway and diabetic kidney damage . we are the first to investigate the role of ficolin b ( which corresponds to ficolin m in human ) in the inflammatory response of diabetic nephropathy . in mice with deficiency of mbl , the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [ 29 , 30 ] . the fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system , but only deficiency of mbl has been shown to protect against diabetic kidney damage . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described . the function of other complement factors in the first parts of the lectin pathway ( e.g. , ficolin a and masps ) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies . one study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae , suggesting a synergetic immunological effect . this emphasises the need for further investigations involving both mouse ficolins . in order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy , an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation in conclusion , this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b.

background . the innate immune system may have adverse effects in diabetes and cardiovascular disease . the complement system seems to play a key role through erroneous complement activation via hyperglycaemia - induced neoepitopes . recently mannan - binding lectin ( mbl ) was shown to worsen diabetic kidney changes . we hypothesize that mouse ficolin b exerts detrimental effects in the diabetic kidney as seen for mbl . methods . we induced diabetes with streptozotocin in female wild - type mice and ficolin b knockout mice and included two similar nondiabetic groups . renal hypertrophy and excretion of urinary albumin and creatinine were quantified to assess diabetic kidney damage . results . in the wild - type groups , the kidney weighed 24% more in the diabetic mice compared to the controls . the diabetes - induced increase in kidney weight was 29% in the ficolin b knockout mice , that is , equal to wild - type animals ( two - way anova , p = 0.60 ) . in the wild - type mice the albumin - to - creatinine ratio ( acr ) was 32.5 mg / g higher in the diabetic mice compared to the controls . the difference was 62.5 mg / g in the ficolin b knockout mice , but this was not significantly different from the wild - type animals ( two - way anova , p = 0.21 ) . conclusions . in conclusion , the diabetes - induced effects on kidney weight and acr were not modified by the presence or absence of ficolin b.

similar experience is seen in malaysia with breast cancer being the most common cancer among women . advances in detection and treatment modalities have improved the survival rate of women with breast cancer . in malaysia , the 5-year survival rate among breast cancer patients has seen improvements over the past decades ; studies revealed a 5-year observed survival from 58.4% ( ci 0.540.63 ) to 75.7% ( ci 0.730.79 ) . because of its high prevalence and relatively good prognosis , the increase in numbers of survivors forms a growing area of clinical interests and research . a diagnosis of cancer can alter a person 's perspective on health and life itself . female breast cancer survivors are often weighed down by issues of physical lethargy , pain , breast sensitivity , and difficulty to concentrate which were associated with diminished physical functioning and emotional well - being . their psychological well - being was found to be affected by fear of cancer spread , recurrence , distress from surgery , fear of second cancer , and future tests . cancer survivors face a wide range of problems during and after their primary treatment which often persists in a chronic , long - term manner . during this period of survivorship , these survivors have multitude of needs which require attention and identification . recognizing these needs early in the cancer care continuum therefore , needs assessment should be carried out as it offers three advantages : ( i ) patient 's perceived needs are directly assessed , ( ii ) the level of need can be identified as well , and ( iii ) individuals or patient subgroups with higher level of needs can be identified . furthermore , understanding unmet needs among cancer survivors , across different age groups , gender , regions , cancer types , stages , survival durations , and various other factors , is in line with making healthcare patient - centered . with the growing number of breast cancer survivors in the milieu of limited healthcare resources , there is a pressing demand to uncover the unmet needs of these survivors so that health systems may prioritize its service delivery in order to be more effective and efficient . therefore , this study aims to describe the prevalence of unmet needs among breast cancer survivors in kuching , sarawak , and to assess relationship between their unmet needs and various associated factors . this descriptive cross - sectional study was conducted among breast cancer survivors recruited from the community - based nongovernmental organization ( ngo ) sarawak breast cancer support group ( sbcsg ) in kuching , sarawak . prior permission was obtained from the center in writing and all survivors in the support group were invited to participate in the study . written informed consent was obtained prior to the interview assisted survey , which was conducted at the center from january 2014 to june 2014 . this community - based approach , in which respondents were recruited , ensured that breast cancer survivors , whether they received treatment from public , private , or mixture of health facilities , were captured in this study . the supportive care framework by fitch recognizes that about 20% of cancer patients within the healthcare system have unmet needs . recent studies showed that cancer patients in taiwan and hong kong had prevalence of supportive care needs between 1733% and 1846% , respectively . therefore , in order to estimate the 20% prevalence of unmet supportive care needs ( moderate to high needs ) with a confidence interval of 95% and a 10% margin of error , an estimate sample size of 62 was required as per the equation of n = zpq / d , where z = 1.96 , p = 0.2 , q = 1 p , and d = 0.1 . the eligibility criteria for inclusion in the study were ( i ) adult malaysian females aged 18 years and above , ( ii ) diagnosed with breast cancer ( all stages ) , and ( iii ) physically and mentally able to participate . sociodemographic characteristics of respondents such as age , ethnicity , marital status , cohabitation status , household income , education , and employment status were included in the survey . medical characteristics such as age at diagnosis , duration of survivorship , cancer stage at time of diagnosis , and current treatment status were collected as well . the 34-item short - form supportive care needs survey ( scns - sf34 ) was adapted and employed in this study to identify the unmet supportive care needs among survivors . respondents have reported their preference for the scns questionnaire over other health - related quality of life questionnaires . furthermore , the scns - sf34 maintained the same constructs which were ( i ) psychological , ( ii ) physical and daily living , ( iii ) sexuality , ( iv ) health system and information , and ( v ) patient care and support , while preserving the psychometric properties of the original long - form scns . validation studies have shown that the cronbach 's alpha of the english version was 0.860.96 . the 34-item supportive care needs survey ( scns - sf34 ) offered an assessment of needs in cancer patients via five analytically derived domains : ( i ) psychological ( 10 items ) , ( ii ) physical and daily living ( 5 items ) , ( iii ) sexuality ( 3 items ) , ( iv ) health system and information ( 11 items ) , and ( v ) patient care and support ( 5 items ) . for each item , the respondents were required to indicate their level of need for help over the past one month in relation to having cancer . the level of need would be scored on a five - point likert scale : 1 , no need , not applicable ; 2 , no need , satisfied ; 3 , low need ; 4 , moderate need ; and 5 , high need . for each item , the respondents are dichotomized to as having moderate to high level of need if they scored options 4 or 5 or no to low level of need if they scored options 1 , 2 , or 3 . the items with highest percentage of respondents reporting the domain score was obtained by summing up the responses to each of the items within the domain and dividing the sum by the number of items in the domain . for the purpose of analysis , the sociodemographic and medical characteristics of respondents were organized and compared with the supportive care needs domain mean scores using independent t - test and one - way analysis of variance ( anova ) . the collected data was then entered and analyzed using the ibm statistical product and service solutions ( spss ) statistics program version 20 . inferential statistics were generated to answer the study objective based on p value of less than 0.05 ( p < 0.05 ) . this study was conducted with approval from the medical ethics committee , faculty of medicine and health science , universiti malaysia sarawak ( unimas ) , malaysia [ unimas / tnc(aa)-03.02/06 - 11 jld . a total of 101 female breast cancer survivors participated in this study . the average age of respondents was 57.9 ( sd 9.53 ) years . most of the respondents stay with 2 to 4 other persons at home ( 56.4% ) and have an average household income of between rm 3001 to rm 5000 ( 43.6% ) . majority of the respondents were unemployed ( 70.3% ) which include survivors who were housewives and retired from employment . the mean age at first diagnosis of breast cancer among the respondents in this study was 49.7 ( sd 8.45 ) years with 54.5% of the respondents diagnosed at the age 50 years and older . nearly two - thirds ( 63.4% ) of the respondents had a survival duration of more than 5 years . approximately 80% of the respondents had early stage ( stages i and ii ) breast cancer at time of diagnosis . among the respondents , 72.3% were no longer undergoing any active treatment at time of study and were only on annual outpatient department follow - ups . the other information on the sociodemographic and medical characteristics of the respondents is depicted in table 1 . domain mean scores are identified by summing up the responses to each of the items within the domain and dividing the sum by the number of items in the domain . a higher score ( maximum 5.00 , minimum 1.00 ) would indicate higher level of needs in the domain . table 2 revealed that health systems and information domain was found to have the highest mean score ( 2.48 ; 95% ci , 2.322.64 ) , followed by psychological domain ( 2.01 ; 95% ci , 1.912.12 ) and patient care and support domain ( 1.93 ; 95% ci , 1.832.03 ) . meanwhile , the sexuality domain has the lowest mean score ( 1.57 ; 95% ci , 1.441.70 ) . the top 10 items that respondents have indicated a moderate to high level of need for help are as depicted in table 3 . overall , 9 out of the top 10 items of unmet needs were from the health systems and information domain with only one item from the psychological domain . the highest ranked items were having one member of hospital staff with whom you can talk about all aspects of your condition , treatment , and follow - up ( 34.7% ) , being given explanations on those tests about which you would like to get explanations ( 29.7% ) , and having access to professional counseling ( e.g. , psychologist , social worker , counselor , and specialist nurse ) if you , family , or friends need it ( 27.7% ) . the ten items with lowest moderate to high level unmet needs among the respondents are as shown in table 4 . overall , the listed items had only less than 5% of the respondents indicated moderate to high level of need for help . five out of 10 of these items are from the psychological domain , followed by sexuality domain ( 2 items ) , patient care and support domain ( 2 items ) , and physical and daily living domain ( 1 item ) . items with the lowest percentage of respondents reporting needs were feeling down or depressed ( 1% ) , feelings of sadness ( 1% ) , changes in sexual feelings ( 1% ) , changes in your sexual relationships ( 1% ) , and more choice about which hospital you attend ( 1% ) . the supportive care needs ( scns ) domain mean scores are compared to the sociodemographic and medical characteristics of the respondents in table 5 . respondents age below 60 years ( n = 57 , 56.4% ) reported significantly higher mean score across physical and daily living domain ( p = 0.009 ) , psychological domain ( p = 0.002 ) , sexuality domain ( p < 0.001 ) , and patient care and support domain ( p = 0.038 ) compared to survivors aged 60 years and older ( n = 44 , 43.6% ) . ethnic malays and sarawak indigenous group recorded higher mean score in the physical and daily living domain ( p = 0.017 ) and sexuality domain ( p = 0.028 ) compared to the chinese respondents . survivors who are married reported higher mean score in the sexuality domain ( 1.69 , 95% ci : 1.541.84 ) compared to those who are never married / widowed / divorced / permanently separated ( 1.11 , 95% ci : 0.971.25 ) ( p < 0.001 ) . respondents with primary education level reported a significantly lower domain mean score compared to those with postsecondary or tertiary education level across the domains of physical and daily living ( 1.71 versus 2.19 , p = 0.012 ) , sexuality ( 1.33 versus 1.95 , p = 0.007 ) , patient care and support ( 1.76 versus 2.21 , p = 0.014 ) , and health system and information ( 2.37 versus 3.00 , p = 0.035 ) . respondents who were unemployed indicated a higher mean score in both the psychological domain ( 2.18 versus 1.94 , p = 0.042 ) and the patient care and support domain ( 2.13 versus 1.85 , p = 0.034 ) compared to those who were still employed . meanwhile , when comparing the scns domain mean scores to the medical characteristics and disease stages of the respondents in table 5 , significantly higher mean scores were reported by respondents with survival duration of 5 years or less compared to those who have survived more than 5 years in the physical and daily living domain ( p < 0.001 ) , psychological domain ( p < 0.001 ) , sexuality domain ( p = 0.019 ) , and patient care and support domain ( p = 0.008 ) . treatment status among respondents showed significant difference in mean scores across all 5 domains with survivors undergoing active treatment indicating a higher mean score compared to those who are not undergoing any current active treatment ( p value range from < 0.001 to 0.019 ) . there were no significant differences in supportive care needs domain mean scores among respondents with various cohabitation statuses , household income level , and age at diagnosis and between respondents who had early stage cancer compared to those with late stage cancer . the present study is a cross - sectional analysis of the perceived needs among breast cancer survivors in kuching , sarawak , malaysia . the survivors recruited were generally less than 60 years old , were mainly chinese , were married , received at least secondary level education , and were unemployed ( table 1 ) . the age at diagnosis ( mean age 49.7 ) and staging ( early stage , 80.6% ) were closely similar to various studies done in malaysia . the mean duration of survivorship which was 8.2 years and was higher in this study compared to 6.7 years in another local study ; this could be attributed to the fact that there were more later stage cases recruited from the hospital - based medical records in that study . the current study ( table 2 ) revealed that survivors indicated a higher mean score ( higher scores representing a higher level of unmet needs in the domain , range 1.005.00 ) in the health system and information domain ( 2.48 , 95% ci : 2.322.64 ) , followed by psychological domain ( 2.01 , 95% ci : 1.912.12 ) , and patient care and support domain ( 1.93 , 95% ci : 1.832.03 ) . meanwhile , sexuality domain was ranked lowest ( 1.57 , 95% ci : 1.441.70 ) . this finding is consistent with recent research among breast cancer survivors in singapore , hong kong , and korea which ranked the health system and information domain with the highest mean score . this tendency towards informational needs has been studied and established , with recent systematic reviews suggesting that asian women reported higher informational needs compared to western women . furthermore , the current finding could suggest a dissatisfaction among survivors with information received during follow - up care . based on individual item ranking ( table 3 ) , there is a greater need expressed within the health system and information domain compared to the other domains such as psychological domain . these findings however were in contrast to multiple australian - based research outcomes , in which cancer survivors ranked the psychological domain as area with highest level unmet needs [ 11 , 19 ] . such difference in prevalence has been established in recent systematic review and could be a reflection of underlying cross - continental cultural difference between survivors of asian compared to australian origin . researchers discovered a shift in perceived needs from informational needs to psychological needs among cancer survivors , citing improvements in information delivery over the years , which have led to the shift . assuming this trend holds true for cancer survivors worldwide , the current trend in this study indicates much needs to be done in addressing the informational needs of our survivors . nevertheless , a less emphasis on psychological domain in this study sample could be influenced by the fact that the study population was from a community - based breast cancer support group , in which their psychological and emotional needs would probably have been addressed . this finding highlights the importance of proper support group among cancer survivors , as well as having breast cancer survivors as support members . younger respondents aged below 60 years old reported higher level of unmet needs across all 5 domains compared to their older counterparts ( table 5 ) , with significant differences seen in the domains of sexuality , psychological , physical and daily living , and patient care and support . this observation is congruent with studies by researchers in singapore , which demonstrated that the level of need is higher among survivors aged below 60 years . similarly australian cancer patients aged 3160 years endorsed higher levels of unmet needs in sexuality , psychological , patient care and support , and health system and information domains compared to those who are 70 years or older . comparable conclusions were drawn from a qualitative study which explored the psychosocial needs of breast cancer survivors in which younger women reported a higher level of needs in terms of support , psychological , practical , physical and information needs . such disparity may reflect the difference in attitudes between younger adults who are more vocal of their unmet needs than older adults who believe that they should have better coping capacity and thus keep things to themselves . additionally , a diagnosis of cancer in younger adults may impart a sense of perceived loss in all areas of life , such as physical , psychosocial , emotional , sexuality , and the need for support , which give rise to higher magnitude of needs compared to older adults who may well have adjusted to changes in their life as they age . there were , however , no significant differences ( p = 0.643 ) in mean scores within the health system and information domain which may suggest that , across all age group , the informational need is ever present once a person is diagnosed with cancer . significant difference in mean domain scores was found between malays and sarawak indigenous groups compared to ethnic chinese survivors in both the physical and daily living ( 2.18 versus 1.84 , p = 0.017 ) and sexuality ( 1.81 versus 1.48 , p = 0.028 ) domains . this variation could be influenced by the fact that almost 75% of the respondents in this study were of ethnic chinese group , and the number of malays and sarawak indigenous groups were inadequate to be representative . in the absence of adequate research information , this finding is inconclusive . there may be an element of cultural background and variation in health beliefs which influence the level of needs in these domains . further exploration into this area is needed to understand these variations . comparison with other studies involving ethnic chinese survivors revealed similar lower scores in sexuality domain [ 9 , 14 ] and physical and daily living domain . lower sexuality domain scores could be due to underreporting of sexuality needs , as it is regarded as an intimate matter and not for discussion within the asian chinese community . specific cultural norms among the chinese could also account for lower physical and daily living needs , as it is believed that personal problems or outcry for help reflects badly on the family and may bring shame to the family name ; therefore , this may lead to a lower report for need in this domain among the chinese - majority sample of this study . higher level of unmet needs in the sexuality domain was endorsed by survivors who are married compared to others ( p < 0.001 ) . this was consistent with a korean study whereby married patients ( p = 0.03 ) were significantly more likely to indicate the need for help in this domain . furthermore , this finding makes intuitive sense , given that married survivors have spouse in whom they need to confront their sexuality needs more frequently , hence giving rise to needs . higher education attainment was associated with greater level of unmet needs among the respondents ( table 5 ) with mean domain scores being significantly different between respondents with primary level compared to postsecondary or tertiary level education in psychological , sexuality , patient care and support , and health system and information domains . this finding is in agreement with other studies which concluded that education level was found to be predictor of higher level of unmet needs among breast cancer survivors [ 25 , 26 ] . survivors with higher level of education are more aware and receptive of their underlying conditions , this has led to their need to actively seek ways to improve their current state of health , and the barriers which they face would be reflected in the domain scores . current study revealed that survivors who were unemployed reported a higher domain mean score across all 5 domains compared to those who were employed ( table 5 ) with significant differences reported in the psychological and patient care and support domains . could be complications from treatment of breast cancer with residual deficits , which would hamper their ability to work and thus be reported as unemployed . such disadvantage may increase their needs in the psychological and patient care and support domain . in this study , shorter duration of survivorship was significantly associated with higher domain mean score across all supportive care needs domains except the health system and information domain ( table 5 ) . this is in agreement with recent systematic review which suggests shorter time since diagnosis predicts higher level of needs . these findings can be attributed to the fact that newly diagnosed cancer patients had greater physical and emotional needs compared to those who were already receiving posttreatment follow - up care . this universal observation illustrates the need for concerted effort at addressing unmet needs across various domains early in cancer survivorship . furthermore , independent of survival duration , informational needs among current survivors are persistent throughout the continuum of survivorship care with no differences seen between younger or older survivors . this could be an indication highlighting the gap in information flow between providers and patients . closing this gap , a systematic approach in information delivery across the period of survivorship with focus on different aspect of needs and its change in relation to survival duration being under active treatment is significantly associated with higher mean scores across all 5 domains in this study ( p value range < 0.001 to 0.019 ) ( table 5 ) . this finding is consistent with other studies whereby patients under treatment reported high level of unmet needs while those in remission reported fewer unmet needs in the psychological , information , daily living , and patient care domains . additionally , recent findings among breast cancer survivors showed that those who were not in remission were associated with unmet needs in health system and information and patient care and support domains suggesting that these groups of survivors are likely to be receiving intermittent treatment and had to deal with symptom management . greatest difference in mean scores was observed in the physical and daily living domain ( 2.33 versus 1.77 , p < 0.001 ) . this difference can be attributed to the fact that survivors receiving active treatment were still coping with physical side effects of treatment modalities such as surgery and chemotherapy and hence had much greater need in this domain compared to those who were not on any active treatment any more . there was no significant difference in mean scores across all 5 domains in relation to cohabitation status of the survivors ( p = 0.0970.866 ) ( table 5 ) . this finding is different from other studies which concluded that generally cancer survivors living alone reported higher level of unmet needs , and breast cancer survivors living alone reported significantly more needs in area of patient care domain ( p = 0.044 ) . this could be influenced by the fact that the sampled survivors were recruited from a community - based breast cancer support group , and these survivors being part of this group have had some of their needs satisfied . household income level did not show any significant difference in any of the 5 domains mean scores . this finding differs from a korean study which concluded that less income predicted higher needs in physical and daily living domain ( p = 0.019 ) . it was suggested that having less income leads to financial barriers in terms of ability to satisfy daily personal care and physical needs . majority of these survivors receive their initial management and follow - up care from the public funded government health facilities which are considered to be relatively free , and therefore the income level within the current healthcare system does not make much impact . current study revealed that , in relation to cancer stage at time of diagnosis , there was no significant difference in mean scores across all domains ( p value range 0.627 to 0.964 ) . this however was different from other studies which reported that tumor size was significant in predicting needs in the psychological and health system and information domains . nevertheless , this study suggests that , independent of cancer staging at time of diagnosis , there are still unmet needs to be addressed , with highest mean score in the health system and information domain , early stage ( 2.48 , 95% ci : 2.302.66 ) and later stage ( 2.42 , 95% ci : 1.982.85 ) . the present finding may highlight a lack of understanding among survivors about the differences of cancer staging which also is indirectly reflected in the high level of unmet needs in the health system and information domain . on the other hand , there may be some form of underlying passive coping mechanism in play , whereby survivors choose to keep themselves occupied and not to think too much about their condition ; therefore the differences in staging ( early or later state ) do not weigh in on their unmet needs mean scores . first , it was conducted among survivors in a community - based support group , whereby participation was based on voluntary basis and representation from late stage ( stages iii and iv ) , that survivors may be less than expected . this could be attributed to the fact that late stage survivors have shorter life span and might not be in the best health to participate regularly in support group activities . second , the chinese - majority ethnic representation in this study , despite not being the majority population in the country , concurs with other local studies and characterizes the higher breast cancer incidence rate among the chinese in malaysia . third , the inherent limitation of being a cross - sectional study means that cause - effect relationships could not be assessed , and changes in unmet needs could not be evaluated over time . despites these limitations , this study has its strength in the fact that samples were drawn from community - dwelling breast cancer survivors which is more likely to represent their needs in the actual lived - in environment compared to samples obtained purely from hospitals . in conclusion , this study provided valuable insights into the characteristics of breast cancer survivors in kuching , sarawak , and their associated unmet supportive care needs with gaps in the informational domain emphasized . efforts should include the systematic delivery of health information which is targeted , culturally sensitive , and linguistically appropriate , especially to breast cancer survivors who were younger , had higher education attainment , were unemployed , had survivorship of 5 years or less , and were undergoing active treatment . future research should recruit samples from both community - based support group and survivors receiving treatment . a longitudinal exploration of the supportive care needs relative to survival duration is also recommended .

background . recognizing the needs of cancer survivors is one of the important aspects in healthcare delivery . this study aimed to determine the prevalence of unmet supportive care needs and its associated factors among the breast cancer survivors of community - based support group in kuching , sarawak . materials and methods . this was a cross - sectional study using supportive care needs survey ( scns - sf34 ) . all the members of community - based breast cancer support groups in kuching were invited . a total of 101 respondents were face - to - face interviewed after the consent was obtained . data was entered and analyzed using spss version 20 . results . the respondents endorsed health system and information domain with the highest mean score ( 2.48 ; 95% ci : 2.322.64 ) . top 10 items with moderate to high level unmet needs had a prevalence of 14.9% to 34.7% of respondents indicating need . significantly higher level of unmet needs was associated with survivors who were younger ( less than 60 years old ) , had higher education attainment , were unemployed , had survival duration of up to 5 years , and were undergoing active treatment . conclusion . systematic delivery of health information which is targeted , culturally sensitive , and linguistically appropriate for addressing younger age , education level , employment status , length of survivorship , and treatment stage should be considered not only at hospital - based setting but also at the community - based support groups .

in recent years there has been a shift toward minimally invasive surgical techniques . included in this shift has been the widespread adoption of laparoscopy as an alternative to the open operative approach in colon and rectal surgery . with the advancement of laparoscopic techniques compared with open surgery , laparoscopy has been shown to reduce intraoperative blood loss , length of incision , and length of hospital stay . as was seen with the movement from purely open surgery to laparoscopy , we are now witnessing a rising popularity and acceptance of robot - assisted procedures in a variety of surgical fields . the approval and increasing use of robotic equipment among surgeons stem somewhat from the technologic advancements that robotic surgery provides over traditional laparoscopic surgery . robotic surgery has been observed to be advantageous in its 3-dimensionally represented operating field , eliminating difficulties associated with depth perception as are seen with standard laparoscopy 's 2-dimensional image . in addition , a surgeon - operated camera allows for full control of the visual operating field , whereas the endowrist function ( intuitive surgical , sunnyvale , california ) provides increased articulation and rotation in a confined space . much of the controversy surrounding the robot pertains to the high cost and longer operative time associated with robotic procedures . the learning curve associated with new technology , as well as the increased time involved in docking ( and redocking in some cases ) , contributes to the overall operative time and , consequently , the cost of the procedure compared with traditional laparoscopy . in a study by park et al , a robotic right colectomy cost the patient > $ 3600 more out of pocket than a laparoscopic right colectomy . although some of the technical limitations of standard laparoscopy may obviously be addressed with the robotic apparatus , whether these advantages are enough to offset the higher cost of the robot are still debatable . furthermore , few studies have conclusively shown whether robot - assisted procedures yield different outcomes . specifically , studies conducted primarily in asia and europe have been published , but there still remains a paucity of studies in the united states comparing outcomes such as length of hospital stay , estimated blood loss , duration of the procedure , and complications between a robotic approach and laparoscopy for colon and rectal operations . the lack of comparative studies between robotic and laparoscopic approaches regarding specific outcomes , combined with the cost of new technology like the da vinci robot ( intuitive surgical ) , is a prohibitive factor for widespread adoption of the robot in many hospital centers . this study aims to compare and contrast our experience with robot - assisted and laparoscopic approaches to colon and rectal procedures to elucidate any differences in outcomes . we performed a retrospective review of 40 patients who underwent either standard laparoscopic or robotic colorectal surgery with the da vinci si robot ( intuitive surgical ) at tulane medical center , new orleans , louisiana , between january 2008 and february 2013 . patients were randomly assigned to undergo either a standard laparoscopic or robotic procedure based on the availability of the robot apparatus . indications for surgery were documented but did not factor into the inclusion or exclusion criteria for the study . the primary data points included operation time , estimated blood loss , length of stay , complications , and whether the procedure was converted to open . there was no standard protocol in place for advancement of the patients ' diet postoperatively . when malignancy was the indication for surgery , additional data points including histologic diagnosis , clinical stage , and number of nodes collected were noted . cases were performed by 1 of 2 authors ( or both ) . the student t test and test ( as well as the fisher exact test where appropriate ) were used to examine the association between each of the independent factors and outcomes for continuous and categorical variables , respectively . given the small sample size and varied procedure types , 2 subanalyses were performed for the aforementioned intraoperative and postoperative outcomes , one excluding procedures with concomitant liver resection and one solely for right hemicolectomy . all statistical analyses were performed by use of spss software ( version 19 ; ibm , armonk , new york ) . all procedures were performed by a single surgeon who had prior experience with the da vinci si robot in liver , spleen , pancreas , stomach , thyroid , and gallbladder procedures . in this series the surgical steps did not differ between the robotic and laparoscopic approaches , as has been described in previously published studies . the differences in port placement between the laparoscopic and robot - assisted procedures are to accommodate the robotic arms , in addition to an extra port(s ) for the surgeon 's assistant . port placement was standard for the type of procedure and for the approach ( laparoscopic vs robot assisted ) , as has been described previously . we performed a retrospective review of 40 patients who underwent either standard laparoscopic or robotic colorectal surgery with the da vinci si robot ( intuitive surgical ) at tulane medical center , new orleans , louisiana , between january 2008 and february 2013 . patients were randomly assigned to undergo either a standard laparoscopic or robotic procedure based on the availability of the robot apparatus . indications for surgery were documented but did not factor into the inclusion or exclusion criteria for the study . the primary data points included operation time , estimated blood loss , length of stay , complications , and whether the procedure was converted to open . there was no standard protocol in place for advancement of the patients ' diet postoperatively . when malignancy was the indication for surgery , additional data points including histologic diagnosis , clinical stage , and number of nodes collected were noted . cases were performed by 1 of 2 authors ( or both ) . the student t test and test ( as well as the fisher exact test where appropriate ) were used to examine the association between each of the independent factors and outcomes for continuous and categorical variables , respectively . given the small sample size and varied procedure types , 2 subanalyses were performed for the aforementioned intraoperative and postoperative outcomes , one excluding procedures with concomitant liver resection and one solely for right hemicolectomy . all statistical analyses were performed by use of spss software ( version 19 ; ibm , armonk , new york ) . all procedures were performed by a single surgeon who had prior experience with the da vinci si robot in liver , spleen , pancreas , stomach , thyroid , and gallbladder procedures . in this series the surgical steps did not differ between the robotic and laparoscopic approaches , as has been described in previously published studies . the differences in port placement between the laparoscopic and robot - assisted procedures are to accommodate the robotic arms , in addition to an extra port(s ) for the surgeon 's assistant . port placement was standard for the type of procedure and for the approach ( laparoscopic vs robot assisted ) , as has been described previously . sixty - five percent of the patients were male patients with a mean body mass index ( bmi ) of 28.0 5.8 , and 37.5% had a history of abdominal surgery . of the study population , 37.5% had a complication related to the operation . in terms of operative technique there were no significant differences between the groups in terms of age , bmi , or history of abdominal surgery . the mean age of patients undergoing laparoscopic surgery versus robotic surgery was 61.1 10.7 years versus 61.1 8.5 years ( p = .997 ) , the mean bmi was 28.9 6.3 versus 26.2 4.2 ( p = .158 ) , and the percentage with a history of abdominal surgery was 44.0% versus 26.7% ( p = .273 ) . there was a significant difference in terms of sex , with more patients being male and undergoing robotic surgery ( 86.7% vs 52.0% , p = .026 ) . there was no significant difference in surgical indication between the 2 groups ( p = .303 ) . eighteen patients underwent right hemicolectomy , 1 underwent a sigmoidectomy , 3 underwent abdominoperineal resection ( apr ) , and 3 underwent low anterior resection ( lar ) performed laparoscopically . in addition , 4 patients in the laparoscopy group underwent concomitant liver resection for metastases . in the robotic group , 7 patients underwent a right hemicolectomy , 2 underwent a left hemicolectomy , 1 underwent apr , and 5 underwent lar . patient characteristics of laparoscopic and robotic groups data are presented as mean ( standard deviation ) unless otherwise indicated . operative time was similar between the laparoscopic and robotic groups ( 190.8 84.3 minutes vs 258.4 170.8 minutes , p = .183 ) , with similar estimated blood loss ( 163.3 249.2 ml vs 96.8 157.7 ml , p = .385 ) . in the laparoscopic group , 16.0% of cases were converted to open compared with 20.0% in the robotic group ( p > .99 ) . the mean time until postoperative passage of stool was 4.6 1.9 days in the laparoscopic group versus 4.2 0.83 days in the robotic group ( p = .427 ) ; the mean time until the initiation of a regular diet ( 4.5 1.5 days vs 5.8 3.2 days , p = .159 ) was not significantly different between the 2 groups . furthermore , the mean length of stay after laparoscopic surgery was 9.6 7.3 days and was not different from the mean length of stay after robotic surgery ( 6.5 3.8 days , p = .091 ) . the rate of complications was similar for patients undergoing the laparoscopic approach versus those undergoing the robotic approach ( 36.0% vs 20.0% , p = .457 ) ( table 2 ) . of note , 1 patient undergoing the robotic procedure had an intraoperative splenic injury , which was repaired with splenorrhaphy . in addition , 1 patient a 67-year - old woman undergoing a robot - assisted apr intraoperative and postoperative outcomes data are presented as mean ( standard deviation ) unless otherwise indicated . the oncologic characteristics of the colorectal operations performed for cancer resection were also noted ( table 3 ) . there was no significant difference in tumor stage ( p = .4882 ) or histologic grade ( p > .99 ) between the 2 groups . in addition , there was no significant difference in the number of lymph nodes harvested between the laparoscopic group ( 14.0 6.5 ) and the robotic group ( 12.3 4.2 , p = .683 ) . pathologic characteristics for colorectal cancer operations the subanalysis for outcomes of procedures excluding concomitant liver resections is shown in table 4 . no liver resections were performed robotically ; thus the data in the subanalysis only varied in the laparoscopic arm . the duration of laparoscopic procedures averaged 175.2 67.6 minutes versus 258.4 170.8 minutes for robotic procedures ( p = .183 ) . furthermore , blood loss in the laparoscopic group ( 69.7 74.2 ml ) was not different from that in the robotic group ( 96.8 157.7 ml , p = .544 ) . for all other parameters rate of conversion to laparotomy , time to passage of stool , time until resumption of a regular diet , length of stay , and complications there remained no statistically significant differences or any large changes to the raw data . intraoperative and postoperative outcomes for operations excluding concomitant liver resection data are presented as mean ( standard deviation ) unless otherwise indicated . the mean operative time was 146.9 50.0 minutes in the laparoscopic group compared with 145.4 39.9 minutes in the robotic group ( p = .945 ) . mean blood loss was likewise not statistically different between the laparoscopic group ( 78.1 79.6 ml ) and robotic group ( 43.6 29.8 ml , p = .288 ) . the times to passage of stool were similar between the laparoscopic group ( 4.1 1.7 days ) and the robotic group ( 4.0 0.6 days ) , and the finding was not significant ( p = .902 ) . the remaining outcomes examined , including conversion to laparotomy , length of stay , and complications , continued to be similar to the aggregate analysis and were not statistically significant . intraoperative and postoperative outcomes for right hemicolectomy data are presented as mean ( standard deviation ) unless otherwise indicated . the subanalysis for outcomes of procedures excluding concomitant liver resections is shown in table 4 . no liver resections were performed robotically ; thus the data in the subanalysis only varied in the laparoscopic arm . the duration of laparoscopic procedures averaged 175.2 67.6 minutes versus 258.4 170.8 minutes for robotic procedures ( p = .183 ) . furthermore , blood loss in the laparoscopic group ( 69.7 74.2 ml ) was not different from that in the robotic group ( 96.8 157.7 ml , p = .544 ) . for all other parameters rate of conversion to laparotomy , time to passage of stool , time until resumption of a regular diet , length of stay , and complications there remained no statistically significant differences or any large changes to the raw data . intraoperative and postoperative outcomes for operations excluding concomitant liver resection data are presented as mean ( standard deviation ) unless otherwise indicated . the mean operative time was 146.9 50.0 minutes in the laparoscopic group compared with 145.4 39.9 minutes in the robotic group ( p = .945 ) . mean blood loss was likewise not statistically different between the laparoscopic group ( 78.1 79.6 ml ) and robotic group ( 43.6 29.8 ml , p = .288 ) . the times to passage of stool were similar between the laparoscopic group ( 4.1 1.7 days ) and the robotic group ( 4.0 0.6 days ) , and the finding was not significant ( p = .902 ) . the remaining outcomes examined , including conversion to laparotomy , length of stay , and complications , continued to be similar to the aggregate analysis and were not statistically significant . intraoperative and postoperative outcomes for right hemicolectomy data are presented as mean ( standard deviation ) unless otherwise indicated . interest in use of the robot in colorectal procedures has increased in recent years . for any new operative technique to become an accepted alternative to traditional methods for instance , studies have emerged since the adoption of laparoscopy for colorectal operations that have shown that it can yield a decreased length of hospital stay , oncologically adequate resection , and no differences in postoperative complications or in - hospital deaths when compared with a traditional open approach . because of studies like these , laparoscopy is now considered an acceptable alternative to an open approach in colorectal resection . our current experience shows many of the similarities between patients undergoing laparoscopic colorectal surgery and those undergoing robotic - assisted colorectal surgery and suggests that robot - assisted colon and rectal surgery is a safe and feasible alternative to the conventional laparoscopic approach . this study was conducted in groups of patients that were similar in terms of demographic characteristics and indications for operation . there was no significant difference between the robotic and laparoscopic groups in terms of age , bmi , or history of abdominal surgery . regarding operative outcomes , no significant difference was noted between the 2 groups in terms of complication rate , estimated blood loss , conversion to open procedure , or length of hospital stay . return of bowel function and resumption of a regular diet were also noted to be similar between the 2 groups . across all operations and approaches , the number and location of ports did not appear to have a clinically observable effect on intraoperative and postoperative outcomes including the need to convert to laparotomy , complications , and length of hospital stay . consequently , this study of well - matched patients establishes the equivalence in outcomes that can be achieved between laparoscopic and robotic approaches to colon and rectal surgery . the 2 subanalyses sought to create better - matched patient groups and decrease the large standard deviations as a result of aggregating a broad range of procedures . two ways of accomplishing this were to ( 1 ) eliminate procedures with concomitant liver resection and ( 2 ) include only right colectomies , the most commonly performed procedure in our study . in the subanalysis of outcomes , excluding cases with concomitant liver resection yielded a large drop in estimated blood loss , as well as duration of the procedure . however , there continued to be no statistically significant difference in any of the outcomes . likewise , although the subanalysis of right colectomies showed nearly identical operative times , greatly decreased blood loss ( laparoscopy - associated blood loss again greater than robotic ) , and smaller standard deviations , none of the differences were significant . thus , despite attempting to better match the patient groups , the results remained similar between the laparoscopic and robotic procedures . our results are similar to those shown in the randomized clinical trial by park et al that compared outcomes of robot - assisted and laparoscopic approaches for right - sided colon cancer . they showed no difference in complications , blood loss , conversion to open surgery , time to passage of flatus , or time to resume a regular diet between the 2 groups . other studies , by rawlings et al and d'annibale et al , have also shown similar findings . in the study by rawlings et al , when examining right colectomy operations , no difference was seen between the laparoscopic and robot - assisted groups in terms of length of hospital stay and estimated blood loss . at present , the literature shows conflicting results in terms of the duration of the operation between the 2 surgical techniques . similar to our findings , d'annibale et al noted no significant variation in operative times between the robotic and laparoscopic groups . in contrast , park et al found that the duration of the operation was significantly shorter in the laparoscopic group compared with the robot - assisted group ( 130 minutes vs 195 minutes , p < .001 ) . likewise , rawlings et al noted a significant difference in operative time ( mean of 169 minutes in laparoscopic group vs 219 minutes in robotic group , p = .002 ) . further well - matched studies are needed to truly elucidate whether there is a significant difference in operative times between the 2 surgical approaches . whether patients had a history of abdominal surgery the fact that patients in both study arms had similar histories of abdominal operations suggests that prior abdominal surgery is not a contraindication to robotic - assisted surgery . in addition , the rate of conversion to an open procedure was not significantly different between the laparoscopic and robot - assisted groups . this finding is comparable with results reported in studies by park et al and trastulli et al , both of which examined patients undergoing robotic right colon resections . in the comparative study by park et al , there was no difference in the number of cases converted to an open procedure : none of the 35 robot - assisted cases were converted to open surgery ; nor were any of the 35 laparoscopic cases . likewise , in the study by trastulli et al examining a consecutive series of 20 patients , none were converted to open surgery . in our study , none of the robotic right colon resections were converted to an open procedure , whereas in the laparoscopic group , only 1 patient , who also underwent concomitant liver resection , was converted to open surgery . in contrast to the aforementioned results , a study of rectal cancer resections in 84 patients ( laparoscopic in 37 and robot assisted in 47 ) by baek et al found a significantly higher rate of conversion to open surgery in the laparoscopic group compared with the robotic group ( p = .020 ) . together , these findings indicate that conversion to an open procedure likely occurs at similar rates between the 2 approaches and that there may in fact be superior outcomes after a robotic approach for colon and rectal surgery . when operations are performed for cancer resection , new techniques must not only be safe but also provide a comparable outcome for oncologic resection . in this study we performed a subanalysis comparing laparoscopic versus robotic surgery that examined oncologic outcome . there was no difference in the number of lymph nodes harvested in cases performed as a result of colorectal malignancy , and both approaches yielded an average number of lymph nodes > 12 , proving the techniques oncologically sufficient . similarly , studies by d'annibale et al and park et al reported no difference between the approaches in harvesting nodes . in these studies , robot - assisted resection obtained , on average , 17 nodes and 29.9 nodes , respectively the series of 20 patients undergoing robotic right colectomy reported by trastulli et al also showed oncologic sufficiency by harvesting an average of 17.6 lymph nodes . although the technical limitations of standard laparoscopy are addressed with the robotic apparatus by using a 3-dimensional field and improved instrument articulation and rotation , there remain certain disadvantages to consider . because robot - assisted operations are still relatively new , the process to set up and dock robotic arms increases the length of time in the operating room . even with an experienced team , the prolonged time is a drawback compared with conventional laparoscopy . perhaps more important than operating room time , however , is the duration of the procedure because it is of greatest importance to the patient that prolonged risks of anesthesia are mitigated . it has been shown that colorectal surgery is associated with cardiac complications that are exacerbated by longer operative times and are associated with mortality rates of 20% to 40% . the single patient death in this study , as a result of cardiac arrest , occurred in a 67-year - old woman undergoing a particularly complicated robotic apr for squamous cell carcinoma of the anal canal that lasted 492 minutes . as was seen in this study , however , as well as other studies of right colectomies , the duration of the operation overall was not significantly different between the laparoscopic group and the robotic group . when the robotic operative time is averaged after excluding this apr case , the time is 240.5 160.3 minutes , a nonsignificant difference from the laparoscopic time . this time is similar to average operative times for robotic colorectal procedures published in the current literature . although some surgeons may question , on the basis of this instance of death after a robotic apr , the safety of the robot as a surgical modality , studies have shown low mortality rates . a systematic review by kanji et al of robot - assisted colorectal procedures encompassing 854 patients yielded no reported deaths . similarly , salman et al , in an assessment of the nationwide inpatient sample , found a mortality rate of 79.1 per 10 000 laparoscopic colorectal procedures compared with a rate of 0.0 per 10 000 robotic cases ( p < .05 ) . thus , despite the single case of death in our study , both laparoscopic colorectal surgery and robotic colorectal surgery have been shown to have low in - hospital mortality rates . multiple studies have shown that robot - assisted surgery is significantly more costly than standard laparoscopic surgery . we did not analyze cost in our study , but prior publications showed a greater expense of thousands of dollars when using the robotic approach compared with traditional laparoscopy . in addition to cost being a limitation to the robotic approach in hospitals , robotic surgery also results in a greater out - of - pocket expense for patients . although many studies , including our study , have shown that in most aspects , outcomes are similar between both modalities , significant advantages and benefits to the robotic approach over the laparoscopic approach may be necessary to justify the use of the robot in some centers . in other centers , however , the standardization of robotics in surgery may lead to cost savings over time . the small sample size and retrospective nature of our study present some inherent limitations . at our institution , the robotic approach to colorectal surgery has not become widely adopted , and as such , there are fewer of these procedures being performed . however , this study and its exploratory nature serve as preliminary evidence in the comparison of laparoscopic and robotic colorectal surgery through an appropriate design . at the same time , this study establishes a basis for larger - scale investigation with wider financial and organizational resources . the study design was limited by the availability of the robot and could be remedied by a future prospective randomized study . as mentioned previously , the additional costs incurred when using the robot are a prohibitive factor , especially at an urban academic medical center such as ours that typically treats underinsured patient populations . furthermore , right hemicolectomy was the predominantly performed operation in this study , a procedure that is technically less challenging than an apr , for example . as such , this has a number of implications regarding operative time , blood loss , and perioperative and postoperative complications . although our study is a small retrospective comparison and thus subject to the limitations of such a study , it presents promising data on the efficacy of robotic surgery at institutions similar to ours . further investigation into the comparative costs and into the magnitude of the effects in a larger patient population is necessary to better elucidate the differences between laparoscopic and robot - assisted techniques in colon and rectal operations . in our early experience , the robotic - assisted technique is a safe and efficacious approach to colorectal surgery , in addition to preserving oncologically sufficient outcomes .

background and objectives : over the years , there has been a continual shift toward more minimally invasive surgical techniques , such as the use of laparoscopy in colorectal surgery . recently , there has been increasing adoption of robotic technology . our study aims to compare and contrast robot - assisted and laparoscopic approaches to colorectal operations.methods:forty patients undergoing laparoscopic or robotic colorectal surgery performed by 2 surgeons at an academic center , regardless of indication , were included in this retrospective review . patients undergoing open approaches were excluded . study outcomes included operative time , estimated blood loss , length of stay , complications , and conversion rate to an open procedure.results:twenty-five laparoscopic and fifteen robot - assisted colorectal surgeries were performed . the mean patient age was 61.1 10.7 years in the laparoscopic group compared with 61.1 8.5 years in the robotic group ( p = .997 ) . patients had a similar body mass index and history of abdominal surgery . mean blood loss was 163.3 249.2 ml and 96.8 157.7 ml , respectively ( p = .385 ) . operative times were similar , with 190.8 84.3 minutes in the laparoscopic group versus 258.4 170.8 minutes in the robotic group ( p = .183 ) , as were lengths of hospital stay : 9.6 7.3 and 6.5 3.8 days , respectively ( p = .091 ) . in addition , there was no difference in the number of lymph nodes harvested between the laparoscopic group ( 14.0 6.5 ) and robotic group ( 12.3 4.2 , p = .683).conclusions : in our early experience , the robotic approach to colorectal surgery can be considered both safe and efficacious . furthermore , it also preserves oncologically sufficient outcomes when performed for cancer operations .

a girl weighing 3,190 g was delivered by a caesarean section at 38 weeks and five days of gestation . at the time of birth , a systolic murmur was noted during a physical examination , and transthoracic echocardiography revealed a peri - membranous ventricular septal defect ( vsd ) with a septal aneurysm , a small patent foramen ovale ( pfo ) , and a small right - sided patent ductus arteriosus ( pda ) from the innominate artery . the vsd was measured as having a diameter of 3.5 mm and a shunt flow less than 2.5 m / sec . due to the presence of neonatal hyperbilirubinemia , echoencephalography was conducted , and no abnormalities were found . five months later , the patient was referred to konkuk university medical center for vsd and pda . her body weight was 6,700 g ( 25th percentile ) , and her height was 61.6 cm ( 10th percentile ) . transthoracic echocardiography revealed a vsd approximately 6 mm in size with a minimal aneurysm , a left - sided pda 3.6 mm in diameter from the right aortic arch and an aberrant left subclavian artery . a subsequent computed tomographic scan demonstrated isolation of the left subclavian artery with a right aortic arch , a left pda , and a vsd ( fig . the intraoperative findings were a perimembranous vsd , a pfo , a mildly patent right ductus arteriosus , and isolation of the left subclavian artery connected to the left pulmonary artery via a left pda . the left subclavian artery was disconnected from the left pulmonary artery and reimplanted to the left common carotid artery by end - to - side anastomosis with monofilament polypropylene 6 - 0 sutures ( fig . ductus arteriosus is usually located on the left side , between the descending aorta and the junction of the main pulmonary artery and left pulmonary artery . however , ductus arteriosus may also be present on the right side or , very rarely , may occur bilaterally in association with aortic arch anomalies or conotruncal anomalies . in such aortic arch anomalies , isolation of the left subclavian artery with right aortic arch is also uncommon . here , isolation refers to the fact that the left subclavian artery connects to the pulmonary artery via either the ligamentum arteriosum or a patent ductus arteriosus without any connection to the aorta . isolation of the left subclavian artery with a right aortic arch is known to be commonly associated with congenital heart disease , but may also occur with normal intracardiac anatomy , although few such cases have been described . isolation of the left subclavian artery with a right aortic arch may be related to the 22q11 deletion . bilateral ductus arteriosus and isolation of the left subclavian artery with a right aortic arch can be explained through the hypothetical double aortic arch plan suggested by edward . regression takes place on two levels in the double aortic arch plan : on one level , regression occurs between the left common carotid artery and the left subclavian artery ; and on the other level , regression occurs at the left dorsal aortic root distal to the left ductus arteriosus . and then right ductus arteriosus remains persistent , left ductus arteriosus connects the left subclavian artery to the left pulmonary artery ( fig . however , the right ductus arteriosus regressed , and only the left ductus arteriosus remained patent . if the left ductus arteriosus is patent , blood may be supplied to the left subclavian artery via the left ductus arteriosus . if the left ductus arteriosus regresses , the blood supply to the left subclavian artery may involve a mediastinal , thoracic anastomosis , or vertebral pathway . isolation of the left subclavian artery usually presents with no apparent symptoms in neonates , but it may present with congenital pulmonary steal syndrome , subclavian steal syndrome , or may even present in adults with late symptoms due to sporadic progression . hayabuchi et al . reported the case of a three - month - old girl with cerebral atrophy and an underdeveloped left arm . reported the case of a 15-year - old boy with an underdeveloped left arm . due to these symptoms and signs , the therapeutic management of isolation of the left subclavian artery remains controversial , especially when it is associated with complicated congenital heart disease . some authors have suggested that adequate collateral circulation must be ensured , meaning that reconstruction of the isolated subclavian artery is optional , regardless of the symptoms and signs . successful results have been reported after ligation or device closure of the pda and ligation of the left subclavian artery . however , reconstruction of the left subclavian artery due to pulmonary steal syndrome after right pda closure in bilateral pda has been reported . in one report , ischemic symptoms in the left arm and vertebrobasilar insufficiency occurred years after ligation of the left subclavian artery . hokari et al . reported that a man with peutz - jeghers syndrome presented with his first vertigo attacks due to subclavian steal syndrome at 29 years of age . our patient presented with no symptoms and signs related to subclavian or pulmonary steal syndrome , and had shown normal findings on an echoencephalography study conducted at our medical center due to neonatal hyperbilirubinemia . however , brain computed tomography angiography performed after surgery revealed hypoplasia of the left vertebral artery . we suggest that this hypoplasia would have led to vertebrobasilar insufficiency or underdevelopment of the left arm without surgical reconstruction . since surgical reconstruction of the isolated left subclavian artery leads to antegrade flow in the left subclavian artery , it can prevent hypoplasia of the left vertebral artery and subclavian / pulmonary steal syndrome . our case shows that early surgical reconstruction is reasonable , regardless of the symptoms , in cases of isolation of the left subclavian artery .

right aortic arch with isolation of the left subclavian artery is a rare anomaly . the incidence of bilateral ductus arteriosus is sporadic , and a right aortic arch with isolation of the left subclavian artery in association with bilateral ductus arteriosus is therefore extremely rare . since the symptoms and signs of isolation of the left subclavian artery can include the absence or underdevelopment of the left arm , subclavian steal syndrome , or pulmonary artery steal syndrome , the proper therapeutic approach is controversial . we report a case in which surgical reconstruction was used to treat isolation of the left subclavian artery with right aortic arch in association with bilateral ductus arteriosus and a ventricular septal defect .

therapeutic hypothermia has been shown to provide neuroprotection against ischemic injury after cardiac arrest in in vitro and in vivo models . in the previous issue of critical care , meybohm and colleagues demonstrate that cardiac arrest triggers the release of cerebral inflammatory cytokines in pigs ' cerebral cortex . . the combination of hypothermia with sevoflurane post - conditioning does not confer additional anti - inflammatory effects compared with hypothermia alone . cardiac arrest remains the leading cause of death in the us and europe , with an out - of - hospital cardiac arrest survival - to - discharge rate of less than 10% . in - hospital cardiac arrest presents a dismal prognosis . according to a large in - hospital registry , the survival - to - discharge rate is 18% , whereas that of a developing country is 6.9% . without prompt care when immediate care is available and victims are successfully resuscitated , the majority of these initial survivors subsequently suffer crippling neurologic injury or die in the few days following the cardiac arrest event . thus , improving survival and brain function after initial resuscitation from cardiac arrest remains a critical challenge . therapeutic hypothermia , introduced more than six decades ago , remains an important neuroprotective factor in cardiac arrest . laboratory studies have demonstrated that cooling after resuscitation from cardiac arrest improves both survival as well as subsequent neurologic and cardiac function and has few side effects . these findings have been reproduced using a variety of cooling techniques in different species , including rats , dogs , and pigs . however , physician use of hypothermia induction in patients resuscitated from cardiac arrest is low . in 2003 , abella and colleagues reported that 87% of us physicians did not use therapeutic hypothermia following cardiac arrest . various reasons for non - use were cited : 49% felt that there were not enough data , 32% mentioned lack of incorporation of hypothermia into advanced cardiovascular life support protocols , and 28% felt that cooling methods were technically too difficult or too slow . in 2002 , a european group demonstrated an improvement in survival - to - discharge rate with favorable neurologic status in cooled patients , compared with normothermic patients surviving after cardiac arrest ( 53% versus 35% , respectively ) , and with no significant adverse events from cooling ; thereafter , induced hypothermia was considered the best practice for patients following cardiac arrest . in 2005 , the american heart association recommended the consideration of therapeutic hypothermia for unconscious adult patients with return of spontaneous circulation following out - of - hospital cardiac arrest due to ventricular fibrillation . in 2008 , binks and colleagues reported that 85.6% of intensive care units in the uk were using hypothermia as part of post - cardiac arrest management . clinical observation demonstrated that tumor necrosis factor - alpha ( tnf ) and interleukin-6 ( il-6 ) protein were increased in cerebrospinal fluid following cardiac arrest . animal studies showed that inflammatory markers were unregulated in rats ' hippocampus tissue and pigs ' serum and myocardial tissue after cardiac arrest [ 8 - 10 ] . meybohm and colleagues go further to demonstrate anti - inflammatory and anti - apoptosis effects of therapeutic hypothermia via the reduction of the upregulation expression of il-1 , il-6 , il-10 , tnf and intercellular adhesion molecule-1 , bcl-2 , and bax mrna and il-1 protein in cerebral cortex after cardiac arrest in a pig model . small reductions in core temperature lead to vaso - constriction and shivering , effectively hindering hypothermia . thus , prevention of vasoconstriction and shivering has become a major goal during induction of therapeutic hypothermia . sevoflurane pre - conditioning and early post - conditioning reduced both cerebral infarct size and neurological defect score , reduced impairment of hippocampus long - term potentiation resulting from myocardial ischemia , and increased nuclear factor inhibitory kappabalpha content in thp-1 cells [ 11 - 13 ] . sevoflurane pre - conditioning preserves myocardial function in patients undergoing coronary artery bypass graft surgery under cardiologic arrest . an in vivo study showed that combination hypothermia with sevoflurane attenuates the inflammatory response during endotoxemia . however , meybohm and colleagues could not provide evidence to support the view that sevoflurane post - conditioning confers additional anti - inflammatory effects in pigs ' cerebral cortex after cardio - pulmonary resuscitation . in summary , meybohm and colleagues provide useful evidence to support the clinical use of therapeutic hypothermia for cardiac arrest , but they did not study the anti - inflammatory effects of sevoflurane in this model . it is even possible that in the setting of clinical practice , anesthetics may not provide significant neuroprotection beyond that which is already being produced by therapeutic hypothermia . thus , at this time , it is difficult to recommend anesthetics for the purpose of neuroprotection in cardiac arrest .

in the previous issue of critical care , meybohm and colleagues provide evidence to support hypothermia as a kind of therapeutic option for patients suffering cardiac arrest . although anesthetics had been used to induce hypothermia , sevoflurane post - conditioning fails to confer additional anti - inflammatory effects after cardiac arrest . further research in this area is warranted .

acromegaly is serious endocrinological derangement which , left untreated , reduces life expectancy and results in physiological derangements and complications that may negatively affect a patient 's quality of life . those patients with residual or recurrent disease are often treated with medication to decrease growth hormone secretion or block its action on peripheral tissues . these treatments are not universally effective for patients and are sometimes contraindicated . as an adjunct treatment , and sometimes as an alternative treatment , radiosurgery has proven to be an attractive therapy . it is noninvasive , has few side effects , and is available in many centers internationally [ 13 ] . in the current paper , we first describe the pathophysiology of acromegaly , the existing surgical and medical treatments , and then introduce radiosurgical methods . in particular , we will focus on gamma knife radiosurgery ( gks ) since it has a broader base of supporting literature than alternative forms of stereotactic radiosurgery . finally , the overall efficacy of gks is described , along with its reported morbidities . acromegaly is a syndrome caused by elevated levels of circulating growth hormone ( gh ) . the most common cause of the disorder , accounting for about 98% of cases , is a gh - secreting pituitary adenoma . rare nonpituitary causes of acromegaly include diverse entities such as hypothalamic hamartomas , small - cell lung cancers , pheochromocytomas , and bronchial carcinoids . among gh - secreting pituitary adenomas , roughly 60% are pure gh - secreting somatotrope adenomas , while the remainder are mixed mammosomatotropes , which secrete both gh and prolactin ( prl ) and sometimes thyroid - stimulating hormone ( tsh ) . the symptoms of increased gh are mediated by both the direct effects of gh binding to the gh receptor , activating the jak / stat pathway , and indirectly via insulin - like growth factor 1 ( igf-1 ) . the combined effects of gh and igf-1 on target tissues lead to bony and soft tissue growth , noted by a characteristic constellation of signs including frontal bossing , prognathism , widely spaced teeth ( due to mandibular growth ) , increased shoe or ring sizes , skin tags , carpal tunnel syndrome , and coarse facial features . these externally recognizable signs in themselves are not as important to overall morbidity as internal changes , including cardiomegaly and visceromegaly [ 4 , 5 ] . diabetes mellitus occurs in roughly 25% of patients , and cardiomyopathy with arrhythmia , hypertension , and diastolic dysfunction occurs in 30% . additionally , colonic polyps are more frequent , as is sleep apnea which occurs in 60% of patients , presumably secondary to soft tissue expansion and macroglossia . multiple studies through the 1920s to 1990s agreed on a prevalence of roughly 60 per million , a mean age of onset of 44 years , symptoms lasting on average 8 years before diagnosis , and no difference in incidence between men and women . however , more recent studies place the prevalence at 86 per million , 124 per million , and a remarkably high 1034 per million . however , in addition to being geographically and demographically restricted ( primary care patients in germany ) , this last study was based on a biochemical definition of acromegaly ( elevated igf-1 and gh ) rather than a syndrome definition , and therefore will include many patients who would otherwise not seek treatment . such a more liberal definition might be one method of discovering at - risk patients earlier in the course of their disease and permitting more time to mitigate the accruing morbidity . surgery is typically the first line of treatment for acromegaly due to pituitary adenomas provided there are no surgical contraindications . residual disease is then managed medically with somatostatin analogues , dopamine agonists , or gh receptor antagonists . most adenomas are resected via the transsphenoidal approach ( transsphenoidal adenomectomy , tsa ) . using either a microscope or endoscope , the surgeon enters the sphenoid sinus transnasally , then penetrates the sella , and finally debulks and removes the tumor , sparing as much of the normal pituitary as possible . for microadenomas ( < 10 mm in diameter ) , tsas lead to correction of gh levels in ~70% of patients . only 50% of patients with macroadenomas achieve normalization . for those patients who do not experience normalization of gh levels after surgery , or who are not surgical candidates , the lar ( long - acting release ) formulation of octreotide uses polymeric microspheres and is injected monthly . lanreotide atg ( autogel ) is the only formulation of lanreotide currently available in the united states and is suspended in aqueous solution in microsyringes for subcutaneous delivery by the patient . the two formulations appear to be equally efficacious and normalize igf - i levels in 5060% of patients [ 1316 ] . somatostatin analogues appear to induce tumor shrinkage in approximately 42% of patients , when data are pooled across studies . interestingly , however , tumor shrinkage appears more pronounced in primarily treated patients ( 52% ) as opposed to patients receiving adjunctive , postsurgical treatment ( 21% ) . dopamine agonists appear to work best in patients whose tumors also secrete prolactin . among dopamine agonists , cabergoline appears to be the most efficacious , resulting in normalization of igf - i levels in 34% of patients . however , no dopamine agonist alone is as effective as a somatostatin analogue , although there might be synergistic effects when used in conjunction with somatostatin analogues [ 2124 ] . pegvisomant is a pegylated growth hormone analogue that is subcutaneously injected by patients . in one study , treatment for 12 months led to normalized igf-1 levels in 97% of patients , and treatment for 24 months led to normalization in 76.3% of patients in a different study . however , the side effects of pegvisomant include transaminitis , lipodystrophy at injection sites , and , most worrisome , possible tumor progression , due to blocking of the normal inhibitory feedback of growth hormone levels of the adenoma [ 2729 ] . simply put , resective surgery is a means of removing unwanted tissue from the body . radiosurgery ( rs ) achieves the identical goal but , rather than directly removing cells , induces cell death instead . this cell death can be induced either directly , via necrosis or apoptosis , or indirectly , by damaging the tissue blood supply . when inducing cell death , rs uses ionizing radiation , wherein charged particles or photons strip electrons from atoms and molecules , thereby damaging dna , proteins , and other molecules within cells and in the extracellular matrix . when this damage can not be repaired , cells undergo either apoptosis or necrosis [ 30 , 31 ] . various types of radiosurgery are available , but each works by emitting charged particles or photons . proton beams , for example , generated by particle accelerators and can be used to target structures deep within the cranium . this is due , in part , to the characteristic peak and subsequent drop - off of radiation intensity in proton beams the bragg peak . the depth of this peak can be modulated and is used to focus the effects of the radiation on particular structures , sparing healthy ones . the drawback to proton therapy , however , is the high cost and relative scarcity of facilities capable of providing therapeutic proton beams . the two major techniques are linear accelerators and radioactive isotopes . linear accelerators , like the cyberknife ( accuray inc . , sunnyvale , ca ) , produce photons which are then aimed toward deep structures within the brain or spine . to limit damage along the path of the beam thus , the target of the beam is constant , always receiving radiation , but the intervening structures are exposed only briefly [ 3032 ] . the gamma knife ( elekta ab , stockholm , sweden ) , developed by lars leksell in 1968 , each source generates a beam of photons as the cobalt decays , and these beams are focused on a central target within the brain through the use of collimators . because each individual beam is weak , intervening tissue is exposed to far less radiation than the central target , which is the common focus of all 201 beams . the gamma knife has been around longer than most linear accelerator radiosurgical devices and is therefore a better - studied tool for use by neurosurgeons , though direct comparisons between the gamma knife and linear accelerators will undoubtedly change the prevailing practices in the future . a large number of small case series have been carried out to evaluate the effects of gamma knife surgery ( gks ) on acromegaly ( table 1 ) . most use remission criterion of a normal igf-1 level and many add the criterion gh < 1 to 2 ng / ml , but it should be noted that remission criteria vary across studies . these criteria are roughly in line with the criteria set forth by the acromegaly consensus group on 2010 ( normal igf-1 and gh < 1 ng / ml ) . also variable between studies is the follow - up time , along with radiation dose , targeting protocol , and , most critically , pre - gks therapy . this last note is particularly important since most series include patients who have already received transsphenoidal surgical resection as an inadequate treatment . unfortunately , the data are not presented in the reviewed papers in such a way as to separate out response rates in patients receiving prior treatment versus those who were treated primarily with gks . overall , however , the results from these studies suggest that gks is an effective treatment for acromegaly . across the 29 studies and 964 cases examined , 43% of patients achieved remission . the time to remission is not reliably reported , but most studies agree that the further out from rs patients are examined , the more likely they are to achieve a cure . for example , vik - mo et al . showed an increase from 58% of patients with normal igh-1 levels to 86% of patients over the time span of 5 to 10 years after rs . how exactly the effects of rs continue to evolve over such a protracted time is unknown , but consistent with how rs affects other diseases , like epilepsy and vestibular schwannoma . interestingly , there is some ( still debated ) evidence that the use of antiacromegalic medicines prior to irradiation attenuates the effects of gks . that is , using somatostatin analogues or gh receptor antagonists has been shown in select studies to decrease a patient 's chances of remission [ 3739 ] . though this has not been thoroughly examined , the mechanism is believed to be suppression of tumor cell cycle , thereby making the cells less prone to radiation - induced damage . there does not appear to be a correlation between radiation dose and rates of remission ( figure 1 ) . however , the heterogeneity in study design , follow - up , and definition of remission makes such conclusions fraught . the most common complication of gks for acromegaly is hypopituitarism , presumably from damage to the normal gland during irradiation , ranging from 0 to 43% ( table 2 ) . however , the degree to which such damage is a pure result of gks versus prior surgery , if done , is unclear . moreover , there is no discernible relationship between radiation dose and the incidence of this complication ( figure 2 ) , though these are very heterogeneous studies and it is possible that better - controlled or larger studies would uncover such relationships if they exist . other complications are inadequately documented but include headache , epilepsy , carotid artery stenosis , and , more frequently , cranial nerve palsies or neuropathies ( including trigeminal neuralgia and visual decline ) ( table 2 ) . the rate of visual disturbances seems to be , in the worst case , 11% [ 40 , 41 ] , though most trials either do not report these adverse events or show them to be on the order of 06% ( table 2 ) . nevertheless , these complications should be viewed in light of the complications inherent to uncontrolled acromegaly or , if being used as an alternative to surgery , the morbidity of an endonasal neurosurgical procedure . while surgical resection remains the first line of treatment , stereotactic radiosurgery is proving itself a feasible alternative therapy ( when surgery might be contraindicated ) and adjunct ( when surgical resection is unsuccessful in leading to complete disease remission ) . ultimately , further studies are needed to delineate which patients are most likely to receive benefit from gks , along with ways to improve gks outcomes .

acromegaly is debilitating disease occasionally refractory to surgical and medical treatment . stereotactic radiosurgery , and in particular gamma knife surgery ( gks ) , has proven to be an effective noninvasive adjunct to traditional treatments , leading to disease remission in a substantial proportion of patients . such remission holds the promise of eliminating the need for expensive medications , along with side effects , as well as sparing patients the damaging sequelae of uncontrolled acromegaly . numerous studies of radiosurgical treatments for acromegaly have been carried out . these illustrate an overall remission rate over 40% . morbidity from radiosurgery is infrequent but can include cranial nerve palsies and hypopituitarism . overall , stereotactic radiosurgery is a promising therapy for patients with acromegaly and deserves further study to refine its role in the treatment of affected patients .

it is estimated to increase the prevalence rate of cancer about 45% by 2025 in developed countries . iran is one of developing countries , in which recently occurred the significant population growth and economic and social status changes . nearly , 38% of these cancer cases in both sexes were due to gastrointestinal tract , of which 65,000 cases suffered from esophagus cancer . of yearly 35,000 death from cancer in iran , 5800 cases were related to this cancer . esophagus cancer rarely occurs in western countries , but it is considered one of the eight most common cancers in the world . the highest incidence rate , 100180/100,000 , was observed in the north of iran and north of china , while in the usa the rate was < 5/100,000 . the lack of early symptoms and the strong bilateral lymph flow of esophagus make the diagnosis to take place in advanced stages . both histological types of esophagus cancer ( squamous cell carcinoma [ scc ] and adenocarcinoma [ adc ] ) are very fatal , with 5 years survival < 10% . in the past , , adc has increased , and recently constitutes 60% of the cancer cases . in developing countries , such as iran and china , recent studies in iran showed that the prevalence of adenocarcinoma ( ads ) is increasing . this may be the improvement of public health , and epidemic of obesity and gastro - intestinal reflex diseases . environmental factors , including western lifestyles , are effective in the incidence and pathology of esophagus cancer , especially in developing countries such as iran . the lack of a comprehensive and population - based study on the incidence , pathological , and clinical aspects of esophagus cancer , this study investigated the epidemiological trend and pathological changes in esophagus cancer in iran . this secondary data analysis study was carried out based on longitudinal program in iran that have national registry of cancer ( ncr ) which is trying to identify all cases of cancer occurring in iran . data used in this study were obtained from ncr , and disease control and prevention of ministry of health and medical education in iran for 20032008 . more details about cancer registry in iran were previously published in the international journal of preventive medicine . in this study , data on the incidence of esophageal cancer were selected according to the international classification of diseases - oncology with the code c15 for age groups and sex . histological data , like squamous cell carcinoma nos , scc keratinizing , scc large cell nonkeratinizing , adenocarcinoma nos , carcinoma nos , also was extracted by sex and years of the study for the cancer . we calculated crude incidence rate and the age - standardized incidence rate ( asir)/100,000 persons . to describe incidence time trends for 6 years studied , we carried out joinpoint regression analysis using the software joinpoint regression program , version 4.1.1.1 october 2014 . as well to evaluate the histological changes , were obtained the percentage allocated for kind of histological types . so to analysis histology change percentage trends for 6 years , we used the software joinpoint regression program and carried out joinpoint regression analysis for data analysis . the test of significance uses a monte carlo permutation method ( i.e. , it finds the best fit line ) . joinpoint regression analysis involves fitting a series of joined straight lines on a log scale to the trends . the final model selected was the most parsimonious of these , with the estimated annual percent change ( apc ) based on the trend within each segment . the terms significant increase or significant decrease signify that the slope of the trend was statistically significant ( p < 0.05 ) . this secondary data analysis study was carried out based on longitudinal program in iran that have national registry of cancer ( ncr ) which is trying to identify all cases of cancer occurring in iran . data used in this study were obtained from ncr , and disease control and prevention of ministry of health and medical education in iran for 20032008 . more details about cancer registry in iran were previously published in the international journal of preventive medicine . in this study , data on the incidence of esophageal cancer were selected according to the international classification of diseases - oncology with the code c15 for age groups and sex . histological data , like squamous cell carcinoma nos , scc keratinizing , scc large cell nonkeratinizing , adenocarcinoma nos , carcinoma nos , also was extracted by sex and years of the study for the cancer . we calculated crude incidence rate and the age - standardized incidence rate ( asir)/100,000 persons . to describe incidence time trends for 6 years studied , we carried out joinpoint regression analysis using the software joinpoint regression program , version 4.1.1.1 october 2014 . as well to evaluate the histological changes , were obtained the percentage allocated for kind of histological types . so to analysis histology change percentage trends for 6 years , we used the software joinpoint regression program and carried out joinpoint regression analysis for data analysis . the test of significance uses a monte carlo permutation method ( i.e. , it finds the best fit line ) . joinpoint regression analysis involves fitting a series of joined straight lines on a log scale to the trends . the final model selected was the most parsimonious of these , with the estimated annual percent change ( apc ) based on the trend within each segment . the terms significant increase or significant decrease signify that the slope of the trend was statistically significant ( p < 0.05 ) . of all cases , 45.72% ( 8311 cases ) were females and 54.28% ( 9866 cases ) males . the most common histological types related to squamous cell carcinoma nos and adenocarcinoma nos were 64.53% and 10.37% , respectively . frequency , crude , and standardized incidence of esophagus cancer by sex , during the years 2003 - 2008 to evaluate the changes in the incidence of esophageal cancer , comparison of asir indicated an increasing trend . in other words , the standardized incidence rate increased from 4.93 to 7.77 for women and from 4.64 to 7.66 for men . results of joinpoint analysis revealed that the trend of annual changes of incidence rate significantly increased in both sexes . the annual percentage changes ( apc ) , the incidence rate was 7.9 ( ci : 3.3 - 12.6 ) for women and 9.6 ( ci : 6.0 - 13.2 ) for men [ figure 1 ] . joinpoint analysis for incidence of esophagus cancer ( male and female ) in iran , 20032008 ; age - standardized rate/100,000 ( using world standard population ) two type of histology ( squamous cell carcinoma nos , and adenocarcinoma nos ) included 87.3%and 86.56% of the histology of esophageal cancer in women and men , respectively . the frequency of five common histological types of esophageal cancer is shown in figure 2 . distribution of histology esophageal cancer in iran the percentage allocated to histology types of esophageal cancer was not constant during years studied . the percentage allocated for histology of squamous cell carcinoma nos ( scc ) , and scc large cell , and nonkeratinizing had a significant decreasing trend in both sexes ( p < 0.05 ) . the type of adenocarcinoma nos increased in both sexes ( p < 0.05 ) [ table 2 ] . joinpoint analyses of cancer percentage allocated trend to the histology data for esophageal cancer in iran ( 2003 - 2008 ) to evaluate the changes in the incidence of esophageal cancer , comparison of asir indicated an increasing trend . in other words , the standardized incidence rate increased from 4.93 to 7.77 for women and from 4.64 to 7.66 for men . results of joinpoint analysis revealed that the trend of annual changes of incidence rate significantly increased in both sexes . the annual percentage changes ( apc ) , the incidence rate was 7.9 ( ci : 3.3 - 12.6 ) for women and 9.6 ( ci : 6.0 - 13.2 ) for men [ figure 1 ] . joinpoint analysis for incidence of esophagus cancer ( male and female ) in iran , 20032008 ; age - standardized rate/100,000 ( using world standard population ) two type of histology ( squamous cell carcinoma nos , and adenocarcinoma nos ) included 87.3%and 86.56% of the histology of esophageal cancer in women and men , respectively . the frequency of five common histological types of esophageal cancer is shown in figure 2 . distribution of histology esophageal cancer in iran the percentage allocated to histology types of esophageal cancer was not constant during years studied . the percentage allocated for histology of squamous cell carcinoma nos ( scc ) , and scc large cell , and nonkeratinizing had a significant decreasing trend in both sexes ( p < 0.05 ) . the type of adenocarcinoma nos increased in both sexes ( p < 0.05 ) [ table 2 ] . joinpoint analyses of cancer percentage allocated trend to the histology data for esophageal cancer in iran ( 2003 - 2008 ) our study investigated the trend of incidence and pathological changes of esophagus cancer during 20032008 in iran . our findings showed that there was an increasing trend of esophageal cancer over 6 years . the decreasing trend was quicker for scc , large cell , and nonkeratinizing than scc . adc , nos had an increasing trend in both sexes . in this study , most cases were scc ( 64.53% ) followed by the ad ( 10.37% ) . there was a significant decreasing trend for scc , while ad is significantly increased in both sexes . ad incidence is enhancing in china . in developing countries , including iran and china , the prevalence of this type of cancer is decreasing in the world . also scc type of esophagus cancer is declining , but adc is increasing in iran . changes in the epidemiology of esophageal cancer in the world and iran occurred two to three decades after health promotion and cultural , economic and social development . obesity and acid reflux from the stomach into the esophagus are possible causes of this cancer . in iran and other developing countries , lifestyle changes and westernization lead to become the incidence of cancer similar to the developed countries . the reported incidence rate of the cancer was 13.8/100,000 in men and 6.5/100,000 in women in developing countries . in developed countries , the rates were 6.5 and 1.3/100,000 in men and women , respectively . our results indicated that the rates were 7.56 and 7.77/100,000 in men and women , respectively . . age - standardized rate ( asr ) for women and men was 43.3 and -36.3/100,000 , respectively , in the province of golestan . asr was 15.4 and 14.4/100,000 in women and men , respectively , in the province of ardebil . in the province of semnan , the lowest in kerman province rate was 3 and 2.1/100,000 in women and men , respectively . the mentioned regions are the belt for esophageal cancer , especially the north east of iran . unlike scc , with equal prevalence in men and women , male factor is considered an important risk factor for adc . according to globacan , our study revealed that the prevalence of ad was 2.64 times more in men than women . a number of case control studies in iran and india showed that drinking hot tea and coffee increases the risk of esophageal cancer . the results of case control studies conducted in golestan showed that drinking too hot has increased the risk of esophageal cancer about 10-fold . however , scientific evidences have not declared that regular consumption of vegetables and fresh fruits may reduce the risk of scc . a study performed in high - risk areas ( such as golestan ) in iran revealed that nitrosamine levels in the saliva of population were 4 times more than german population . there is a relationship between oral disease and tooth loss , and higher rates of mouth and stomach cancers . in china , latin america , eastern europe , and japan , similar to iran , inadequate oral hygiene was considered as a precursor of esophageal squamous dysplasia . strong risk factor for ad may be obesity and the absence of helicobacter pylori infection . smoking and acid reflux from the stomach into the esophagus increases the risk of ad . , this type of esophagus cancer dramatically increases in iran because of obesity and overweight epidemic associated with acid reflux from the stomach into the esophagus and reducing the amount of helicobacter pylori infection . improving the cancer registration system may be one of the reasons of increasing the cancer . a study on the trend of skin cancer incidence performed in italy declared that advances in diagnostic techniques and the development of cancer registration lead to a large proportion of the increase in the incidence of skin cancer . according to the report of iranian ministry of health , the cancer registry in the country hence , in interpreting the results of studies regarding all cancers , the problems and of cancer registration should be considered . in other words , the share of the development of cancer registration and high proportion of the most difficult areas should be distinguished from each other . the lack of population - based data regarding the prevalence and mortality is one of the major problems in esophageal cancer . in some low - income countries , there are the primary barriers such sanctions and racial and cultural beliefs , except for the diagnosis and effective management of cancer . researchers believe that advances in cancer treatment , even in countries with limited resources , can helpful because early detection increases the effectiveness of treatment . according to this study , the trend of asir of esophagus cancer in iran is rising . hence to prevent and control this cancer , it is necessary to investigate related risk factors and implement prevention programs in iran .

background : esophageal cancer is the sixth cause of death in the world , there was a lack of population - based information on the trend and incidence rate of esophagus cancer , so this study aimed to determine the incidence and pathological changes of esophagus cancer in iran.methods:in this study , data were extracted from annual cancer registry reports of iranian ministry of health between 2003 and 2008 . standardized incidence rates were calculated using the world standard population , and incidence rate was calculated by age groups , sex , and histological type . data on epidemiologic trend and histology were analyzed using joinpoint software package.results:in this study , there were 18,177 recorded cases of esophagus cancer . of all cases , 45.72% were females and 54.28% were males . sex ratio was 1.19 . the most common histological types related to squamous cell carcinoma nos and adenocarcinoma nos were 64.53% and 10.37% , respectively . the trend of annual changes of incidence rate significantly increased in both sexes . the annual percentage changes , the incidence rate was 7.9 ( 95% confidence interval [ ci ] : 3.312.6 ) for women and 9.6 ( 95% ci : 6.013.2 ) for men . the histology type of scc , large cell , nonkeratinizing and scc , keratinizing and scc , nos had a significant decreasing trend in total population ( p < 0.05).conclusions : according to this study , the trend of age - standardized incidence rate of esophagus cancer in iran is rising . hence , to prevent and control this cancer , it is necessary to investigate related risk factors and implement prevention programs in iran .

gene expression is well regulated by the epigenetic modification of dna and histone tails independent of their primary sequences . in particular , cytosine methylation , in which the c5 position of the cytosine base is methylated enzymatically , plays a crucial role in the regulation of chromatin stability , gene regulation , parental imprinting , and x - chromosome inactivation in females [ 14 ] . therefore , detection of cytosine methylation is very important , and much effort has gone into developing a simple reaction for 5-methylcytosine ( c ) detection . for the evaluation of the methylation status of genes , several conventional methods have so far been used , such as a cleavage assay with methylation - insensitive restriction enzymes [ 57 ] , hydrolysis and sequencing with a bisulfite salt [ 810 ] , and immunofluorescence with anti-5-methylcytosine antibody [ 11 , 12 ] . although the conventional methods have many merits , there are many disadvantages , and methylation detection assays must be further improved through another approach . the existence of a more rapid and selective chemical reaction capable of distinguishing between methylcytosine and unmethylated cytosine on a chip has promise as a good method for efficiently analyzing the status of cytosine methylation at a specific site in a gene . the sequence - selective dna methylation - detection probe , icon ( interstrand complexes formed by osmium and nucleic acids ) , may be effective for the development of an on - chip analysis of dna methylation [ 1315 ] . in the presence of osmium oxidants and a bipyridine ligand , 5-methylcytosine forms a stable osmium - centered complex , in contrast to unmethylated cytosine ( figure 1 ) [ 1619 ] . icon probes form a crosslink with a specific 5-methylcytosine in the probe - hybridizing dna mediated by osmium - centered complex formation . this function will be effective for the capture of methylated dna on a chip for sequence - selective methylation analysis . in this paper , development of an on - chip analyzing method for typing of dna methylation at a specific cytosine icon probes fixed to the bottom of microwells assisted the on - chip detection of the methylation status of a specific cytosine in the target dna . artificial dna was synthesized by the conventional phosphoramidite method using an applied biosystems 392 dna / rna synthesizer or an nts h-6 dna / rna synthesizer . the phosphoramidite form of bipyridine - modified adenine ( b ) was prepared according to the synthetic protocol described in a previous paper . the 5-amino end was attached using the phosphoramidite of 5-aminomodifier c12 ( glen research ( http://www.glenresearch.com/index.php ) ) . synthesized dna was purified by reverse phase hplc on a 5-ods - h column ( 10 mm 150 mm , elution with a solvent mixture of 0.1 m triethylammonium acetate ( teaa ) , ph 7.0 , linear gradient over 30 min from 5% to 20% acetonitrile at a flow rate of 3.0 ml / min ) . a 100 l solution of synthetic dna ( 100 nm ) in te buffer ( ph 7.0 ) or deionized water in the presence of 10 mm 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide , and 10 mm 1-methylimidazole was put into each well of nucleolink strips ( nalge nunc ( http://www.nalgenunc.com/ ) ) . after incubation at 50c for 5 h , the reaction mixture was removed from the well , and the well was rinsed three times with a solution of 100 mm tris - hcl ( ph 7.5 ) , 150 mm sodium chloride , and 0.1% tween20 , and then three times with deionized water . the target dna sequence p53(n n ) was 5-tgt gca gct gtg g gtt gat t cga cac ccc c gcc cgg cac c cgc gtc cgc g cca tgg cca t cta caa gca g tca cag cac a tga ngg agg t tgt gag g ng c tgc ccc cac c-3 ( n , a 25 l solution of dna ( 8 nm ) in 50 mm tris - hcl buffer ( ph 7.7 ) , 0.5 mm edta , and 1 m sodium chloride was added to each probe - attached well at 0c . the reaction mixture was incubated at 0c for 5 min , and then the solution was removed from the wells . a 25 l solution of 5 mm potassium osmate(vi ) and 100 mm potassium hexacyanoferrate(iii ) in 50 mm tris - hcl buffer ( ph 7.7 ) , 0.5 mm edta , and 1 m sodium chloride was incubated at 0c for 5 min or at 25c for 10 min . the wells were rinsed seven times with 0.4 m sodium hydroxide , 0.25% tween20 ( 130 l / well ) . after further rinsing with deionized water twice , the wells were coated with 10 mg / ml bsa in 100 mm tris - hcl ( ph 7.5 ) , 150 mm sodium chloride , and 0.1% tween20 . the process of pcr amplification was performed in a reaction solution of 1 u takara ex taq hs , 10 buffer , 2.5 mm dntp mix , and 1 m primer mix ( forward , 5-agctgd514gggttgattc-3 for the exciton primer method , 5-tgtgcagctgtgggttgattc-3 for the sybr green i method ; reverse , 5-actgcttgtagatggccatg-3 ; d514 in an exciton primer is a hybridization - sensitive fluorescent nucleotide ( figure 1 ) ) . in the case of using the stain method with sybr green i fluorescence for monitoring the amplification , sybr green i dye was also added to the reaction mixture in advance . amplifications were performed in microwells as follows : after heating at 95c for 60 s , 35 cycles of denaturation at 95c for 5 s , annealing with fluorescence monitoring at 52c for 20 s , and extension at 72c for 20 s on the corbett rotor - gene , the amplification process was monitored by the fluorescence of d514 or sybr green i through an sybr green i ( 470 nm/510 nm ) filter . for the on - chip study , we adopted a nucleolink strip , because it is a microwell strip in which the amino - modified icon probe can be attached to well bottoms with covalent bonds . the target dna was a fragment of the human p53 gene exon 5 including mutation hotspots at cpg dinucleotides . the sequence of the icon probe was designed to form a crosslink with one of the methylated cytosines in the target dna . the icon probe was modified with an amino end for attaching to the plate and an alkyl linker and a poly c sequence for introducing enough distance between the probe and the plate . this icon probe was synthesized by the conventional phosphoramidite method by using a dna autosynthesizer . the phosphoramidite form of bipyridine - modified adenine ( b ) was prepared according to the synthetic protocol described in a previous paper . synthetic dna was fixed to the bottom of each well of nucleolink strips in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-methylimidazole ( figure 2 ) . oxidative osmium complexation using icon probes is a rapid and mild reaction for detection of methylated dna , and it is not accompanied with nonspecific strand damage , in contrast to conventional bisulfite methods . therefore , this reaction would be effective for on - chip analysis of dna methylation . the target dna was put into the wells and hybridized with the icon probes fixed in the wells . the dna samples in the wells were incubated at 0c for 5 min then at 25c for 5 min in the presence of 5 mm potassium osmate(vi ) and 100 mm potassium hexacyanoferrate(iii ) in 50 mm tris - hcl buffer ( ph 7.7 ) , 0.5 mm edta , and 1 m sodium chloride . after reaction , the wells were rinsed with 4 m sodium hydroxide and then coated with bsa . the fixed dna was detected using pcr amplification of a part of the crosslinking dna strand . the pcr primers were designed for the region without icon binding . for one of the primers , a hybridization - sensitive fluorescent dna was used , containing a fluorescent nucleotide d514 ( figure 1 ) [ 2124 ] . this fluorescent dna shows very weak fluorescence in the unhybridized state , whereas it shows strong fluorescence after hybridization with the complementary nucleic acids . this fluorescence switching is controlled by an intramolecular excitonic interaction between dyes tethered to the dna . this dna shows weak fluorescence emission , whereas the pcr mixture emits strong fluorescence after pcr amplification . this system has been applied to the detection of single nucleotide polymorphisms in genome dna samples . the process of pcr amplification was performed in a reaction solution of takara ex taq hs polymerase in the presence of a mixture of dntp and primer mix . amplifications were performed in microwells and the change in the fluorescence intensity monitored using a real - time pcr system . in the experiment for p53(c c ) and p53(c c ) , the methylation of the target cytosine was determined from the increase in the fluorescence signal associated with the exponential growth of the pcr product . c ) started first , and then the amplification of p53(c c ) started several cycles later ( figure 3 ) . washingout of uncrosslinked sample dna brought about this lag in the starting point of amplification . the amplification curve observed for p53(c c ) almost overlapped that for the osmium - untreated p53(c c ) , suggesting that the amplification curve for unmethylated dna is due to amplification of the dna nonspecifically adsorbing to the well surface . on - chip capture of methylated dna by the icon probe at the methylation site made possible the sequence specific detection of methylation through pcr amplification . a prototype for icon - based on - chip methylation analysis makes possible sequence - specific amplification . we prepared four dna strands with different methylation sites , p53(c c ) , p53(c c ) , p53(c c ) , and p53(c c ) . the sample dna was added to the wells , in which the icon probe targeting only the c of the sample dna 5 side was fixed . after osmium treatment and bsa coating , the crosslinked dna was amplified by pcr in the presence of unlabeled primers and sybr green i. after 15 cycles of the amplification reaction , the fluorescence intensity of sybr green i was quantified on a microplate reader ( figure 4 ) . c ) and p53(c c ) exhibited higher fluorescence intensities compared with those from wells containing p53(c the icon probe fixed on the well bottom distinguished 5-c from 3-c and detected only methylation of 5-c regardless of methylation of 3-c . we have described a new , high - value aspect of on - chip methylation analysis through osmium - dna complexation . an icon probe fixed onto a microwell formed a crosslink with the target 5-methylcytosine and assisted the detection using pcr amplification . although there remain further aspects to be examined toward realizing an easier - to - use methylation analysis , such as optimization of pcr conditions suitable for icon , this on - chip assay supported by the chemical basis could be an important component of the next generation of high - throughput methylation analyses .

the development of a reaction for detecting the presence / absence of one methyl group in a long dna strand is a chemically and biologically challenging research subject . a newly designed chemical assay on a chip for the typing of dna methylation has been developed . a methylation - detection probe fixed at the bottom of microwells was crosslinked with methylated dna mediated by osmium complexation and contributes to selective amplification of methylated dna .

in order to maintain cellular homeostasis , the amount of proteins in cells is selectively controlled not only in protein synthesis but also in protein degradation . the ubiquitin proteasome pathway is essential for multiple physiological systems via selective degradation of target proteins ( hershko and ciechanover , 1998 ) . the proteins designated for proteasome - mediated degradation are conjugated with polypeptide of ubiquitin , which are then targeted to 26s proteasome complex ( hochstrasser , 1995 ) . ubiquitination of target protein is regulated through multi - enzyme processes in an atp - dependent manner . first , the e1 protein , ubiquitin - activating enzyme , activates ubiquitin , which is then transferred to the e2 protein . the ring finger - containing e3 ligase binds to its substrate and the ubiquitinated e2 protein and then directly transfers the ubiquitin from the e2 protein to the substrate ( lipkowitz and weissman , 2011 ) . on the other hand , the hect domain - containing e3 ligase can also receive ubiquitin from the e2 protein first through an active - site cysteine of its hect domain then interacts with its substrate to catalyze the conjugation of the activated ubiquitin to the substrate ( kee and huibregtse , 2007 ) . since the specificity of the target proteins for proteasome - mediated degradation is dependent on the interaction between the e3 ligases and their targets , the e3 ubiquitin ligases are critical for regulating the expression levels of key short - lived proteins . cancer is a genetic disease that is caused by multiple genetic mutations . in cancer cells , oncogenic drivers that are frequently mutated or overexpressed activate the signaling pathways to promote cell proliferation , growth , and survival while tumor suppressors that are commonly inactivated by mutation or deletion inhibit these pathways . it has been demonstrated that the expression levels of some key oncoproteins and tumor suppressors are under the control of ubiquitin proteasome system with some e3 ligases that function as oncogenic factors or tumor suppressors . for example , mdm2 and skp2 ubiquitinate and inhibit tumor suppressors via proteasomal degradation , and thereby function as oncogenic factors ( marine and lozano , 2010 ; wang et al . , 2011 ) . in contrast , other e3 ligases such as the anaphase promoting complex / cyclosome ( apc / c ) and f - box and wd repeat domain - containing 7 ( fbw7 ) serve as tumor suppressors by downregulating oncogenic factors ( crusio et al . , 2010 ; wasch et al . , 2010 ) . , bortezomib is a specific proteasome inhibitor that is currently used for the treatment of multiple myeloma . moreover , the inhibitors for specific e3 ligases have been also considered as potential anti - cancer drugs . mdm2 is the primary ubiquitin ligase for tumor suppressor protein , p53 , which induces apoptosis or senescence in response to oncogenic stress or dna damage . the p53 pathway is frequently inactivated in human cancer cells , and the small molecules that block the interaction between p53 and mdm2 to inhibit p53 degradation have been tested in clinical trials ( brown et al . , 2009 ) . protein kinases and phosphatases catalyze the protein phosphorylation and dephosphorylation , respectively , which are essential for maintaining signal transduction . when cells receive extracellular signaling and stress , the signals are primarily transduced to the nucleus via protein phosphorylation , resulting in the alteration of gene expression . for example , epidermal growth factor ( egf ) stimulates cell proliferation by binding to its receptor , egf receptor ( egfr ) , and activating it . egfr is a receptor tyrosine kinase that phosphorylates and activates multiple downstream targets and promotes cell growth and survival . egfr , considered as an oncogene , is frequently overexpressed or mutated in multiple human cancers and promotes tumor progression , metastasis , and drug resistance ( nicholson et al . , 2001 ; hynes and lane , 2005 ; quatrale et al . , indeed , many oncoproteins include protein kinases , and these oncogenic kinases phosphorylate downstream targets to promote tumor growth , metastasis , and/or angiogenesis . the serine / threonine kinase akt is one of the major downstream kinases activated by growth factor signaling such as egfr , platelet - derived growth factor receptor ( pdgfr ) , and insulin - like growth factor receptor ( igfr ) . akt has three isoforms , akt1 , akt2 , and akt3 , and their activities are frequently elevated in multiple human cancers , which contribute to cancer cell survival and growth ( altomare and testa , 2005 ) . akt is activated by phosphatidylinositol-3 kinase ( pi3k ) that converts phosphatidylinositol ( 3,4)-bisphosphate ( pip2 ) to phosphatidylinositol ( 3,4,5)-trisphosphate ( pip3 ) in plasma membrane through lipid phosphorylation ( altomare and testa , 2005 ; yuan and cantley , 2008 ) . akt and phosphoinositide dependent kinase 1 ( pdk1 ) are then recruited to the plasma membrane where akt is directly phosphorylated and activated by pdk1 ( chan et al . , 1999 ) . activated akt phosphorylates various substrates involved in cell metabolism ( gsk3 , tsc2 ) , survival ( bad , foxo ) , and cell cycles ( p21 , p27 , mdm2 ; cross et al . , 1995 ; datta et al . , 1997 ; 2001a , b ; liang et al . , 2002 ; manning et al . , 2002 ) , and then inhibits apoptosis and promotes cell growth . therefore , the pi3k akt pathway is a potential drug target , and several pi3k or akt inhibitors have been actively tested in numerous clinical trials ( garcia - echeverria and sellers , 2008 ; wong et al . , 2010 ; chappell et al . , extracellular signal - regulated kinase ( erk ) is another critical downstream kinase in growth factor signaling and plays an essential role in cancer cell proliferation ( robinson and cobb , 1997 ; sebolt - leopold , 2000 ) . erk pathway is a well - characterized signaling pathway and is commonly activated in multiple human cancers . studies have shown that ras and raf are frequently mutated in various human cancer types and responsible for cancer progression ( adjei , 2001 ; davies et al . , 2002 ) . therefore , this signaling pathway has been considered a drug target for cancer therapy , and a variety of inhibitors have been developed , including mek or raf inhibitors ( davies et al . , 2007 ; bollag et al . , 2010 ; hatzivassiliou et al . , 2010 ; maurer et al . , ib kinases ( ikks ) are the primary regulator of nf-b , which plays a key role in immune response , cell proliferation , and survival ( baldwin , 2001 ; luo et al . , 2005 ) . ikks are activated in response to various cytokines and inflammatory stimuli such as tumor necrosis factor ( tnf)- , interleukin-1 , and lipopolysaccharide . the ikk family includes ikk , ikk , ikk , ikk , and tank - binding kinase 1 ( tbk1 ) . interestingly , ikk has no kinase activity and functions as an adaptor protein for the canonical ikk complex ( hacker and karin , 2006 ) . ikks are involved in two distinct pathways for nf-b activation : the canonical and non - canonical pathways . ikk plays a dominant role in the canonical pathway and ikk in the non - canonical pathway ( perkins , 2007 ; israel , 2010 ) . in the canonical pathway , ikk , ikk , and ikk form a kinase complex that phosphorylates ib , an inhibitor protein of nf-b , and induces the ubiquitination and subsequent proteasome - dependent degradation of ib. in the non - canonical pathway , ikk forms a homodimer and phosphorylates p100 , and generates p52 by partial processing of p100 , resulting in the activation of p52/relb . in contrast to ikk and ikk , ikk and tbk1 play a role in the induction of interferon signaling in response to viral infection ( shen and hahn , 2011 ) . although nf-b has been known to be involved in the progression of various cancers , increasing evidence suggests that ikks also play vital roles in cancer independently of nf-b ( lee and hung , 2008 ; baud and karin , 2009 ; shen and hahn , 2011 ) . for example , ikk phosphorylates both estrogen receptor ( er ) and co - activator src3 and enhances er transcriptional activity while ikk phosphorylates a tumor suppressor , tuberous sclerosis 1 ( tsc1 ) , and inhibits its function ( park et al . , 2005 ; ikk also phosphorylates cbp and the phosphorylated cbp preferentially interacts with nf-b rather than p53 , resulting in nf-b activation as well as p53 inactivation ( huang et al . , 2007 ) . inactivation of foxa2 results in the decrease of numb expression , and subsequent notch activation ( liu et al . , 2012 ) . considering that inhibition of nf-b may affect inflammatory responses , ikks may be the potential alternative drug targets for cancer . the above - mentioned three survival kinases play critical roles in cancer cell survival , metabolism , proliferation , and growth . interestingly , these kinases have many common targets that they directly or indirectly regulate . moreover , we and others have identified several common targets of these three kinases that are regulated by the ubiquitin we will discuss these proteins one by one in the later next section of the review . the forkhead box o ( foxo ) family proteins are critical transcription factors that are involved in the regulation of cell proliferation , cell death , cell metabolism , and dna repair ( tran et al . , 2003 ; arden , 2008 ) . foxo family includes foxo1 , foxo3 , foxo4 , and foxo6 , and is conserved from c. elegans to mammals ( burgering , 2008 ; calnan and brunet , 2008 ) . foxo family proteins directly activate multiple gene expression involved in cell cycles , apoptosis , metabolism , and dna damage repair , such as p27kip , bim , fasl , mnsod , gadd45 ( dijkers et al . , 2000 ; kops et al . , 2002 ; tran et al . , 2002 ; ciechomska et al . , 2003 ; sunters et al moreover , it has been shown that foxo proteins are dysregulated in multiple human cancers such as breast , prostate , leukemia , and glioblastoma ( hu et al . , 2004 ; seoane et al . , 2004 ; conditional knockout mice of foxo1 , 3 , and 4 develop thymic lymphomas and hemangiomas ( paik et al . it has been shown that ikk , akt , and erk directly phosphorylate foxo and induce foxo ubiquitination and degradation . among the three kinases , akt was first identified as a foxo kinase that phosphorylates foxo3 at t32 , s253 , and s315 . phosphorylated foxo3 protein is excluded from entering the nucleus and binds to 14 - 3 - 3 in the cytoplasm ( brunet et al . , 1999 ) in addition to foxo3 , foxo1 , and foxo4 are also phoshorylated by akt ( tzivion et al . , 2011 ) . akt - phosphorylated foxo1 and foxo3 then undergo degradation in a proteasome - dependent manner ( plas and thompson , 2003 ) . specifically , foxo1 phosphorylated by akt translocates to the cytosol where it is ubiquitinated by skp2 and subjected to proteasome - dependent degradation ( huang et al . , 2005 ) . we found that ikk directly phosphorylates foxo3 at s644 and induces its ubiquitination and degradation ( hu et al . , 2004 ) . recently , e3 ligase -transducing repeat - containing protein ( -trcp ) is reported to interact with foxo3 and induces ubiquitination and degradation in an ikk-mediated - phosphorylation - dependent manner ( tsai et al . , 2010 ; su et al . , 2011 ) . furthermore , we and others demonstrated that erk phosphorylates foxo3 and foxo1 , respectively ( asada et al . , 2007 ; yang et al . , 2008 ) . we also showed that foxo3 is phosphorylated by erk at s294 , s344 , and s425 , which then undergoes mdm2-mediated ubiquitination , followed by proteasome - dependent degradation ( yang et al . , 2008 ) . mdm2-mediated ubiquitination and degradation is also observed with foxo1 , which is dependent on the akt - mediated phosphorylation ( fu et al . , 2009 ) . interestingly , mdm2 induces mono - ubiquitination of foxo4 , which promotes nuclear localization of foxo4 , and subsequent polyubiquitination by skp2 and degradation ( brenkman et al . , 2008 ) . taken together , the ubiquitin proteasome system plays an essential role in regulating foxo transcription factors by akt , erk , and ikk , and mdm2 , skp2 , and trcp are e3 ligases for foxo ubiquitination ( figure 1a ) . ( a ) akt , erk , and ikk phosphorylates foxo3 at different sites and induces its ubiquitination and subsequent degradation via skp2 , mdm2 , and -trcp , respectively . gsk3 phosphorylates catenin and induces its ubiquitination and subsequent degradation via -trcp . both akt and ikk phosphorylate -catenin and stabilize it . -catenin is the key protein in both cadherin junction and wnt pathway and plays an important role in development and adult homeostasis as well as tumorigenesis ( cadigan , 2008 ; stepniak et al . , 2009 ) . in the wnt signaling pathway , -catenin functions as a transcription co - factor and is involved in the transactivation of several oncogenic proteins such as c - myc , cyclind1 , and matrix metalloproteases ( he et al . , 1998 ; lin et al . , 2000 glycogen synthase kinase-3 ( gsk3 ) and casein kinase 1 ( ck1 ) are the major protein kinases regulating the -catenin stability . in the absence of wnt ligand , -catenin forms a complex with axin , apc , gsk3 , and ck1 and is phosphorylated by these kinases . once phosphorylated , -catenin undergoes -trcp - mediated ubiquitination and subsequent degradation . when wnt binds to its receptor , frizzles , and co - receptor , lrp5/6 , the receptor complex recruits axin gsk3 complex to cell membrane , releasing -catenin from the complex for its translocation to the nucleus where it activates gene transcription with t - cell factor ( tcf ) and lymphocyte enhancer factor ( lef ) . akt has been shown to directly phosphorylate gsk3 and inhibits it ( cross et al . , 1995 ) , and therefore , akt seems to indirectly inhibit -catenin degradation and inactivation through inhibition of gsk3 ( monick et al . , 2001 ) . in addition to the indirect mechanism , akt directly mediates the -catenin stability by phosphorylating -catenin at s552 . once phosphorylated by akt , -catenin binds to 14 - 3 - 3 and is stabilized ( tian et al . in addition to akt , we also demonstrated that erk upregulates -catenin via inhibition of gsk3 ( ding et al . , activated erk directly interacts with gsk3 and phosphorylates it at t43 . after the phosphorylation by erk , gsk3 is primed for subsequent phosphorylation by p90rsk at s9 , which inactivates it . furthermore , ikk has been shown to be directly involved in the -catenin regulation , and ikk but not ikk phosphorylates -catenin and prevent its ubiquitination and degradation ( lamberti et al . , 2001 ; albanese et al . , 2003 ; carayol and wang , 2006 ) . collectively , -catenin is another common target of akt , erk , and ikk , demonstrating that the ubiquitin myeloid cell leukemia-1 ( mcl-1 ) is a member of anti - apoptotic bcl-2 family proteins which are the central regulators of apoptosis signaling pathway ( inuzuka et al . , 2011a ) . the bcl-2 family consist of pro - apoptotic and anti - apoptotic proteins that regulate the release of apoptogenic proteins such as cytochrome c and smac from mitochondria ( youle and strasser , 2008 ) . pro - apoptotic bcl-2 family proteins include the bh3-only proteins such as bim , bid , bik , and bad and multi - bh domain like bax and bak . bax and bak are able to form channels on the surface of mitochondria from which cytochrome c and smac are released . anti - apoptotic bcl-2 family proteins , which include bcl-2 , bcl - xl , and mcl-1 , prevent apoptotic cell death by inhibiting the activation and channel formation of bax and bak . in contrast , the bh3-only proteins directly bind to anti - apoptotic bcl-2 family proteins and inhibit their functions . thus , the balance of anti - apoptotic and pro - apoptotic proteins determines cell survival and death , and the expression of anti - apoptotic bcl-2 family protein is the critical for cell survival . among the anti - apoptotic bcl-2 family proteins , 2003 ; adams and cooper , 2007 ) but its expression is enhanced in various cancer types ( placzek et al . , 2010 ) . the primary kinase that regulates mcl-1 stability is gsk3. we and another group reported that gsk3 interacts with and phosphorylates mcl-1 at s155 , s159 , and t163 ( maurer et al . mule is the bh3 domain - containing e3 ligase that was the first identified as the mcl-1 ubiquitin ligase by biochemical purification ( zhong et al . , 2005 ) . later , we found that -trcp can ubiquitinate and induce mcl-1 degradation in gsk3-mediated - phosphorylation - dependent manner ( ding et al . , 2007 ) . recently , a tumor suppressor protein , fbw7 , is shown to function as a mcl-1 ubiquitin ligase ( inuzuka et al . fbw7-induced ubiquitination of mcl-1 is also dependent the phosphorylation induced by gsk3. although both -trcp and fbw7 recognize the same gsk3-mediated phosphorylation sites in mcl-1 , the exact roles of these two ubiquitin ligases under various apoptotic stresses are unknown . akt and erk have been shown to upregulate mcl-1 transcription ( wang et al . , 1999 ; in addition , both pi3k / akt and mek / erk pathways have also been shown to enhance mcl-1 protein stability ( derouet et al . , 2004 ) . because akt inhibits gsk3 as described above , akt increases mcl-1 stability , at least in part , by inhibiting gsk3. indeed , the inhibition of pi3k induces s159 phosphorylation of mcl-1 and subsequent ubiquitination and degradation of mcl-1 , which are blocked by gsk3 inhibition ( maurer et al . , 2006 ) . in addition to gsk3 , it has been show that erk phosphorylates mcl-1 at t92 and t163 ( domina et al . in particular , we showed that erk - phoshorylated mcl-1 can interact with pin1 ( ding et al . , 2008 ) . prolyl cis / trans isomerase that binds to specific ps / t - p motifs and then isomerizes its substrates , resulting in their conformational changes . interestingly , pin1 stabilizes mcl-1 protein after the phosphorylation by erk , and the expression of mcl-1 correlates with pin1 in multiple human cancer cell lines ( ding et al . , 2008 ) . regarding ikk , there is no evidence that ikk is directly involved in mcl-1 or gsk3 phosphorylation and/or ubiquitination . however , it has been shown that nf-b is required for egf - induced mcl-1 induction , suggesting that the ikk nf-b pathway plays a role in mcl-1 expression or stability ( henson et al . , 2003 ) . thus , mcl-1 is a critical apoptosis regulator that is controlled by the three kinases at the post - translational as well as transcriptional level ( figure 2a ) . gsk3 phosphorylates mcl-1 and induces its ubiquitination and subsequent degradation via -trcp , mule , and/or fbw7 . erk also directly phosphorylates mcl-1 , and phosphorylated mcl-1 interacts with pin1 , resulting in its stabilization . gsk3 phosphorylates snail and induces its nuclear exclusion , ubiquitination and subsequent degradation via -trcp . nf-b pathway inhibits snail via upregulation of csn2 , which interferes with the gsk3snail interaction and ubiquitination of snail . epithelial mesenchymal transition ( emt ) is an important physiological process that converts epithelial cells to mesenchymal cells which plays an essential role in embryonic development and tissue repair ( nieto , 2009 ) . epithelial cells lose cell cell contacts and gain migratory properties during emt . during cancer progression , cancer cells undergo emt , resulting in increased motility , invasiveness , and aggressive behavior ( kalluri and weinberg , 2009 ) . snail is a zinc - finger transcription repressor that is one of the emt regulators . snail family contains snail1 ( snail ) , snail2 ( slug ) , and snail3 ( smuc ; de herreros et al . , 2010 ) . like other emt regulators in addition to emt , snail is also involved in cell death , survival , stem cell , and immune regulation by controlling multiple target genes ( wu and zhou , 2010 ) . downregulation of snail reduces tumor growth and invasiveness in xenograft animal model ( olmeda et al . , 2007 ) . moreover , snail is overexpressed in multiple human cancers , and expression of snail is associated with poor cancer prognosis ( peinado et al . , 2007 ) . snail stability is primarily regulated by gsk3 through the pi3k akt pathway ( song et al . , 2009 ) . we have shown that gsk3 can phosphorylate snail at six serine residues in which two of them are responsible for snail stability while the other four are involved in its nuclear localization ( zhou et al . , 2004 ) . first , gsk3 phosphorylates snail at four serine residues to allow its export from the nucleus . then , gsk3 phosphorylates the other two sites , resulting in -trcp - mediated ubiquitination and degradation of snail . specifically , nf-b induces cop9 signalosome 2 ( csn2 ) , which disrupts the interaction between gsk3 and snail , resulting in the inhibition of snail ubiquitination and subsequent degradation ( wu et al . , 2009 ) . however , erk does play a role in c - myc - induced emt via the inhibition of gsk3 , and there by stabilizing snail ( cho et al . , 2010 ) . furthermore , erk upregulates snail gene transcription though activation of ap-1 transcription factor ( hudson et al . independently of kinases , wild type p53 but not mutant p53 has been shown to interact with and induce snail and slug ubiquitination and degradation ( wang et al . , 2009 ; lim et al . , moreover , f - box and leucine - rich repeat protein 14 ( fbxl14 ) has been shown to interact with and induce snail ubiquitination and degradation ( vinas - castells et al . , 2010 ) . so far , no other kinases have been reported to be involved in mdm2- or fbxl - mediated snail ubiquitination . thus , the three kinases ( akt , erk , and ikk ) seem to be involved in emt , at least in part , by regulating snail expression through ubiquitination ( figure 2b ) . recent advances in signal transduction studies have identified many key oncogenic kinases and their substrates in cancer progression , and the signaling pathways associated with these kinases have been recognized as promising drug targets . indeed , several kinase inhibitors have been developed and used in clinic that show high efficacy and low toxicity ( sharma and settleman , 2010 ) . however , several clinical studies have emerged showing that some patients exhibit little or no response to these targeted drugs , and those who originally responded the drugs eventually developed resistance . although the detailed mechanisms underlying drug resistance are not fully understood , there is evidence to support that alternative pathways are being activated in resistant cells to compensate for the survival signal blocked by targeting agents . for example , egfr tyrosine kinase inhibitors ( tkis ) are effective drugs for egfr mutant lung cancer , but c - met amplification or k - ras mutation causes the resistance to tkis by bypassing the inhibition of survival signaling ( bean et al . , 2007 ; linardou et al . , we also showed that cancer cells with high akt signaling pathway exhibit resistance to erk inhibitors by inhibiting foxo3 ( yang et al . , 2010 ) . therefore , in order to develop effective personalized cancer therapy , it would be essential that we understand the cross - talk among the multiple oncogenic signaling pathways . in this review , we introduced akt , erk , and ikk as the key survival kinases for cancer progression and survival . we also mentioned that these three pathways have several common targets that are critical for cancer cell proliferation , survival , and emt . because there are many other pathways that are activated in human cancers , we believe that they likely also contribute to the same targets we described here . these three kinase - signaling pathways may have other common targets that are critical for cancer progression . we also introduce some examples of the signaling pathways that are controlled via the ubiquitin proteasome system , which seems to play an essential role in signaling pathway like phosphorylation . as we described above , inhibition of protein ubiquitination moreover , blockade of specific ubiquitination may exhibit less toxicity because inhibition of upstream molecules in key oncogenic signaling pathways may affect numerous signaling pathways and induce unfavorable side effects . clearly , further studies for signaling pathways in cancer including post - translational modifications are required for the development of effective personalized cancer therapies . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the ubiquitin proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer . activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development , and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors . three kinases , akt , extracellular signal - regulated kinase , and ib kinase , we refer to as oncokinases , are activated in multiple human cancers . we and others have identified several key downstream targets that are commonly regulated by these oncokinases , some of which are regulated directly or indirectly via ubiquitin - mediated proteasome degradation , including foxo3 , -catenin , myeloid cell leukemia-1 , and snail . in this review , we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic .

the use of various imaging techniques in post - treatment assessment of head neck cancer is gradually on the rise , though clinical examinations at periodic intervals still remains the mainstay of follow - up . 18f fdg pet / ct is increasingly used in the post - treatment setting to diagnose residual / recurrent disease early so that appropriate treatment can be initiated . the effects of multimodality treatment such as surgery , radiation , and chemotherapy can distort normal anatomical landmarks induce inflammation and infection , which can mask disease . it is important to understand the anatomical and functional effects as well as complications produced by these treatment modalities in order to diagnose the resultant imaging pitfalls on fdg pet / ct . knowledge of the common and unusual patterns of loco - regional recurrent disease and distant metastases on fdg pet / ct is also crucial in the post - treatment assessment of head - neck cancer patients . radiation therapy alone or in combination with chemotherapy is often used to treat head - neck cancer patients . there are several tissue changes , which are a result of early reaction to radiation . these include thickening of the skin and platysma , stranding of the subcutaneous fat , pharyngeal and laryngeal wall thickening and increased vascularity of the major and minor salivary glands . edema , inflammation , and increased vascularity in these structures lead to a pattern of fdg uptake which is low grade , diffuse , symmetrical , and restricted to the radiation field [ figure 1 ] . these findings are seen during the course or within 3 months of radiation therapy and are often reversible . several muscle groups within the radiation field also demonstrate an elevated fdg activity which can be potentially confused with neoplastic uptake . however , absence of a mass lesion on ct and reduction over time differentiate an inflammatory or physiological uptake from disease . axial pet and fused pet / ct images show diffuse symmetrical fdg uptake in the tongue and oropharynx ( arrows in the b and c ) and in the floor of mouth ( arrows in e and f ) . diffuse low intensity fdg uptake is also seen in the neck muscles ( arrowheads in b , c , e , f ) . diffuse uptake is seen within a few days of radiation and can last up to 810 weeks timing of pet / ct after radiation therapy is an important and often debated clinical issue . performing pet / ct as early as possible to detect recurrent disease seems logical as salvage treatment can be initiated early enough to derive the best possible clinical benefit . however , studies performed too early may lead to false positive as well as false negative results . possible mechanism for false negative studies has been attributed to radiation induced vascular damage , which temporarily prevents concentration of radiotracer in the viable tumor cells . imaging after a further few weeks delay in such cases may allow accumulation of tracer in the viable cells leading to a true positive pet result . waiting too long to image might result in a loss of therapeutic window and also a more complicated surgical procedure due to fibrosis setting in . in order to strike a balance between misleading pet results when study is performed early and the clinical drawbacks of imaging late , a time interval of 12 weeks after completion of radiation therapy is generally recommended . absence of fdg uptake at both the primary and nodal sites has a high negative predictive value in ruling out residual disease [ figure 2 ] . studies have shown that a negative pet scan can potentially defer planned neck dissections after chemo - radiation therapy . pretreatment coronal fused pet / ct ct studies show fdg avid right sided hypopharyngeal mass ( arrow in a and b ) . study performed 8 weeks after completion of chemo - radiation therapy show complete metabolic and morphologic response ( arrow in c and d ) which rules out the possibility of residual disease curative resection involves complex surgical procedures , which result in loss of symmetry and anatomical landmarks . various reconstructive techniques are used to close the surgical defects and restore function which produces characteristic well - recognizable imaging findings . procedures such as glossectomy , marginal or segmental mandibulectomy , maxillectomy leave large surgical defects , which are closed by flaps and grafts . pectoralis myocutaneous flap is an example of a composite flap which is often used for reconstruction of large surgical defects in the face , tongue , and skull base . they can be recognized by the well marginated soft tissue / muscle density at the site of the defect and is often accompanied by fat density [ figure 3a , b ] produced by a denervation atrophy and fatty replacement of the muscle . normal symmetric pattern of tracer concentration is disturbed due to absence of physiological fdg uptake at the resected and reconstructed site [ figure 3b and d ] . in patients undergoing total laryngo - pharyngectomy , a neopharynx is reconstructed which can be recognized as a simple tubular structure sans the accompanying complex anatomy of hypopharynx and larynx [ figure 4 ] . the neopharynx is often constructed by using a jejunal free flap , which is recognised recognized by the mesenteric fat along with its native vessels . axial fused pet / ct images show myocutaneous flap with fat density used to reconstruct the defect after hemimandibulectomy ( arrow in a ) and mandibular symphysectomy ( arrow in b ) performed for buccal cancer absence of normal physiological fdg uptake in the reconstructed flap ( arrowheads in a and b ) tissue changes after surgery . axial ct shows a reconstructed neopharynx after a laryngopharyngectomy seen as a tubular structure ( arrow in a ) . diffuse low grade physiological uptake is seen around the neopharynx ( arrow head in b ) during or after the course of treatment pitfalls in image interpretation can occur due to several factors . asymmetric nature of physiological uptake , inflammations / infective processes , and treatment - related complications are some of the common causes of false positive results leading to imaging pitfalls . sometimes , as a consequence of late effects of radiation therapy , there is a reduction of the normal physiological fdg uptake in structures such as the salivary glands and pharyngeal mucosal lining on the side of the therapy , leading to an appearance of asymmetric increased uptake in the normal tissues on the contralateral side [ figure 5 ] . coronal and axial ( pet ) and fused pet / ct images show reduction in physiological uptake ( arrows in a , b , d ) on the left side due to long term effects of radiation therapy . note the relative increase in physiological uptake in the parotid and submandibular glands and the oropharynx on the contralateral normal side which can mimic pathology ( arrowheads in a and d ) absence of physiological fdg uptake in the region of the surgically removed part and persistence of physiological uptake in the normal contralateral side produces an appearance of asymmetric tracer concentration that can mimic disease [ figure 6 ] . occasionally , after partial resection of an organ such as the tongue , the remnant portion retains its physiological uptake , which appears focal in nature mimicking disease [ figure 7 ] . after extensive jaw surgeries and reconstructive procedures , the altered mechanics of mastication can result in physiologically increased focal uptake in the adjacent masticator muscles that can be mistaken for disease [ figure 8 ] . axial ct shows changes of right hemiglossectomy with a myocutaneous flap containing fat ( arrowhead in a ) . axial pet and fusion pet / ct show physiological uptake in the remnant tongue which is focal and asymmetric in nature ( arrows in b and c ) and can potentially mimic disease pitfalls of due asymmetric physiological uptake . coronal pet and fusion pet / ct show physiological uptake along the right side of the oral cavity and the floor of mouth which is asymmetric in nature ( arrows in b and c ) and can potentially mimic disease . note the absence of normal physiological uptake on the left side ( arrowheads in b and c ) . this pattern was seen more than a year after treatment pitfalls of due asymmetric physiological uptake . axial pet and fusion pet / ct show focal asymmetric uptake in the left masseter which is physiological in nature ( arrows in a and b ) and is produced due to altered mechanics of mastication secondary to jaw surgery physiological uptake in the tongue tip and oral cavity arising due to close approximation of tongue and palate , buccal and gingival mucosae can be better resolved by performing certain maneuvers like puffing the cheek and placing a gauzegauze that can help mouth opening . surgical complications occur early and include serous collections , infections , abscess and fistula formation , flap necrosis . benign serous collections are self - limiting and should be distinguished from infected abscesses that might need a drainage procedure . benign serous collections also called seromas usually do not show any fdg avidity unless they get infected . on fdg pet / ct abscesses show a rim of tracer uptake in the periphery with a photopenic center which corresponds to a hypodense collection on ct with air pockets seen occasionally . linear fdg uptake is seen along the fistulous communications associated with abscesses [ figure 9 ] . these complications occur at the site of surgical resection and reconstructed flaps in the head neck region . rarely , one can come across infections / inflammations at the site of the donor flap in the chest or the abdominal wall and focal tracer uptake in this region can potentially mimic metastatic disease [ figure 10 ] . axial fused pet / ct shows an intense focus of fdg uptake in the infratemporal fossa mimicking disease recurrence ( arrow in b ) . corresponding contrast ct image shows a hypodense collection with an air pocket ( arrow in a ) suggestive of an abscess . note the linear fdg uptake along the enhancing fistulous tract opening on the skin surface . ( arrowheads in a and b ) pitfalls due to treatment related complications ( surgery ) . coronal mip image shows an intense focus of fdg uptake in the left hemithorax ( arrow in a ) which appears to be metastatic disease . fused pet / ct shows focal uptake in the anterior chest wall ( arrow in c ) which was the site for the pmmc flap . arrowhead in b ) radiation toxicity can lead to intense inflammatory changes in the mucosal structures and the soft tissues of the neck that can cause intense fdg accumulation [ figure 11 ] . coronal pet ( b ) and fused pet / ct ( c ) shows intense fdg uptake in the naso , oro , and hypopharyngeal structures and the soft tissue of the neck bilaterally , with associated ill - defined fat stranding ( a arrowhead ) . such intense non - infective inflammation is seen on rare occasions after radiation and should not be confused with recurrence radiation induced necrosis can occur in bones ( osteoradionecrosis [ orn ] ) , cartilage ( chondronecrosis / laryngeal necrosis ) and even in irradiated soft tissues . osteoradionecrosis ( orn ) occurs due to devitalization of irradiated bone which gets exposed through the skin and mucosa and remains without healing for at least 3 months . the risk of orn is greatest at 6 - 12 months after radiation therapy and it is uncommon to see orn at radiation doses below 60 gy . lytic destruction , cortical erosion and fragmentation of the mandible with associated fistulae and soft tissue thickening are some of the features seen on ct scan [ figure 12 ] . increased fdg avidity is seen in areas affected by orn [ figure 12 ] , but its specificity in differentiating viable tumor from radionecrosis is not reported to be very high . orn can occur at rare sites such as the hyoid bone and increased fdg avidity may lead to the erroneous diagnosis of viable disease unless the physician is aware of its occurrence [ figure 13 ] . characteristics osseous changes of orn and absence of associated soft tissue lesion make the diagnosis of radiation necrosis more likely axial ct in soft tissue ( a ) and bone window settings ( b ) show soft tissue thickening , erosion and fragmentation of the mandible ( arrows ) . coronal fused pet / ct shows intense fdg uptake restricted to right hemimandible ( arrowhead in c and d ) . findings suggest the diagnosis of orn pitfalls due to treatment related complications - osteoradionecrosis [ orn ] . axial fused pet / ct shows intense fdg uptake along the hyoid bone ( arrowhead in a and b ) . axial ct shows subtle erosion of the hyoid bone along with a small collection and air pockets ( arrows in c and d ) . sometimes , as a consequence of late effects of radiation therapy , there is a reduction of the normal physiological fdg uptake in structures such as the salivary glands and pharyngeal mucosal lining on the side of the therapy , leading to an appearance of asymmetric increased uptake in the normal tissues on the contralateral side [ figure 5 ] . coronal and axial ( pet ) and fused pet / ct images show reduction in physiological uptake ( arrows in a , b , d ) on the left side due to long term effects of radiation therapy . note the relative increase in physiological uptake in the parotid and submandibular glands and the oropharynx on the contralateral normal side which can mimic pathology ( arrowheads in a and d ) absence of physiological fdg uptake in the region of the surgically removed part and persistence of physiological uptake in the normal contralateral side produces an appearance of asymmetric tracer concentration that can mimic disease [ figure 6 ] . occasionally , after partial resection of an organ such as the tongue , the remnant portion retains its physiological uptake , which appears focal in nature mimicking disease [ figure 7 ] . after extensive jaw surgeries and reconstructive procedures , the altered mechanics of mastication can result in physiologically increased focal uptake in the adjacent masticator muscles that can be mistaken for disease [ figure 8 ] . axial ct shows changes of right hemiglossectomy with a myocutaneous flap containing fat ( arrowhead in a ) . axial pet and fusion pet / ct show physiological uptake in the remnant tongue which is focal and asymmetric in nature ( arrows in b and c ) and can potentially mimic disease pitfalls of due asymmetric physiological uptake . coronal pet and fusion pet / ct show physiological uptake along the right side of the oral cavity and the floor of mouth which is asymmetric in nature ( arrows in b and c ) and can potentially mimic disease . note the absence of normal physiological uptake on the left side ( arrowheads in b and c ) . this pattern was seen more than a year after treatment pitfalls of due asymmetric physiological uptake . axial pet and fusion pet / ct show focal asymmetric uptake in the left masseter which is physiological in nature ( arrows in a and b ) and is produced due to altered mechanics of mastication secondary to jaw surgery physiological uptake in the tongue tip and oral cavity arising due to close approximation of tongue and palate , buccal and gingival mucosae can be better resolved by performing certain maneuvers like puffing the cheek and placing a gauzegauze that can help mouth opening . surgical complications occur early and include serous collections , infections , abscess and fistula formation , flap necrosis . benign serous collections are self - limiting and should be distinguished from infected abscesses that might need a drainage procedure . benign serous collections also called seromas usually do not show any fdg avidity unless they get infected . on fdg pet / ct abscesses show a rim of tracer uptake in the periphery with a photopenic center which corresponds to a hypodense collection on ct with air pockets seen occasionally . linear fdg uptake is seen along the fistulous communications associated with abscesses [ figure 9 ] . these complications occur at the site of surgical resection and reconstructed flaps in the head neck region . rarely , one can come across infections / inflammations at the site of the donor flap in the chest or the abdominal wall and focal tracer uptake in this region can potentially mimic metastatic disease [ figure 10 ] . axial fused pet / ct shows an intense focus of fdg uptake in the infratemporal fossa mimicking disease recurrence ( arrow in b ) . corresponding contrast ct image shows a hypodense collection with an air pocket ( arrow in a ) suggestive of an abscess . note the linear fdg uptake along the enhancing fistulous tract opening on the skin surface . ( arrowheads in a and b ) pitfalls due to treatment related complications ( surgery ) . coronal mip image shows an intense focus of fdg uptake in the left hemithorax ( arrow in a ) which appears to be metastatic disease . fused pet / ct shows focal uptake in the anterior chest wall ( arrow in c ) which was the site for the pmmc flap . arrowhead in b ) radiation toxicity can lead to intense inflammatory changes in the mucosal structures and the soft tissues of the neck that can cause intense fdg accumulation [ figure 11 ] . coronal pet ( b ) and fused pet / ct ( c ) shows intense fdg uptake in the naso , oro , and hypopharyngeal structures and the soft tissue of the neck bilaterally , with associated ill - defined fat stranding ( a arrowhead ) . such intense non - infective inflammation is seen on rare occasions after radiation and should not be confused with recurrence radiation induced necrosis can occur in bones ( osteoradionecrosis [ orn ] ) , cartilage ( chondronecrosis / laryngeal necrosis ) and even in irradiated soft tissues . osteoradionecrosis ( orn ) occurs due to devitalization of irradiated bone which gets exposed through the skin and mucosa and remains without healing for at least 3 months . the risk of orn is greatest at 6 - 12 months after radiation therapy and it is uncommon to see orn at radiation doses below 60 gy . lytic destruction , cortical erosion and fragmentation of the mandible with associated fistulae and soft tissue thickening are some of the features seen on ct scan [ figure 12 ] . increased fdg avidity is seen in areas affected by orn [ figure 12 ] , but its specificity in differentiating viable tumor from radionecrosis is not reported to be very high . orn can occur at rare sites such as the hyoid bone and increased fdg avidity may lead to the erroneous diagnosis of viable disease unless the physician is aware of its occurrence [ figure 13 ] . characteristics osseous changes of orn and absence of associated soft tissue lesion make the diagnosis of radiation necrosis more likely ( b ) show soft tissue thickening , erosion and fragmentation of the mandible ( arrows ) . coronal fused pet / ct shows intense fdg uptake restricted to right hemimandible ( arrowhead in c and d ) . findings suggest the diagnosis of orn pitfalls due to treatment related complications - osteoradionecrosis [ orn ] . axial fused pet / ct shows intense fdg uptake along the hyoid bone ( arrowhead in a and b ) . axial ct shows subtle erosion of the hyoid bone along with a small collection and air pockets ( arrows in c and d ) . fdg pet / ct has been used to detect recurrent disease in the head and neck cancer patients . it can confirm the site of clinically suspected local recurrence and determine its true extent . recurrences at the site of the treated primary disease , subtle disease in the reconstructed flaps and nodal / soft tissue recurrence in the irradiated neck , the clinical assessment of which is difficult can be seen on pet / ct as enhancing lesions with intense fdg avidity [ figures 14 and 15 ] . at the same time , it can pick up distant failures with a good accuracy and thus is performed before salvage surgery can potentially change the intended treatment plan . in addition to common sites of distant metastases like the lungs and bones , pet / ct has the potential to unmask second primaries as well as certain unusual and rare metastatic sites such as the subcutaneous tissue , muscles , and feeding stomies [ figures 16 and 17 ] . recurrent disease . operated case of buccal cancer . coronal mip shows intense foci of fdg uptake on the left side of the face and neck ( arrowhead and arrows in a ) . axial fused pet / ct shows fdg avid recurrent nodule in the pmmc flap ( arrowhead in b and c ) and a metastatic cervical lymph node ( arrow in d and e ) recurrent disease . sagittal mip shows foci of fdg uptake in the face ( arrows in a ) . axial fused pet / ct ( arrowhead in b and c ) shows subtle uptake at the cut margin of the mandible due to local recurrence as well as metastatic deposit in the pterygopalatine fossa ( arrows in d and e ) distant metastases . fusion pet / ct images show metastatic deposits in spleen ( b ) , lungs ( c ) , liver ( d ) , bone ( e ) , subcutaneous region ( f ) and muscle ( g ) distant metastases ( unusual sites ) . operated case of base tongue cancer with feeding gastrostomy . sagittal pet and pet / ct images show a fdg avid mass in the anterior abdominal wall at the site of the gastrostomy ( arrows in a - b ) s / o metastatic deposit . local recurrence in the tongue base fdg pet / ct is a useful modality in the post - treatment setting of head and neck cancers . knowledge of certain characteristic imaging appearances and a few commonly encountered treatment related complications is important to avoid pitfalls in post - treatment imaging . understanding the role of fdg pet / ct in detecting local recurrence and distant metastases plays a crucial role in deciding salvage treatment .

majority of patients with head and neck cancer are treated with combined treatment regimes such as surgery , radiation therapy , and chemotherapy . the loss of structural symmetry and imaging landmarks as a result of therapy makes post - treatment imaging a daunting task on conventional modalities like computed tomography ( ct ) scan and magnetic resonance imaging ( mri ) as well as on 18 fluorine - fluorodeoxyglucose positron emission tomography / computed tomography ( 18f fdg pet / ct ) . combined multimodality treatment approach causes various tissue changes that give rise to a spectrum of findings on fdg pet / ct imaging , which are depicted in this atlas along with a few commonly encountered imaging pitfalls . the incremental value of fdg pet / ct in detecting locoregional recurrences in the neck as well as distant failures has also been demonstrated .

split - liver transplantation ( slt ) is an attractive alternative procedure to expand the donor pool in patients waiting for liver transplantation . the standard split - liver procedure for a child and an adult ( adult / pediatric split liver ttransplantation , a / p slt ) , by splitting segment ii - iii for pediatric recipient and segment i - iv - v - vi - vii - viii for an adult , is an accepted surgical option with good results both for the adult and for pediatric recipient . biliary complications continue to be the major cause of morbidity after slt with a reported high incidence ranging between 10% and 32% [ 1 , 2 ] . as a consequence of bile duct anatomic variations , slt requires a precise knowledge of the liver anatomy [ 3 , 4 ] . the challenge of this procedure is represented by a preoperative radiological assessment of the biliary anatomy often unavailable at the donor 's hospital . thus the risk of biliary duct injury during the splitting procedure is usually considered higher than during living donor procedure . in this report we describe an uncommon late biliary complication that occurred after slt and was successfully treated by a multidisciplinary approach . a 63-year - old male with hepatitis c - related cirrhosis was referred for liver transplantation to our institution . we performed a conventional a / p slt with in situ technique providing the left lateral segment for a child ( segments ii - iii ) and leaving the right lobe graft ( segment i - iv - v - vi - vii - viii ) for an adult recipient . the celiac trunk was left on the left graft while the right hepatic artery remained on the right graft . the patient was transplanted using the piggy - back technique without a veno - venous bypass . the biliary tract was reconstructed performing a duct - to - duct anastomosis using a t - tube by our standard technique previously described . a cholangiography through the t - tube was performed on postoperative day 14 , and the t - tube was clamped before patient discharge . three months after a / pslt the t - tube was removed after a cholangiography with normal findings . one year after transplant the patient showed abnormal liver function tests , hyperbilirubinemia , leukocytosis , and elevated g - glutamyl transpeptidase ( ggt ) , and mild elevation in alanine transaminase ( alt ) . the patient underwent a doppler ultrasound that showed ( a patent hepatic artery ) an intrahepatic bile duct dilatation and an anastomotic biliary stricture . the anastomotic stricture was treated by stenting the main biliary duct during an endoscopic retrograde cholangiopancreatoghaphy ( ercp ) without any evidence of intrahepatic biliary dilatation . after this procedure the patient was submitted to a percutaneous transhepatic cholangiography ( ptc ) showing a complete obstruction of segments vi and vii biliary branches near the duct - to - duct biliary anastomosis ( figure 1 ) . the patient was discharged leaving the external biliary drain open allowing bile drainage and an easy access for repeated radiologic treatment and an internal stent in the common bile duct . three months later the patient underwent a surgical revision because of repeated episodes of cholangitis . during surgery an intraoperative cholangiography was performed through the biliary drain confirming a bile duct dilatation at the level of segments vi and vii . it was impossible to cross the biliary stricture by a torque catheter and by hydrophilic guide wire ( figure 2 ) . we supposed that the segmentary bile duct branch of posterior segments vi and vii draining in the left bile duct was unidentified and tied at the time of the in situ split - liver procedure during the parenchymal transection . a primary biliary reconstruction by biliary repair or biliodigestive anastomosis was considered at high risk because of the presence of postsurgical adhesions sand due to fibrotic tissue replacing the parenchymal transected area . a liver resection of the dilated segments vi and vii was considered at high risk of leaving an inadequate liver mass . we decided to perform a permanent intraoperative obliteration of the dilated intrahepatic ducts by a percutaneous embolization using a nonresorbable agent . with a fluoroscopic guidance through the transhepatic access we positioned a 5-french polyethylene catheter inside the ducts , preliminary flushed by a nonionic dextrose solution . we then injected the tissue adhesive agent n - butyl cyanoacrylate ( nbca , glubran 2 , gem , viareggio , italy ) mixed with ionized oil ( lipiodol , guebert , aulnay - sous - bois , france ) for opacization in a ratio of 1:5 . this solution completely filled the biliary duct , and the occlusion was totally accomplished in a few seconds ( figure 3 ) . during the first 3 days after the chemical bile duct embolization , the patient had a low fever with a slightly abnormal liver function test . a computed tomography ( ct ) scan performed 6 months later showed no sign of hepatic abscesses , and the bile duct dilatation was completely occupied by the nbca - lipiodol mixture . one year after the procedure patient showed normal liver function tests without no episodes of cholangitis . chemical bile duct embolization treatment could represent a valuable solution to treat uncommon biliary complications . these tissue adhesive glues are low - viscosity liquid monomers that undergo rapid polymerization and solidification when they come into contact with organic fluids such as bile . nbca is a permanent liquid embolic material that produces long - term occlusion in vessels of various size through an inflammatory tissue response resulting in vessel thrombosis or tissue atrophy . little is known about the use of cyanoacrylate compounds , and unlike european countries the use of glubran has not been approved by the food and drug administration yet . other authors have described the efficacy of biliary duct ablation by nbca in patients with persistent postsurgical bile leaks after lobectomy or cholecystectomy . vu et al . treated six patients with persistent postsurgical bile leaks as a complication after hepatic lobectomy or cholecistectomy using nbca glue for the obliteration of isolated segmental bile ducts in four cases . endoscopic treatment of biliary leakage by nbca has been described by seewald in nine patients in whom primary stent placement or nasobiliary drain was unsuccessful . described the use of a cyanoacrlylate in the treatment of a pancreatic fistula after distal pancreatectomy . the percutaneous interventional technique represents an effective valuable approach to reduce mortality and morbidity in the treatment of biliary complications after liver transplantation . the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection above all in high - risk patients with posttransplant bile duct injuries , decreasing the morbidity associated with chronic external biliary drainage . futher studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and costs .

biliary complications continue to be a major cause of morbidity after split - liver transplantation ( slt ) . in this report we describe an uncommon late biliary complication . one year after slt the patient showed an intrahepatic bile dicy dilatation with severe cholangitis episodes . the segmentary bile duct of hepatic segment vi - vii draining in the left duct was unidentified and tied at the time of the in situ split - liver procedure . we perform a permanent obliteration of the dilated intrahepatic ducts by a percutaneous embolization using an n - butyl cyanoacrylate ( nabc ) . the management of biliary complications after slt requires a multidisciplinary approach . the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection and decreasing the morbidity associated with chronic external biliary drainage . further studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and cost .

sevoflurane has a low blood / gas partition coefficient and does not irritate the airways . inhalation induction should provide rapid loss of consciousness to ensure patient 's comfort and satisfaction and to avoid adverse effects such as excitations , breath holding , coughing and/or laryngospasm . several adjuvants and techniques have been proposed to ensure a rapid induction of anesthesia with sevoflurane , such as intravenous ( iv ) midazolam , clonidine , fentanyl , breathing different gas flows , or ventilation with larger tidal volumes . the effect of nitrous oxide ( n2o ) when added to the inhalation of sevoflurane in oxygen has been controversial . n2o added to oxygen was reported to make shorter the induction time with sevoflurane , an effect similar to that produced by clonidine . these results are not consistent with the results obtained from other studies , which conclude that the addition of n2o to the sevoflurane / oxygen mixture is not advantageous regarding the speed of induction of anesthesia . in contrast , results from previous studies point out that the addition of n2o to the sevoflurane / oxygen mixture may be associated with more , though minor , excitatory phenomena . our hypothesis was that the speed of inhalation induction with sevoflurane may benefit from the pretreatment with n2o , and subsequent volume contraction before sevoflurane administration . the aim of the present study was to investigate the effect of pretreatment with 50% n2o in oxygen on the speed of inhalation induction of anesthesia with sevoflurane in women undergoing minor gynecological procedures . the study was approved by the institutional review board ( n-79/04 - 04 - 06 ) . eighty patients american society of anesthesiology physical status i or ii scheduled for hysteroscopy under general anesthesia , who accepted an inhalation induction with no premedication , gave their written informed consent to participate in the study protocol . patients with body weight exceeding the ideal body weight by 25% , with central nervous system disease , respiratory disease , hyper - or hypothyroidism , intake of sedatives , hypnotics , and antidepressants were excluded from the study before randomization . patients were randomly allocated to the air ( fio2 = 0.21 ) or n2o ( n2o / o2:50/50 , fio2 = 0.5 ) group . randomization was done by means of odd and even numbers retrieved from a computer - generated table ( http://www.randomizer.org ) corresponding to the n2o and to the air group respectively . the pretreatment gas was administered via n2o - oxygen flow meters or an air flow meter connected to the wall pipelines , all covered by drapes so the investigators were blinded to the gas administered . group allocation to intervention was done by an independent anesthesiologist not participating in the study who arranged the gases before induction ( air or n2o / o2 ) , covered the flow meters with drapes and recorded the group each patient was assigned . in the operating room monitoring consisted of pulse oximetry , electrocardiogram , heart rate , noninvasive arterial pressure , end - tidal carbon dioxide ( etco2 ) , inspired and end - tidal sevoflurane concentrations , tidal volume and respiratory rate ( s/5 anesthesia monnitor , datex - ohmeda division , datex - ohmeda , helsinki , finland ) and bispectral index ( bis xp a-2000 monitor , system rev 3.21 , aspect medical systems , leiden , the netherlands ) . according to the group allocation , in the preinduction period patients were fully awake , breathing spontaneously either 50% n2o in oxygen ( fio2 = 0.5 ) or air ( fio2 = 0.21 ) for 10 min via a mapleson c breathing system and a tightly fitting face mask . the anesthetic circuit was primed with 8% sevoflurane : an oxygen flow 6 l / min was allowed to pass through the circle anesthetic system with the sevoflurane vaporizer dial being set at 8% for 5 min . an endotracheal tube was attached to the end of the anesthetic system with the distal end tightly surrounded by a plastic bag . after the completion of the 10 min administration of the n2o / oxygen or air , the face mask was connected to the sevoflurane primed anesthetic circuit and all patients were allowed to breathe spontaneously sevoflurane in oxygen with the sevoflurane vaporizer set at 8% and an oxygen flow rate 6 l / min . after a 5 min period of sevoflurane inhalation , the study protocol and data collection were completed . a short acting opioid was given iv and a supraglottic airway device was inserted for the ventilation of patients during surgery . during the 10 min preinduction period , bis , respiratory rate , tidal volume , etco2 , heart rate and oxygen saturation ( spo2 ) were recorded every minute . the same parameters were recorded every 30 s during the first 5 min induction period with the patient breathing sevoflurane in oxygen . during the induction period inspired and end - tidal sevoflurane concentrations were also recorded every 30 s. arterial blood pressure was recorded only before starting the pretreatment with the gas and after completion of measurements to avoid stimulus during cuff inflation affecting bis measurements . we hypothesized that the speed of inhalation induction with sevoflurane will be enhanced by n2o pretreatment , and possible subsequent volume contraction occurring before sevoflurane administration . bis was the primary outcome measure , the rest variables being secondary outcome measures . at each time point , three consecutive bis values were read and the median one was selected . initial sample size estimation showed that approximately 40 patients are needed in each group to detect a clinically relevant reduction of bis after induction of anesthesia by 30% with a power of 0.80 and level of significance of 5% . to assess differences in patients characteristics between the two groups , two independent samples t - tests for normally distributed responses and mann - whitney tests for nonnormally distributed responses were carried out . overall group differences in all variables were assessed by using repeated measures anova . a paired samples t - test or a mann - whitney test was used to assess differences at each time point wherever appropriate . all analyses were performed using the statistical package for social sciences ( spss ) software , version 11.0 ( spss inc . , chicago , il , usa ) . in the operating room a peripheral vein was catheterized and a ringer 's solution infusion was started . monitoring consisted of pulse oximetry , electrocardiogram , heart rate , noninvasive arterial pressure , end - tidal carbon dioxide ( etco2 ) , inspired and end - tidal sevoflurane concentrations , tidal volume and respiratory rate ( s/5 anesthesia monnitor , datex - ohmeda division , datex - ohmeda , helsinki , finland ) and bispectral index ( bis xp a-2000 monitor , system rev 3.21 , aspect medical systems , leiden , the netherlands ) . according to the group allocation , in the preinduction period patients were fully awake , breathing spontaneously either 50% n2o in oxygen ( fio2 = 0.5 ) or air ( fio2 = 0.21 ) for 10 min via a mapleson c breathing system and a tightly fitting face mask . the anesthetic circuit was primed with 8% sevoflurane : an oxygen flow 6 l / min was allowed to pass through the circle anesthetic system with the sevoflurane vaporizer dial being set at 8% for 5 min . an endotracheal tube was attached to the end of the anesthetic system with the distal end tightly surrounded by a plastic bag . after the completion of the 10 min administration of the n2o / oxygen or air , the face mask was connected to the sevoflurane primed anesthetic circuit and all patients were allowed to breathe spontaneously sevoflurane in oxygen with the sevoflurane vaporizer set at 8% and an oxygen flow rate 6 l / min . after a 5 min period of sevoflurane inhalation , the study protocol and data collection were completed . a short acting opioid was given iv and a supraglottic airway device was inserted for the ventilation of patients during surgery . during the 10 min preinduction period , bis , respiratory rate , tidal volume , etco2 , heart rate and oxygen saturation ( spo2 ) were recorded every minute . the same parameters were recorded every 30 s during the first 5 min induction period with the patient breathing sevoflurane in oxygen . during the induction period inspired and end - tidal sevoflurane concentrations were also recorded every 30 s. arterial blood pressure was recorded only before starting the pretreatment with the gas and after completion of measurements to avoid stimulus during cuff inflation affecting bis measurements . we hypothesized that the speed of inhalation induction with sevoflurane will be enhanced by n2o pretreatment , and possible subsequent volume contraction occurring before sevoflurane administration . bis was the primary outcome measure , the rest variables being secondary outcome measures . at each time point , three consecutive bis values were read and the median one was selected . initial sample size estimation showed that approximately 40 patients are needed in each group to detect a clinically relevant reduction of bis after induction of anesthesia by 30% with a power of 0.80 and level of significance of 5% . to assess differences in patients characteristics between the two groups , two independent samples t - tests for normally distributed responses and mann - whitney tests for nonnormally distributed responses overall group differences in all variables were assessed by using repeated measures anova . a paired samples t - test or a mann - whitney test was used to assess differences at each time point wherever appropriate . all analyses were performed using the statistical package for social sciences ( spss ) software , version 11.0 ( spss inc . , chicago , il , usa ) . the n2o and the air groups did not differ in age , body weight , and height . similarly , they did not differ in systolic and diastolic arterial pressure before or after the end of measurements [ table 1 ] . patient characteristics , sap and dap blood pressures before and after the measurements of the 80 patients recruited for the study , 43 patients were assigned to the n2o and 37 to the air group . four patients in the n2o group and one patient in the air group did not accept the facemask from the very beginning and no data were collected from these patients . one patient in the n2o group presented excitatory movements and removed the mask after n2o inhalation for 7 min and no further data were collected . in another patient in the air group bis values were not at any time point obtained due to technical reasons , but all the other variables were recorded and analyzed [ figure 1 ] . the flow diagram of the study during sevoflurane inhalation in the n2o group the study was interrupted in one patient at 120 s due to persisting cough and salivation , in one patient at 180 s due to tachypnea , tachycardia and upper airway obstruction , in one patient at 210 s due to tachypnea and tachycardia , in one patient at 240 s due to upper airway obstruction , and in one patient at 270 s due to prolonged apnea . the incidence of complications during pretreatment with n2o or air followed by sevoflurane induction in the n2o and air groups during the 10 min period of breathing 50% n2o in oxygen or air , the bis , etco2 , tidal volume , respiratory rate and heart rate did not differ between the group given n2o and that given air , nor was there a trend to a difference . spo2 was slightly but significantly greater in patients given n2o , consistent with the higher inspired oxygen concentration of oxygen breathed . during sevoflurane induction bis values were lower in the n2o group [ table 3 and figure 2 ] . at the time points 60 , 90 , 120 and 150 s , the bis values in the n2o versus air group were : 77 19.0 versus 92 8.6 ( p < 0.001 ) , 44 19.8 versus 73 21.4 ( p < 0.001 ) , 36 12.4 versus 55 24.7 ( p = 0.002 ) and 32 11.0 versus 45 20.8 ( p = 0.014 ) , respectively . etco2 and tidal volume values were lower and respiratory rate higher in the n2o group when compared to the air group . spo2 , heart rate , inspired and end - tidal sevoflurane concentration did not differ significantly between the two groups during the first 300 s of sevoflurane inhalation [ table 3 ] . bis , etco2 , vt , rr , spo2 , hr and fi and fe sevoflurane concentrations in the n2o- and air - breathing groups during induction of anesthesia with sevoflurane bispectral index values ( mean standard deviation ) during inhalation with sevoflurane in the nitrous oxide and air groups the results of the present study suggest that pretreatment with n2o in 50% oxygen for 10 min before inhalation induction with sevoflurane enhances induction as assessed by decreased bis values . these results are not consistent with other studies when n2o was administered concomitantly with sevoflurane for inhalation induction . nevertheless , these studies were based on clinical criteria , which are subjective and therefore more difficult to be quantified . found that the addition of 50% n2o to the sevoflurane / oxygen mixture had no effect on the speed of induction and was associated with more excitatory effects . the investigators defined induction time as the time needed for the raised arm to fall to the horizontal level . also , according to siau and liu , 66% n2o added to oxygen did not decrease the induction time with 8% sevoflurane when compared to the induction time of 8% sevoflurane in oxygen alone . these investigators administered higher concentrations of n2o than those administered in the present study but did not prime the circle breathing circuit with sevoflurane . in a previous study , we have shown that pretreatment with 100% oxygen enhances the inhalation induction with 7% sevoflurane as assessed by bis monitoring . replacing nitrogen in the lungs with a more soluble gas like oxygen , results in lung collapse , decreased functional residual capacity and increased alveolar concentration of the inhaled anesthetic , that is , sevoflurane . in contrast to oxygen , in the present study the n2o was discontinued during induction . it is worthy to note that during induction , the end - tidal ( fe ) sevoflurane concentration ( though no statistically significant ) was higher during the first 120 s. the higher fe is consistent with increased ventilation in the n2o group , as indicated by the higher respiratory rates and the lower co2 values . , oxygen is also expected to enhance the sevoflurane uptake and to diminish the difference between the n2o and air groups as it is more soluble than nitrogen and part of the lungs will collapse to some extent ( absorption atelectasis ) . similarly , n2o - being 32 times more soluble than nitrogen - is rapidly up taken by the blood , resulting in lung contraction and increased alveolar concentration of sevoflurane . however , eventually alveolar collapsing will be associated with pulmonary shunt which will delay sevoflurane uptake . the time points at which bis values differ significantly in the present study are between 60 and 150 s. the inspired and expired sevoflurane concentrations did not differ between the n2o and the air groups during the 300 s period of induction , though the end - tidal / inspired concentration ( f e / fi ) ratio for sevoflurane was consistently higher by 2 - 7% in the n2o group . sevoflurane due to its low blood gas partition coefficient might not benefit from the second gas effect phenomenon . however taheri and eger have nicely demonstrated the second gas effect for desflurane , an agent even less blood soluble . demonstrated a higher ratio of alveolar / inspired ( f a / fi ) sevoflurane concentration when n2o was added to the inspired mixture . peyton et al . reported a two to three times more powerful second gas effect of n2o on arterial versus the end - expired sevoflurane pressures and attributed this effect to ventilation perfusion inhomogeneity by general anesthesia . in both studies , our data were collected with the patients breathing spontaneously and receiving solely sevoflurane without other anesthetics or adjuvants . pretreatment with n2o may not ensure an overall faster inhalation induction , but highlights the failure of previous studies to demonstrate enhancement of sevoflurane induction by concomitant n2o administration . previous studies failed to show a more rapid induction of anesthesia with sevoflurane for various reasons . as sevoflurane is an agent with low solubility , it may not benefit the very first minutes of induction by the concomitant administration of n2o . so we remain with our hypothesis that this difference between the n2o and air groups may be attributed to the pretreatment rather than the co - administration of n2o during sevoflurane induction of anesthesia . a very early increased concentration effect due to volume reduction and before the shunt effect , as n2o is taken up by the pulmonary capillaries or increased ventilation , as shown by the lower etco2 , in the n2o group may have influenced the speed of the sevoflurane uptake . in clinical practice , fast inhalational induction may be beneficial for both patients and hospitals ; it may shorten the second stage of anesthesia , thus reducing the risk of agitation / adverse respiratory events , and increase patient 's satisfaction , while - depending on the time saved - it may also decrease the overall operating room time and facilitate fast turnover . our measurements are limited by the sensitivity of the device we used to measure sevoflurane and n2o , thus the infrared analyzer , as the gold standard to measure concentrations of inhaled anesthetics is the gas chromatography or the mass spectrometry . other limitations of the study are the number of dropouts , which may have an impact on the study power and the relatively long period of n2o pretreatment which weakens the clinical implementation of our results and shorter pretreatment periods like 2 or 3 min may be clinically applicable . under the present study design , breathing of 50% n2o in oxygen before induction with sevoflurane enhances the speed of induction in the very early period as assessed by the bis monitor .

background and aims : inhalation anesthesia with sevoflurane may be enhanced by several drugs or techniques . the aim of the present study was to investigate the effect of nitrous oxide ( n2o ) pretreatment on the speed of anesthesia induction with sevoflurane.material and methods : eighty patients scheduled for hysteroscopy under general anesthesia were randomly assigned to inhale for 10 min before induction 50% n2o in oxygen or air via a facemask . anesthesia was induced with 7 - 8% sevoflurane in oxygen via a facemask . bispectral index ( bis ) , end - tidal carbon dioxide ( etco2 ) tidal volume , respiratory rate , oxygen saturation ( spo2 ) , and heart rate were recorded every minute during the 10 min pretreatment periods and every 30 s during the first 300 s of induction with sevoflurane . during induction of anesthesia inspired and end - tidal sevoflurane concentrations were also recorded.results:during the 10 min of inspired 50% n2o or air bis , etco2 , tidal volume , respiratory rate and heart rate values did not differ between the two groups except for the spo2 , which was higher in the n2o group ( p < 0.001 ) . during induction of anesthesia the n2o group exhibited lower bis values ( p = 0.001 ) , being significant at 60 - 150 s ( p < 0.001 , p < 0.001 , p = 0.002 , p = 0.014 ) as well as at 270 s ( p = 0.004 ) . etco2 and tidal volume were consistently lower in the n2o group ( p = 0.001 , p = 0.041 respectively ) and respiratory rate was higher ( p = 0.007).conclusion : our results show that pretreatment of the patients with 50% n2o for 10 min enhances the speed of induction with sevoflurane as assessed by the bis monitoring .

the 2 huvec cell lines used in the study were derived from male caucasian donors genotyped as heterozygotes for the 9p21 cad associated snps . the 4 ebv transformed lymphoblastoid cell lines ( lcl ) were selected for their genotypes ( 2 homozygous cad risk or 2 homozygous cad non - risk ) using the hapmap data27 . experiments were performed within 24 passages by treating cells with 100 ng / ml of ifn ( r&d ) for 24 hrs . treated and untreated cells were subjected to rt - pcr ( huvec ) , chip ( huvec and lcl ) , 3c analysis ( huvec ) or fish ( huvec ) . probe design : we designed 12 acceptor probes in the interval chr9:22100523 - 22126469 ( hg18 ; spanning from ecad7 to the et2d2 enhancer ) , on both strands immediately 3 of the 6 bamhi sites in the region . we designed 290 donor probes on both strands immediately 5 of the 145 bamhi sites in the interval chr9:21035934 - 22494089 ( hg18 ; except where acceptor probes were designed ) . a universal sequence added to the probes is compatible with illumina ga adapters for direct sequencing . the 12 acceptor probes and 290 donor probes ( supplemental table 8) were pooled in equimolar amounts , separately . 3d - dsl sequencing : the dsl ligation products were prepared as described in kwon et al.23 . 3c was performed as per lieberman - aiden et al 28 and the products were sheared by sonication . donor and acceptor probes pools were annealed to the biotinylated 3c samples and the biotinated dna was bound on to streptavidin magnetic beads . the 5 phosphate of acceptor probes and 3 oh of donor probes were ligated using taq dna ligase . the ligated products were washed and eluted from the streptavidin magnetic beads , followed by pcr amplification and deep sequencing on the illumina ga2 ( supplemental information ) . the 2 huvec cell lines used in the study were derived from male caucasian donors genotyped as heterozygotes for the 9p21 cad associated snps . the 4 ebv transformed lymphoblastoid cell lines ( lcl ) were selected for their genotypes ( 2 homozygous cad risk or 2 homozygous cad non - risk ) using the hapmap data27 . experiments were performed within 24 passages by treating cells with 100 ng / ml of ifn ( r&d ) for 24 hrs . treated and untreated cells were subjected to rt - pcr ( huvec ) , chip ( huvec and lcl ) , 3c analysis ( huvec ) or fish ( huvec ) . probe design : we designed 12 acceptor probes in the interval chr9:22100523 - 22126469 ( hg18 ; spanning from ecad7 to the et2d2 enhancer ) , on both strands immediately 3 of the 6 bamhi sites in the region . we designed 290 donor probes on both strands immediately 5 of the 145 bamhi sites in the interval chr9:21035934 - 22494089 ( hg18 ; except where acceptor probes were designed ) . a universal sequence added to the probes is compatible with illumina ga adapters for direct sequencing . the 12 acceptor probes and 290 donor probes ( supplemental table 8) were pooled in equimolar amounts , separately . 3d - dsl sequencing : the dsl ligation products were prepared as described in kwon et al.23 . 3c was performed as per lieberman - aiden et al 28 and the products were sheared by sonication . donor and acceptor probes pools were annealed to the biotinylated 3c samples and the biotinated dna was bound on to streptavidin magnetic beads . the ligated products were washed and eluted from the streptavidin magnetic beads , followed by pcr amplification and deep sequencing on the illumina ga2 ( supplemental information ) .

genome wide association studies ( gwas ) have identified snps in the 9p21 gene desert associated with coronary artery disease ( cad)14 and type 2 diabetes ( t2d)57 . despite evidence for a role of the associated interval in neighboring gene regulation810 , the biological underpinnings of these genetic associations to cad or t2d have not yet been explained . here we identify 33 enhancers in 9p21 ; the interval is the second densest gene - desert for predicted enhancers and 6 times denser than the whole genome ( p<6.55 1033 ) . the cad risk alleles of snps rs10811656/rs10757278 are located in one of these enhancers and disrupt a binding site for stat1 . lymphoblastoid cell lines ( lcl ) homozygous for the cad risk haplotype exhibit no binding of stat1 , and in lcl homozygous for the cad non - risk haplotype binding of stat1 inhibits cdkn2bas expression , which is reversed by sirna knock - down of stat1 . using a new , open - ended approach to detect long - distance interactions ( 3d - dsl ) , we find that in human vascular endothelium cells ( huvec ) the enhancer interval containing the cad locus physically interacts with the cdkn2a / b locus , the mtap gene and an interval downstream of infa21 . in huvec , ifn activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus , including stat1 binding , long - range enhancer interactions and altered expression of neighboring genes . our findings establish a link between cad genetic susceptibility and the response to inflammatory signaling in a vascular cell type and thus demonstrate the utility of gwas findings to direct studies to novel genomic loci and biological processes important for disease etiology .

intravesical instillation of bacillus calmette - guerin ( bcg ) is an established option for the treatment and prophylaxis of non - muscle - invasive bladder cancer . low - grade fever and irritative bladder symptoms are common side - effects of intravesical bcg instillation therapy . penile lesions or balanitis as complications of bcg treatment , we present a patient who underwent intravesical treatment with bcg and subsequently developed penile lesions and bilateral inguinal lymphadenopathy . the pathologic diagnosis was pta , g2 transitional cell carcinoma and carcinoma in situ ( who 1973 ) . he was offered six weeks of bcg instillation ( oncotice , tice strain , administered intravesically after reconstitution in 50 ml saline ) . the patient was unable to complete the treatment cycles consecutively due to severe irritative symptoms ; hence at the second and fourth week , treatment cycles were cancelled for a period of one week and his symptoms were treated with antibiotics and antispasmodics . he was administered five cycles of bcg at full dose , atraumatically , by a staff urologist using a 12fr hydrophilic nelaton catheter . one week after the last instillation the patient reported penile swelling and tenderness , itching at suprapubic areas . two days later , he presented with a penile lesion and indurated lesions at the suprapubic area . physical examination demonstrated two areas of painless indurated lesions at the pubic area and shallow ulceration on the penil shaft [ figures 1a and b ] and bilateral palpable inguinal lymphadenopathy . chest x - ray was unremarkable and computed tomography ( ct ) of abdomen revealed bilateral inguinal adenopathy ( maximal length of diameter 1 cm on the left and 1,5 on the right side ) . laboratory data showed slight elevation of c - reactive protein and erythrocyte sedimentation rate , and otherwise blood chemistry was normal . polymerase chain reaction ( pcr ) for urine tubercle bacilli was negative , and thereafter urine cultures were also negative for mycobacterium . ( a , b ) penile ulcerated lesion and papule at pubic area before treatment ; ( c , d ) appearance at three months ( c ) and six months ( d ) after treatment the patient underwent diagnostic biopsy from the papules of pubic areas and cystoscopy at the same time . cystoscopy showed several erythematous areas in the bladder and cold - cut biopsy was obtained from these areas . pathology of the bladder biopsies revealed acute and chronic inflammation with granulomatous changes ; there was no evidence of malignancy . the pathologic examination of biopsy samples from endurated pubic lesions demonstrated granulomatous inflammation with caseous necroses [ figure 2 ] and multinucleated giant cells of langhans type , which was compatible with bcg - induced granulomatous skin infection . histological appearance of granulomatous inflammation with caseous necroses ( h and e , 10 ) intravesical bcg instillation therapy was discontinued and the patient was given a three - drug antitubercular regimen including rifampicin , isoniazid , and pyrazinamide . the total treatment course was six months , with discontinuation of pyrazinamide after two months . complete resolution of penile , pubic lesions and inguinal adenopathy was achieved at six months of treatment [ figures 1c and d ] . follow - up cystoscopy showed no evidence of tumor recurrence and ct of abdomen and chest x - ray were unremarkable . side - effects of bcg usually occur when bcg is given immediately after transurethral resection of a bladder tumor , or when the patient had a traumatic catheterization or concurrent cystitis during intravesical instillation . use of immunosuppressive agents or genetic factors may also play significant role . during the instillation cycles of bcg , urine culture was negative however this may not preclude urinary tract infection and may have predisposed to bcg side - effects . although catheterization was atraumatic in the present case unnoticed trauma during catheterization could have played a role in the bcg toxicity determined in our patient . although our patient had no co - morbidities such as diabetes or immunosupression , his older age may have been a factor for the progression of side - effects . granulomatous reaction associated with bcg treatment is known to occur in the bladder , epididymis , prostate , kidney , lung and liver . however , occurrence of balanitis is uncommon and only a few cases of balanitis associated with bcg instillation have been reported . granulomatous balanitis symptoms include penile edema , papules and ulcers , occasionally associated with inguinal lymphadenopathies as in our case . therefore , clinical diagnoses are made in the majority of cases by histopathology and by the history of bcg therapy . although the stain and culture were negative for mycobacterium , given the recent history of intravesical instillation of bcg , these lesions likely represent dissemination of inoculums . the mechanism of infection presumably occurred via direct inoculation of the penis during the instillation of bcg , followed by dissemination to the superficial inguinal lymph nodes . histological appearances of these changes typically show epithelioid granuloma with central necroses and giant cells of langhans type as determined in our case . as bcg - induced penile ulcer and adenopathy is a rare complication of intravesical bcg therapy , treatment for this disease is not yet standardized . however , modern short - course anti - tuberculosis drug regimens for genitourinary tuberculosis are also recommended for the treatment of such cases . according to the world health organization anti - tuberculous drug treatment is based on an initial two - month intensive phase of treatment with three or four drugs with rifampicin , isoniazid , pyrazinamide , and ethambutol to destroy almost all tubercle bacilli . treatment is followed by a four - month continuation phase with only two drugs , rifampicin and isoniazid . in our case , adverse effects of bcg intravesical administration affect several organs in the genitourinary system and can disseminate to distant organs . the clinician must be alert to any complaints the patient has after treatment , and investigate accordingly . physicians should be aware of the possibility of bcg - induced granulomatous balanitis and concomitant adenopathy . when these clinical features appear in patients , adequate therapy should be started as soon as possible . we consider that immediate antituberculous therapy should be commenced to achieve prompt resolution and management of clinical findings .

intravesical bacillus calmette - gurin ( bcg ) is widely used as an adjuvant therapy in the treatment of non - muscle - invasive bladder cancer . bcg is generally well tolerated , though localized and systemic infectious complications may occur . infection of the glans and inguinal adenopathy are rare local complications of intravesical bcg therapy . traumatic urethral catheterization is one of the main causes . we report the case of a 75-year - old male who developed granulomatous balanitis and enlarged inguinal lymph nodes after five cycles of intravesical bcg treatment for transitional cell carcinoma of the bladder . histology revealed giant cell granuloma . oral antituberculous treatment was initiated with subsequent full recovery of penile lesions and adenopathy . physicians who administer bcg must be familiar with the possible complications and their adequate management and should inform patients about the side - effects accordingly .

cd47 ( originally named integrin - associated protein ( iap ) ) is a cell surface protein of the immunoglobulin ( ig ) superfamily , which is heavily glycosylated and expressed by virtually all cells in the body . cd47 was first recognized as a 50 kda protein associated and copurified with the v 3 integrin in placenta and neutrophil granulocytes and later shown to have the capacity to regulate integrin function and the responsiveness of leukocytes to rgd - containing extracellular matrix proteins [ 14 ] . soon after this integrin - associated protein was cloned , it was shown to be identical to the erythrocyte cell surface antigen cd47 . the fact that cd47 is also expressed by cells like erythrocytes , that do not express integrins , indicates that it can be more appropriate to refer to this protein as cd47 than using its original name integrin - associated protein ( iap ) . the protein is fairly well conserved between species and has about 6070% similarity in the amino acid sequence when comparing human cd47 with that of mouse , rat , and bovine cd47 . cd47 has also been shown to be identical to the oa-3/ovtl3 antigen highly expressed on most ovarian carcinomas [ 6 , 7 ] . it also shows homology to a protein family of variola and vaccinia viruses [ 1 , 5 , 6 ] , the significance of which is still unclear . cd47 consists of an extracellular igv domain , a five times transmembrane - spanning domain , and a short alternatively spliced cytoplasmic tail . in both humans and mice , the cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids , showing different tissue expression patterns . the 16 amino acid form 2 , which is by far the predominant isoform , is expressed in all cells of hematopoietic origin , as well as in endothelial and epithelial cells . in contrast , the 36 amino acid form 4 is expressed primarily in neurons , intestine , and testis . expression of the 4 amino acid form 1 is found in epithelial and endothelial cells , while the expression pattern of the 23 amino acid form 3 resembles that of form 4 . despite a study showing that cd47 form 3 and 4 could be associated with memory retention in rats , and that form 2 is predominating in astrocytes , little is known as to what these splice variants mean in terms of possible difference in the functionality of the protein . a new twist in the understanding of cd47 comes from a recent finding that cd47 can be expressed as a proteoglycan with a molecular weight of > 250 kda , having both heparin and chondroitin sulfate glycosaminoglycan ( gag ) chains . this form of cd47 was found to be expressed in both the human jurkat t cell line and in murine primary t cells , as well as in human umbilical vein endothelial cells ( huvecs ) , murine lung endothelial cells , and in human smooth muscle cells . functionally , the gag chains at cd47 ser were found to be crucial to inhibit t cell receptor signaling following the ligation of cd47 by its ligand thrombospondin-1 . early studies of cd47 were based on the use of monoclonal antibodies ( mabs ) raised against the cd47 protein purified from placenta [ 2 , 11 ] , showing a role of cd47 in mediating an enhanced igg - mediated phagocytosis response in the presence of rgd - containing ligands , such as fibronectin , fibrinogen , vitronectin , or collagen type iv [ 2 , 11 ] . the same mabs were also found to block neutrophil transendothelial migration stimulated by interleukin 8 ( il-8 ) or the bacterial peptide n - formyl - methionyl - leucyl - phenylalanine ( f - met - leu - phe ) and to inhibit neutrophil migration across tumor - necrosis - factor-- ( tnf- ) stimulated endothelial cells , where cd47 on both the neutrophils and the endothelial cells was found to be important . generation of other anti - cd47mabs , raised against epithelial membrane preparations , showed that cd47 is present at the basolateral membrane of epithelial cell monolayers , that mabs blocking cd47 on either neutrophils or the epithelial cells delay neutrophil trans - epithelial migration , and that efficient neutrophil chemotaxis correlates with an increased neutrophil cell surface expression of cd47 . the basement membrane protein entactin , which contains an rgd sequence , was also found to stimulate neutrophil adhesion and chemotaxis in a cd47-dependent manner in vitro . generation of cd47-deficient mice further proved the importance of this protein in regulating neutrophil inflammatory responses , by showing an increased sensitivity to bacterial infection due to a delayed neutrophil accumulation in bacterial peritonitis . cd47-deficient neutrophils also show a strongly impaired rgd - stimulated neutrophil adhesion , phagocytosis , and respiratory burst . for v 3 integrin - mediated cellular responses to the extracellular matrix protein vitronectin , cd47 was found to be required for v 3-mediated binding to vitronectin - coated beads , but not v 3-mediated adhesion to vitronectin - coated surfaces . in addition to its original association with v 3 integrins , cd47 has also been shown to interact with and regulate the integrins 2 1 and iib 3 on platelets [ 16 , 17 ] , the 2 1 integrin on smooth muscle cells , the 4 1 integrin on sickle red blood cells and b lymphocytes [ 19 , 20 ] , the 6 1 integrin in microglia , and the 5 integrin in chondrocytes ( figure 1 ) . thrombospondin-1 ( tsp-1 ) is the prototypic member of the thrombospondin family of extracellular matrix glycoproteins , which are implicated in regulating cell motility , proliferation , and differentiation . the extracellular igv domain of cd47 was found to be a receptor for the c - terminal cell - binding domain ( cbd ) of tsp-1 , since the expression of cd47 in otherwise cd47-deficient cells promotes adhesion to tsp-1 or its cbd , and a functional blocking mab against cd47 can block endothelial cell chemotaxis against tsp-1 or the cd47 binding cbd - peptide 4n1k . it was later shown that tsp-1 , its cbd , or the 4n1k peptide stimulates v 3 integrin - mediated cell spreading on vitronectin in a cd47-dependent manner . in platelets , tsp-1 , the tsp-1 cbd , or 4n1k activates the platelet iib 3 integrin and induces platelet spreading on fibrinogen , platelet aggregation , and increased focal adhesion kinase ( fak ) tyrosine phosphorylation , which are all dependent on interaction between cd47 and integrin iib 3 . furthermore , cd47 was found to mediate a synergistic effect of soluble type i collagen and tsp-1 or 4n1k , which enhance 2 1 integrin - mediated platelet activation or vascular smooth muscle cell chemotaxis [ 17 , 18 ] . early experiments also suggested that cd47 regulates tsp-1-induced cell spreading or platelet activation by affecting signal transduction in a pertussis toxin - sensitive way via a heterotrimeric gi protein [ 16 , 24 ] . cd47 was later shown to functionally associate with heterotrimeric gi , to suppress camp levels , and mediate the inhibition of erk in platelets and smooth muscle cells . more recently , cd47 ligated by tsp-1 was found to inhibit nitric oxide ( no ) signaling in vascular cells and to oppose no / cgmp - mediated inhibition of integrin activation to facilitate platelet aggregation . in addition to signaling through gi proteins , cd47 has been shown to signal via the 3 integrin cytoplasmic tail . although it is not entirely clear how much these two signaling pathways overlap , accumulation of cd47/ 3-integrin - complexes in cholesterol - rich lipid rafts , which appears to depend on both the cd47 igv domain , the multiple transmembrane - spanning domain , and a long range disulfide bond between cys in the igv domain and cys in the transmembrane domain , engage in gi signaling . cd47 has also been shown to be involved in the regulation of intracellular ca ( [ ca]i ) , exemplified by its regulation of an integrin - dependent increase in [ ca]i in endothelial cells and tumor cells , and that cd47 synergizes with t cell receptor - stimulated elevations of [ ca]i in t lymphocytes [ 33 , 34 ] . the cytoplasmic tails of the form 2 and form 4 splice variants of cd47 were found to bind to the cytosolic ubiquitin - related proteins plic-1 and plic-2 ( plic , proteins linking iap to cytoskeleton ) . in b lymphocytes , a role of cdc42 and rac has also been suggested in cd47-dependent regulation of neuronal development and neurite formation [ 36 , 37 ] . adhesion of intestinal epithelial cells to collagen i induces the association of cd47 with 2 integrins , and cd47 is necessary for collagen i - induced cyclooxygenase-2 ( cox-2 ) expression and epithelial cell migration , which is mediated by g i3 ( figure 1 ) . sirp proteins belong to the ig family of cell surface glycoproteins , where the first member identified was sirp ( also known as shps-1 , cd172a , bit , mfr , or p84 ) [ 3944 ] . sirp is highly expressed in myeloid cells and neurons , but also in endothelial cells and fibroblasts , and has three extracellular ig - like domains , one distal igv - like domain , and two membrane proximal igc - like domains [ 41 , 42 ] . in addition , an alternatively spliced form having only one igv domain has also been reported . in its intracellular tail , sirp has two immunoreceptor tyrosine - based inhibitory motifs ( itims ) , which when tyrosine phosphorylated can bind the src homology 2 ( sh2 ) domain - containing protein - tyrosine phosphatases shp-1 and shp-2 . additional cytoplasmic binding partners for sirp are the adaptor molecules src kinase - associated protein of 55 kda homolog / skap2 ( skap55hom / r ) , fyn - binding protein / slp-76-associated phosphoprotein of 130 kda ( fyb / slap-130 ) , and the tyrosine kinase pyk2 . sirp is also a substrate for the kinase activity of the insulin , egf , and bpdgf receptors , and the overexpression of sirp in fibroblasts decreases proliferation and other downstream events in response to insulin , egf , and bpdgf . since sirp is also constitutively associated with the m - csf receptor c - fms , sirp overexpression partially reverses the v - fms phenotype . two other family members have also been identified , sirp ( also known as cd172b ) [ 42 , 47 ] and sirp ( also known as cd172 g or sirp2 ) , whose extracellular ig - like domains are similar to that of sirp. however , the cytoplasmatic regions of sirp and sirp are different from that of sirp. sirp has a very short cytoplasmatic tail with no signaling motifs . instead , the transmembrane region contains a positively charged lysine residue , which can bind the immunoreceptor - tyrosine - based - activating - motif- ( itam- ) carrying adaptor protein dnax activation protein 12 ( dap12/karap ) [ 49 , 50 ] . sirp has no recognizable signaling motif or capability to interact with cytoplasmic signaling molecules and is therefore unlikely to generate intracellular signals . cd47 has been shown to be a ligand for sirp [ 52 , 53 ] and sirp [ 54 , 55 ] , but does not bind sirp . the cd47/sirp interaction regulates not only a multitude of intercellular interactions in many body systems , such as the immune system where it regulates lymphocyte homeostasis [ 56 , 57 ] , dendritic cell ( dc ) maturation and activation , proper localization of certain dc subsets in secondary lymphoid organs [ 5961 ] , and cellular transmigration [ 62 , 63 ] , but also regulates cells of the nervous system ( reviewed in [ 64 , 65 ] ) . an interaction between these two proteins also plays an important role in bone remodeling [ 66 , 67 ] . cellular responses regulated by the cd47/sirp interaction are many times dependent on a bidirectional signaling through both receptors [ 51 , 64 , 65 ] ( figure 1 ) . the finding that cd47 on host cells can function as a marker of self and regulate phagocytosis by binding to sirp will be further described in a subsequent section . the interaction between cd47 and sirp has proven to be very specific species , as shown by the relatively weak binding of cd47 from mouse , rat , or cow to human sirp [ 69 , 70 ] . in addition , the glycosylation of cd47 or sirp does not seem to be necessary for their interaction , but the level of n - glycosylation of sirp has , however , an impact on the interaction such that over glycosylation reduces the binding of cd47 . the long range disulfide bond between cys in the cd47 igv domain and cys in the transmembrane domain is also important to establish an orientation of the cd47 igv domain that enhances its binding to sirp . ligation of cd47 by anti - cd47 mabs was found to induce apoptosis in a number of different cell types . this phenomenon was first described in jurkat t cells , in anti - cd3 activated but not in resting primary t cells , and in b - cell chronic lymphocytic leukemia ( b - cll ) cells [ 72 , 73 ] . cd47-induced apoptosis can be induced by several different mabs ; however , while some of these ( e.g. , ad22 , 1f7 , or mabl ) show potent apoptosis induction in suspension [ 7376 ] , others ( e.g. , b6h12 and 2d3 ) need to be immobilized to a surface to promote cell death [ 72 , 77 ] . of the two sirp - family members known to bind the cd47 igv domain ( sirp and sirp ) , sirp as a soluble fc - fusion protein does not induce cd47-dependent apoptosis , while sirp or sirp bound onto the surface of beads induces apoptosis through cd47 in jurkat t cells and the myelomonocytic cell line u937 . in addition , tsp-1 or the cd47-binding tsp-1 cbd - peptide 4n1k also induces cd47-dependent apoptosis [ 74 , 75 , 78 , 79 ] . indeed , mice deficient in cd47 or tsp-1 sustain oxazolone - induced inflammation significantly longer than wild - type mice due to a deficiency in t cell apoptosis . this form of cell death was initially described to be characterized by cell shrinkage , reduction in mitochondrial transmembrane potential , and exposure of phosphatidylserine ( ps ) on the cell surface , but to be independent of fas ( cd95 ) or tnf receptor signaling . classical apoptosis , and it is independent of cysteinyl aspartate protease ( caspase ) activation [ 72 , 73 ] ( figure 2 ) . furthermore , inhibitors of actin polymerization or mitochondrial electron transfer prevent cd47-induced ps exposure . in support of a role for the actin cytoskeleton , peripheral blood mononuclear cells ( pbmcs ) from wiskott - aldrich syndrome ( was ) patients , where mutations in the was protein ( wasp ) results in defective cdc42-induced regulation of the actin cytoskeleton , are resistant to cd47-induced apoptosis . although the mitochondrial transmembrane potential is affected in cd47-induced apoptosis , it does not involve the mitochondrial release of cytochrome c or apoptosis - inducing factor ( aif ) , but does involve the production of reactive oxygen species ( ros ) . in jurkat t cells , it was shown that the inhibition of gi signaling with pertussis toxin can counteract cd47-induced apoptosis , that ligation of cd47 reduces intracellular camp levels , and that camp elevating agents prevents apoptosis by cd47 ligands . this signaling pathway , which likely also involves reduced signaling through protein kinase a ( pka ) , is not only described in t cells , but also in several breast cancer cell lines . in the latter cell type , it was shown that epidermal growth factor can inhibit the cd47 death pathway via protein kinase c ( pkc ) . a yeast two - hybrid screen , where cd47 was used as bait , identified the pro - apoptotic bcl-2 family member bcl-2-homology-3- ( bh3- ) only protein 19 kda interacting protein-3 ( bnip3 ) . in t cells , bnip3 was found to physically associate with cd47 , which prevents its degradation in proteasomes and sensitizes t cells to cd47-induced apoptosis [ 78 , 79 ] . ligation of cd47 induces the translocation of bnip3 to mitochondria , and attenuation of bnip3 activity inhibits cd47-induced apoptosis , which together suggests that bnip3 is crucial as a mediator of this cell death pathway . moreover , bnip3 gene expression was found to be increased and regulated by hypoxia - inducible factor-1 ( hif-1 ) following the ligation of cd47 by single - chain fragments of an anti - cd47 mab which kills b - cll cell lines both in vitro and in vivo , where the knockdown of hif-1 represses cd47-induced cell death ( figure 2 ) . the finding that a jurkat t cell clone lacking cd47 is resistant to fas- ( cd95- ) induced apoptosis , but that expression of cd47 restores the sensitivity to fas - ligation , suggested that cd47 can augment fas - induced apoptosis via a mechanism that requires neither cd47 signaling nor its association with lipid rafts . in fact , the lack of cd47 impairs important proapoptotic events downstream of fas , such as loss of mitochondrial membrane potential , cytochrome c release , caspase activation , poly(adp - ribose ) polymerase ( parp ) cleavage , and dna fragmentation . this function of cd47 is likely also important in primary cells , since t cells from cd47-deficient mice are protected from fas - induced apoptosis . in hematopoietic cells , cd47-induced apoptosis has been described in hematopoietic tumor cells [ 54 , 7274 , 7678 , 81 , 82 , 84 , 85 ] and in activated primary t or b cells [ 73 , 74 , 77 , 79 ] . however , whether apoptosis can be induced through cd47 in nonactivated leukocytes is still somewhat unclear . the only situation where nonactivated t or b cells have been found to undergo cd47-induced apoptosis is when immobilized anti - cd47 mab has been used , but not when using soluble cd47 ligands or mabs known to induce apoptosis in activated cells or tumor cells [ 73 , 74 , 79 ] . surprisingly , although cd34 hematopoietic progenitors express cd47 , they are resistant to cd47-induced apoptosis by either immobilized or soluble mab [ 76 , 77 ] . in addition , immature human monocyte - derived dendritic cells ( idcs ) were described as resistant to cd47-induced apoptosis , following incubation with an immobilized cd47 mab for 18 hours . however , another study showed that freshly isolated human monocytes or human monocyte - derived idc undergoes a rapid ( within 60 min ) cell death in response to the cd47-ligand 4n1k . this cell death , which was described to occur in a subset of cells and where monocytes or idc not affected by 4n1k remain viable in culture , is associated with cellular features previously described for cd47-induced apoptosis , such as not only ps exposure , increased plasma membrane permeability , reduced mitochondrial membrane potential , caspase independence , but also included dna fragmentation . thus , these findings suggest that although a subset of idc may undergo cd47-induced cell death at an early time point , this may not be detectable at later time points . however , it raises the question if specific subsets of monocytes or idcs are sensitive to this form of rapid cell death whereas others are resistant and maintain their viability in culture . in addition to hematopoietic cells , overexpression of cd47 can induces cell death of cultured cerebral cortical neurons , which is enhanced by the coexpression of sirp and prevented by brain - derived neurotrophic factor ( bdnf ) when cd47 and sirp are coexpressed . apoptosis in neurons overexpressing cd47 , however , is dependent on caspases and apoptotic cells have condensed apoptotic nuclei with fragmented dna . it has also been shown that endothelial cells incubated under static conditions in the absence of flow increase their expression of tsp-1 , which uses the cd47/ v 3 integrin as a receptor to trigger endothelial cell apoptosis . this mechanism also appears to be involved in endothelial cell apoptosis during proatherogenic turbulent flow conditions and in mechanosensitive induction of apoptosis in fibroblasts . tsp-1-mediated apoptosis , mapped to the type-3 repeat / c - terminal domain of tsp-1 , in promyelocytic leukemia cells ( nb4-lr1 ) has also been suggested to depend on the engagement of both cd47 and the v 3 integrin . in contrast to the proapoptotic effects of cd47 described above , it was reported that the tsp-1-derived peptide 4n1 could abolish c2-ceramide - induced apoptosis in primary porcine thyroid cells by preventing reduction in intracellular camp levels , an effect blocked by the functional blocking antihuman cd47 mab b6h12 . a similar effect of 4n1 peptide was also found to inhibit the cytotoxic effects of the anticancer drugs camptothecin and doxorubicin in thyroid carcinoma cells . although it is unclear how these effects of cd47-ligation can be explained in relation to the proapoptotic effects of this molecule , it is interesting to note that tumor cell tsp-1 overexpression has been linked to disease recurrence and decreased survival [ 9496 ] , and it was suggested that this pathway could be one explanation behind drug resistance in thyroid cancers . mature erythrocytes express high levels of cd47 , but do not express integrins , which early indicated that other important functions of cd47 could be expected in these cells . the fact that individuals with the rhnull phenotype , which do not express any of the proteins of the rh protein complex , only express about 25% of normal levels of cd47 suggested a close relation between cd47 and erythrocyte rh proteins [ 5 , 97 ] . in the erythrocyte cell membrane , rh polypeptides associate in a complex with many other proteins ( e.g. , rh associated glycoprotein ( rhag ) , glycophorin b , lw , and cd47 ) . another erythrocyte membrane protein complex is formed by the band 3 anion exchanger and several other proteins ( e.g. , glycophorin a , protein 4.2 , and ankyrin ) . the latter multiprotein complex mediates the anchorage of the erythrocyte membrane to the spectrin cytoskeleton [ 99 , 100 ] . in addition , it has been suggested that the rh complex and the band 3 complex may in fact be associated in the erythrocyte membrane . mutations in band 3 , or complete band 3 deficiency , in human erythrocytes results in reduced expression of rh polypeptides and rhag and results in a virtual lack of cd47 . moreover , human erythrocytes deficient in protein 4.2 also show a marked deficiency of cd47 as well as an altered glycosylation of rhag . when combining these finding , a hypothesis was put forward , which suggests that cd47 of the rh complex may indeed form a link to the band 3 complex by binding to protein 4.2 ( figure 3 ) . studies of protein mobility within the erythrocyte membrane have shown that cd47 is associated with the erythrocyte cytoskeleton as being a part of the rh complex , but that cd47 is also present as a noncytoskeleton anchored pool which is more mobile in the erythrocyte membrane . thus , cd47 in erythrocytes may serve several different functions depending on its grade of mobility in the plasma membrane . it has been suggested that the freely mobile cd47 pool may be of importance to accumulate cd47 in specific membrane areas upon cell - cell contacts , for example , in order to efficiently interact with sirp on other cell types such as phagocytic cells . importantly , data from studies of murine erythrocytes have shown remarkable differences as compared with human erythrocytes , and the link between cd47 , the rh complex , and protein 4.2 in murine erythrocytes is not well understood . first , there are little or no rh polypeptides in erythrocytes from band 3-deficient mice , while the expression of cd47 is only slightly reduced . second , erythrocytes from cd47-deficient mice were found to contain normal amounts of murine rh and rhag polypeptides . taken together , the total picture suggests that cd47 seems to interact with rh polypeptides and protein 4.2 in human erythrocytes , whereas the interaction between cd47 , rh polypeptides , and proteins of the band 3 complex in murine erythrocytes is still unclear . a progressive decrease in the cd47 expression level has been observed on human erythrocytes stored under blood bank conditions at + 4c for more than 14 days [ 107109 ] and in a mouse model of erythrocyte storage which tried to mimic human blood bank conditions . however , the magnitude of reduction in cd47 expression was rather different , from a modest reduction of up to 6% at day 42 of storage , around 30% reduction on day 28 of storage , to a more than 50% reduction on day 14 of storage . furthermore , a recent study indicated that the cd47 levels of erythrocytes stored for 35 days were not different from that of fresh cells . the discrepancy between these findings may have several explanations , such as the exact storage conditions or methods for the quantification of erythrocyte cd47 expression levels . in addition , the loss of cd47 may also be sensitive to leukocytes remaining in the erythrocyte concentrates . indeed , a gradual loss of cd47 from erythrocytes during storage was observed in stored erythrocytes irrespective of whether the buffy coat was removed or not before storage , although buffy coat removal resulted in an increased expression level of cd47 at all time points tested , showing a significant correlation between the number of remaining leukocytes and erythrocyte cd47 levels . a study of cryopreserved leukoreduced erythrocytes was unable to detect any effects on erythrocyte cd47 expression levels . microparticles released in the blood from blood cells or endothelial cells have recently gained interest due to their possible role in regulating a variety of normal or pathological biological functions . such microparticles are also released from erythrocytes during cryopreservation or storage at + 4c , and these microparticles do among other erythrocyte membrane proteins also carry cd47 [ 112 , 114 ] . although the presence of cd47 on these microparticles suggests a mechanism for the observed loss of cd47 from stored erythrocytes , soluble cd47 has also been detected in the supernatants of erythrocytes stored at + 4c . in addition to a possible loss of cd47 on stored erythrocytes , a recent study also indicated that storage as well as experimental aging in vitro results in a conformational change of the cd47 protein . this modification can be detected as a selectively increased binding of the anti - cd47 mab 2d3 , which recognizes an epitope different from the epitope involved in the binding of tsp-1 or sirp and recognized by mab b6h12 . however , the use of mouse models has been informative to further understand if the level of cd47 is changing during erythrocyte aging . in these models , biotin is injected intravenously to label all blood cells at one or two specific time points [ 115 , 116 ] . analyses of circulating erythrocytes at later time points will then allow for discrimination between older biotin - positive and younger biotin - negative circulating erythrocytes [ 115 , 116 ] . using this approach , it has been found that cd47 is gradually lost from the surface of circulating murine erythrocytes , where the oldest erythrocytes may have up to 30% lower cd47 expression levels as compared with younger erythrocytes [ 115 , 117 ] . sickle cell disease , caused by a mutation in the hemoglobin chain and a formation of insoluble intracellular aggregates of mutated hemoglobin , resulting in characteristic sickle shaped erythrocytes , is associated with severe vasoocclusive crisis [ 118 , 119 ] . one important mechanism behind this pathology is the enhanced adhesion of sickle erythrocytes to vascular endothelium [ 118 , 119 ] . cd47 was shown to play a role in the pathologic sequestration of sickle erythrocytes to vascular endothelium under shear stress by binding to tsp . it has been found that the reticulocyte - enriched fractions of sickle erythrocytes are most efficiently binding to endothelial cells , that this function requires heterotrimeric g proteins and tyrosine kinase activity , and is mediated by 4 1 integrins on the reticulocytes . interestingly , sickle erythrocytes also show an increased expression of the cd47 epitope recognized by mab 2d3 . in gaucher 's disease , a sphingolipidosis caused by glucocerebrosidase deficiency , macrophages accumulate glucosylceramide following the excess phagocytosis of erythrocytes , which converts splenic macrophages to pathogenic gaucher cells . erythrocytes from patients with untreated gaucher 's disease have been found to have reduced levels of cd47 , which can be reversed upon enzyme - replacement therapy . it has been suggested that the anemia associated with the untreated disease can in part be explained by a combination of reduced cd47 levels together with other morphological erythrocyte abnormalities observed in this disease . the distinction between self and non - self is central to the maintenance of integrity in a multicellular organism and allows for a powerful and successful elimination of potentially dangerous pathogens , while carefully preserving healthy host cells and tissues . this distinction has been well studied in the adaptive immune response , which is specialized in the recognition of foreign peptides by virtue of small modifications to major histocompatibility complex ( mhc ) molecules made by these peptides . for the innate immune system , recognition is thought to be based on a large extent on the recognition of specific microbial structures , pathogen - associated molecular patterns ( pamps ) . the innate immune system of the host organism has through the evolution developed a group of receptors , pattern recognition receptors ( prrs ) , which serves to specifically recognize pamps . in this way , the host may utilize a certain number of receptors , encoded in the genome , for the recognition of various evolutionary stable molecular pathogen - associated structures . while some of the prrs ( like the mannose receptor ( mr ) ) recognize pamps directly , others ( like complement receptors ( crs ) ) are specialized to detect products generated secondary to pamp recognition . however , a system based on specific recognition of foreign is clearly flawed as it prevents the recognition of anything that is also present on the organism 's own cells . this can be very elegantly circumvented by a defense system that has very broad recognition , when it is combined with specific molecules that mark host cells and tissues as self . thus , recognition of self will inhibit the activation of innate immune cells , whereas the recognition of missing self allows for an immune response to proceed . in other words , a system where the own cells express a unique marker of self , not present on foreign cells , would make the distinction between self and foreign very simple . in that way , it would not matter if the organism 's own cells express substances or ligands that are similar to those found on foreign cells / particles . innate immune cells such as macrophages would only look for the presence of the self marker on the recognized particle , where the presence of self would release the recognized particle but the absence of self would allow for activation and destruction . this would significantly simplify the recognition process , as there would only be a need for a few broad specific recognition receptors instead of a countless number of foreign - specific receptors . it would also fulfill the criteria for a defense system that effectively recognize and destroy foreign objects but at the same time reduces the risk of damaging the organism 's own structures . such a system for macrophage activation would be analogous to the well - established missing - self hypothesis for natural killer ( nk ) cell activation . nk cells recognize target cells by a range of activating receptors which also recognize ligands on many normal cells . however , in the nk cell system expression of self - mhc class i will protect the recognized cell via the ligation of nk cell inhibitory receptors specific for it is , however , important to note , that nk cell inhibitory receptors are now described , which seem to have ligands other than mhc class i . upon the binding of self - mhc class i , ligated nk cell inhibitory receptors will recruit and activate the phosphatases shp-1/shp-2 that mediate the inhibition of nk cell activation . nk cells thus spare cells whenever they express markers of normal self , and eliminate them when these markers are absent or inadequately expressed . other leukocytes also express molecules related to nk cell inhibitory receptors , further suggesting that similar mechanisms are operative also , for example , in macrophage activation . many of these inhibitory receptors recognize mhc class i , but the marker of self could in principle be any ubiquitously expressed surface molecule . a new chapter in the understanding of cd47 and its functions started when cd47 was found to bind sirp [ 52 , 53 ] . at the time , sirp was regarded as one of several immunoreceptors with cytoplasmic itim motifs , generally suggested to be involved in negative regulation of cellular functions , mediated by the recruitment of the tyrosine phosphatases shp-1 and/or shp-2 or the inositol phosphatase ship to the tyrosine phosphorylated itims . in addition , sirp was found to be highly expressed in primary macrophages and macrophage cell lines , as well as in other myeloid cells such as monocytes , granulocytes , and dendritic cells . in macrophages , sirp was found to negatively regulate signaling through tyrosine kinase - dependent signaling pathways ( e.g. , fcri ) . although both shp-1 and shp-2 can bind to the phosphorylated sirp itims , only shp-1 has been associated with the inhibitory function of sirp in macrophages , similar to that of the nk cell inhibitory receptors , whereas shp-2 associated with sirp leads to a phosphatase - dependent enhancement of the signal in many situations . when studying cd47-deficient mice , we were struck by the fact that cd47-deficient bone marrow can not reconstitute and engraft in lethally irradiated syngeneic wild - type recipient mice , while it engrafts normally in cd47-deficient recipient mice . our work on trying to understand this controversy first involved experiments where different leukocyte populations were transferred from cd47-deficient mice into wild - type recipient mice . however , we later simplified the system by studying transfusion of erythrocytes instead of leukocytes . the reason for this is that erythrocytes in contrast to many leukocyte populations have a long half - life in circulation , they do not divide , do not express mhc class i , and they do not home to extravascular tissues or organs . these studies showed that fresh erythrocytes isolated from the blood of cd47-deficient mice , labeled with a fluorescent cell tracker dye and transfused into wild - type recipient mice , have markedly reduced survival , whereas their half - life is normal in cd47-deficient recipients . the clearance rate of such freshly isolated cd47-deficient erythrocytes is remarkably fast with complete clearance within 24 hours . to put this in perspective , the average lifespan of murine erythrocytes in circulation is somewhere between 45 and 60 days , depending on the mouse strain investigated [ 135 , 136 ] . the rapid clearance of cd47-deficient erythrocytes from the circulation of wild - type recipient mice does not require complement , since cd47-deficient erythrocytes are also cleared from the circulation of complement factor 3- ( c3- ) deficient mice . neither is there a requirement for lymphocytes or antibodies to enable the clearance of cd47-deficient erythrocytes from the circulation , since clearance is normal in rag1-deficient mice , which lack mature t and b lymphocytes . rather , the transfused cd47-deficient erythrocytes that are eliminated from the circulation of wild - type mice are recognized and cleared by splenic red pulp macrophages , and the removal of these macrophages by splenectomy or by treatment with macrophage - toxic clodronate liposomes abrogates the elimination of cd47-deficient erythrocytes . furthermore , macrophage sirp is tyrosine phosphorylated upon contact with cd47 on erythrocytes , and when sirp on isolated splenic macrophages is blocked , it increases the level of phagocytosis of wild - type erythrocytes to that seen with cd47-deficient erythrocytes , whereas phagocytosis of the cd47-deficient erythrocytes is unaffected by the antibody treatment . in addition , studies in sirp-mutant mice , where the cytoplasmic signaling domain of the receptor is deleted , have shown a shorter half - life of normal cd47-expressing erythrocytes in these mice , which also present with mild anemia . such spontaneous anemia is , however , not seen in cd47-deficient mice , suggesting that cd47 could also be needed on the macrophages to facilitate the clearance of erythrocytes in the spleen . however , lack of cd47 on platelets also results in very rapid clearance when transfused into wild - type recipients , and both cd47-deficient mice and sirp-mutant mice have a mild spontaneous antibody - independent thrombocytopenia [ 139 , 140 ] . altogether , these findings indicated that all erythrocytes can be phagocytosed by splenic red pulp macrophages when sirp is blocked or cd47 is missing , and that these macrophages must have a receptor for erythrocytes . indeed , we have identified ldl receptor - related protein ( lrp-1 ) , which by recognizing calreticulin on the surface of normal erythrocytes can mediate phagocytosis of untreated cd47-deficient erythrocytes [ 141 , 142 ] . by showing that macrophages in the splenic red pulp can recognize normal circulating erythrocytes , but that these macrophages do not phagocytose erythrocytes as long as they display cd47 on their surface , these findings were the first to prove that macrophages are perfectly capable of recognizing normal host cells and rely on self recognition for proper function . in addition , it also demonstrated major similarities between mechanisms for self recognition in nk cells and macrophages . based on the findings that unopsonized erythrocytes lacking cd47 can be phagocytosed by splenic macrophages , and that the cd47/sirp interaction can potently inhibit the prophagocytic mechanism operating in that situation , we next hypothesized that the cd47/sirp interaction might as well be able to negatively regulate phagocytosis of opsonized erythrocytes , and that for the cells of the innate immune systems front line defense ( i.e. , macrophages and dc ) there would be a balance between signals from activating receptors ( e.g. , fcr or cr ) and the inhibitory signal from sirp ligated by target cell cd47 . indeed , the cd47/sirp interaction can also regulate phagocytosis of igg - opsonized or complement opsonized erythrocytes as well as other opsonized host cells , making cd47-deficient cells severely sensitive to being phagocytosed [ 139 , 143145 ] . in addition , cd47-deficient mice are severely sensitive to experimental autoimmune hemolytic anemia ( aiha ) and experimental thrombocytopenia [ 139 , 146 ] . on an autoimmune background prone to develop spontaneous aiha , lack of cd47 results in a more rapid and lethal aiha . using motheathen viable ( me / me ) mice , which only have about 20% of normal levels of shp-1 , but normal levels of shp-2 [ 147149 ] , we could pinpoint the role of these phosphatases in mediating the phagocytosis inhibitory effect of the cd47/sirp interaction in vivo . when transfused into me / me mice , igg - opsonized wild - type erythrocytes are cleared with the same rapid kinetics as seen with equally opsonized cd47-deficient erythrocytes , showing that at that particular signaling strength through prophagocytic fc receptors , the level of cd47 on normal erythrocytes was not enough to prevent phagocytosis if the shp-1 level was reduced by about 80% . in contrast , the prophagocytic signaling mediating phagocytosis of unopsonized cd47-deficient erythrocytes ( presumably mediated by lrp-1 ) is likely much weaker , since unopsonized wild - type erythrocytes are not cleared with the same rapid kinetics as cd47-deficient cells in me / me mice . however , while unopsonized cd47 heterozygous erythrocytes ( expressing about 50% of the cd47 found on wild - type erythrocytes ) show normal half - life when transfused into wild - type recipients , they were found to be cleared more rapidly when transfused into me / me mice . these findings thus proposed that a reduction of the cd47 level to 50% of normal , in combination with a strongly reduced level of shp-1 in the macrophages , together weakened the inhibitory signaling through sirp such that clearance of unopsonized erythrocytes was allowed . furthermore , the rate of phagocytosis of igg - opsonized erythrocytes is distinctly regulated by the amount of cd47 present on the surface of the erythrocytes both in vivo and in vitro . thus , activation of phagocytosis in a macrophage in contact with a target erythrocyte ( or any other host cell ) can be viewed as a balance between signals from activating receptors ( i.e. , fcr , cr , or lrp-1 ) and the inhibitory signal from sirp ligated by target cell cd47 . in the macrophage , neither signal appears to be dominant , but rather the decision to phagocytose a target host cell is based on an integration of positive prophagocytic signals and inhibitory cd47/sirp signaling ( figure 4(a ) ) . the same functional regulation also seems to be operating in dcs and in microglia . in hemophagocytic lymphohistiocytosis , hematopoietic stem cells are phagocytosed by bone marrow macrophages as a result of systemic inflammation . in this disease , hematopoietic stem cells were found to express reduced levels of cd47 , which shows that pathological conditions may occur where a combination of inflammatory macrophage activation and reduced expression of cd47 results in a severe loss of critical cell types . however , not all prophagocytic receptors seem to be regulated by the cd47/sirp interaction . although fc receptor - mediated phagocytosis of igg - opsonized oxidatively damaged erythrocytes is strongly inhibited by the cd47/sirp interaction , scavenger receptor - mediated uptake of unopsonized oxidized erythrocytes turned out to be insensitive to this inhibitory mechanism . the mechanism whereby recruitment of shp-1 to sirp can inhibit phagocytosis has been suggested to involve the tyrosine kinase syk and phosphoinositide 3 kinase ( pi3 kinase ) . more recently , it was suggested that shp-1 mediates dephosphorylation of nonmuscular myosin iia at the phagocytic synapse between the phagocyte and a host cell , as a result of the interaction between macrophage sirp and cd47 on the host cell , which brings further insight into the mechanism behind phagocytosis inhibition by the cd47/sirp interaction . based on the seemingly important interaction between cd47 and sirp to prevent phagocytosis of host cells , and the fact that cd47-deficient cells are rapidly phagocytosed when transfused into wild - type mice , it is clearly a puzzle why cd47-deficient mice do not present with a more severe phenotype where the macrophages phagocytose a large fraction of the cd47-deficient cells in those mice . however , this phenomenon is rather similar to what is described in 2 microglobulin deficient mice , which lack expression of mhc class i but where the nk cells still do not attack and destroy host cells and also show hyporeactivity to cells carrying ligands for activating nk cell receptors , cells which are efficiently killed by wild - type nk cells . one explanation proposed for this function in nk cells is that inhibitory nk cell receptors need to interact with licenced and able to become activated and kill target cells lacking or expressing reduced levels of self- mhc class i . by investigating the ability to phagocytose cd47-deficient cells in mice where cd47 was expressed by hematopoietic cells , nonhematopoietic cells , or both , the phagocytic tolerance to cells lacking cd47 in cd47-deficient mice it was found that macrophages developing in an environment where nonhematopoietic cells lack cd47 become tolerant to cells lacking cd47 , which allowed cd47-defcient leukocytes to avoid clearance . curiously , this did not involve erythrocytes , which were cleared by splenic macrophages also in the bone marrow chimeras where nonhematopoietic cells lacked cd47 and macrophages had become the latter phenomenon has so far not been explained , but may suggest that phagocytosis of erythrocytes is regulated differently from that of leukocytes . although cd47-deficient macrophages express normal amounts of sirp , which can also become tyrosine phosphorylated upon contact with cd47 expressing cells ( oldenborg et al . , unpublished observations and ) , it is possible that the tolerance to cd47-deficient cells that develop in cd47-deficient mice has to do with tuning of intracellular signaling pathways . in favor of such a hypothesis is the observation that transfused igg - opsonized wild - type erythrocytes are cleared from the circulation at a significantly slower rate in cd47-deficient mice , as compared with that seen in wild - type recipient mice ( oldenborg et al . this is unlikely due to a different expression of fc receptors in cd47-deficient mice , since cd47-deficient igg - opsonized erythrocytes are cleared with the same kinetics in both wild - type and cd47-deficient recipient mice ( oldenborg et al . apoptosis is a physiological process of programmed cell death which is important for embryologic development , maintenance of homeostasis , and elimination of damaged cells . an important event related to this process is the rapid uptake of apoptotic cells or apoptotic bodies by phagocytic cells . the efficacy of this process in the body is shown by the fact that apoptotic cells are apparently removed with such extremely high efficiency that apoptotic cells are very hard to detect in tissues under normal physiological conditions . it has even been suggested that if apoptotic cells are in fact detected in vivo , this may indicate a possibility of defects in their clearance or the presence of a large overload of apoptotic cells [ 159162 ] . the largest part of apoptotic cell phagocytosis is mediated not only by professional phagocytes like macrophages and dcs , but also to some extent by nonprofessional phagocytes such as fibroblasts ; epithelial cells and stromal cells are equipped with this function . one example of the latter phenomenon is the phagocytosis of apoptotic mammary epithelial cells by bystander epithelial cells during mammary gland involution [ 163 , 164 ] . when cd47 was identified as a cell surface protein on host cells that can negatively regulate their phagocytosis through sirp , one important question was to understand if this signaling pathway is altered during apoptosis to facilitate uptake of apoptotic cells . one hypothesis would be that apoptotic cells downregulate the amount of cd47 on their surface , which together with an increased amount of prophagocytic ligands exposed during apoptosis would result in phagocytosis of the dead cells . indeed we found a reduced expression of cd47 on apoptotic fibroblasts and neutrophils , but curiously not on apoptotic jurkat t cells or apoptotic murine thymocytes . this issue became even more confusing when apoptotic murine t cells lacking cd47 , in contrast to the solid data showing that cd47 on host cells inhibits phagocytosis by macrophages , were found to be phagocytosed less efficiently by macrophages than equally apoptotic cd47 wild - type t cells [ 143 , 165 ] . in apoptotic murine t cells , cd47 was rather found to be important in mediating tethering of the apoptotic cells to the phagocyte [ 143 , 165 ] . in addition , cd47 is redistributed into patches on the apoptotic cell surface , areas of the plasma membrane which are different than those harbouring clusters of proteins that function as ligands for prophagocytic receptors [ 141 , 143 ] . thus , one possibility would be that the segregation of cd47 away from these phagocytosis promoting ligands could allow for tethering mediated by the cd47/sirp interaction , but that sirp would be too far away from the prophagocytic receptors to be involved in negative regulation ( figure 4(b ) ) . another very interesting hypothesis was recently suggested , which shed new light on this process . as described above in section 4.2 experimentally oxidized erythrocytes , as well as erythrocytes stored in blood bank conditions , showed enhanced expression of the cd47 epitope recognized by mab 2d3 , suggesting a conformational change in the cd47 igv domain . importantly , this is associated with an increased binding of tsp-1 to cd47 , which together could generate a new binding site for sirp that induces a prophagocytic signal instead of the normal inhibitory cd47/sirp signal . however , this very interesting hypothesis still has to also be investigated in the context of macrophage phagocytosis of apoptotic nucleated cells such as neutrophils or t cells . thus , cd47 may operate in several different ways during cellular senescence in order to switch its normal phagocytosis - preventing role to rather facilitate and promote the phagocytosis of senescent cells . lrp-1 is a multifunctional scavenger receptor , shown to be involved in mediating uptake of apoptotic cells [ 141 , 166 , 167 ] , and as already mentioned it can also bind to calreticulin on viable erythrocytes to induce phagocytosis if the inhibitory cd47/sirp interaction is not strong enough [ 141 , 142 ] ( figure 4(b ) ) . glucocorticoids are powerful in treatment of inflammatory conditions , which can be attributed to their ability to downregulate production of and cellular responses to proinflammatory cytokines and their ability to inhibit the recruitment of inflammatory cells [ 168 , 169 ] . however , another mechanism whereby glucocorticoids may also reduce inflammation is to stimulate phagocytosis of apoptotic cells [ 170 , 171 ] . interestingly , glucocorticoid treatment of macrophages results in increased macrophage expression of lrp-1 and increased phagocytosis of apoptotic cells or viable cd47-deficient erythrocytes . since cd47 can inhibit phagocytosis of host cells , and the amount of cd47 on the cell surface is clearly important in determining the phagocytosis efficiency of macrophages , this also raised the question on whether cells such as tumor cells can also increase their cd47 expression levels in order to escape macrophage elimination . indeed , early studies showed that ovarian carcinoma cell lines express increased levels of cd47 [ 6 , 7 ] , the functional significance of which was unclear at that time . more recently , an enhanced expression of cd47 has been reported for murine myeloid leukemias , as well as human normal and leukemic hematopoietic stem cells and many human solid tumors [ 172174 ] , and that increased cd47 expression is associated with reduced patient survival in aml and solid tumors [ 173 , 174 ] . to challenge the hypothesis that cd47 on the leukemic cells protects from phagocytosis by macrophages and supports tumor cell growth , a xenogenic mouse model was used to study a particular human aml clone ( molm-13 ) with very low endogenous cd47 expression . expression of murine cd47 in molm-13 cells and transplantation of molm-13 clones expressing low or high levels of cd47 showed an enrichment and spreading of the cd47-high expressing clones to many bones of recipient mice , while the cd47-low clones showed minimal engraftment . depletion of macrophages in recipient mice allows for engraftment of cd47-low clones , and macrophage phagocytosis of aml cells both in vitro and in vivo is selective for tumor cells expressing low amounts of cd47 . importantly , by blocking sirp on macrophages , phagocytosis of cd47-high aml cells is increased to that seen with cd47-low clones . thus , these findings strongly suggest that an increased cd47 expression level on tumor cells could serve to avoid macrophage clearance and promote dissemination of tumor cells ( figure 4(c ) ) . it was also recently shown that treatment serving to block the cd47/sirp interaction can result in elimination of aml stem cells in a xenogenic model . furthermore , inline with our original finding that the cd47/sirp pathway also potently inhibits fc receptor - mediated phagocytosis of igg - opsonized host cells [ 139 , 145 , 146 , 150 ] , it has been shown that antibody - mediated blocking of cd47/sirp signaling promotes phagocytosis of non - hodgkin lymphoma cells treated with rituximab and antibody - dependent cellular cytotoxicity ( adcc ) against her2/neu - positive breast cancer cells treated with traztusumab . thus , blocking the cd47/sirp interaction may prove to be a powerful tool in the treatment of various tumors . while organ transplantation is an important procedure to treat end - stage organ failure , it is hampered by a shortage of organ donors . to solve this problem , it has been suggested that pigs could serve as donors of organs , tissues , or cells to be transplanted into humans ( i.e. , xenotransplantation ) [ 158 , 178 ] . both the innate and adaptive immune system participates in the rejection of xenogenic transplants , the exact details of which is outside the scope of the present paper . however , of particular interest here are the findings suggesting that even in the absence of a functional adaptive immune system and in the absence of nk cells ( both of which react to cell surface determinants of xenogenic cells recognized as non - self ) , rejection of xenogenic cells and tissues is seen , which suggests that macrophages could play an important role in this process . indeed , in studies of transplantation of pancreatic islets [ 179 , 180 ] or hematopoietic cells from pigs to mice , macrophage depletion significantly enhances engraftment . the ability of macrophage sirp to inhibit phagocytosis by binding to cd47 on a target cell , and the fact that this interaction is rather species specific suggested that this interaction could play a role in macrophage - mediated clearance of xenogenic cells . indeed , it was found that porcine cd47 can not bind to murine sirp and induce tyrosine phosphorylation of the sirp itims , which could explain the rapid clearance of porcine hematopoietic cells by macrophages both in vitro and in vivo [ 181 , 182 ] . however , expression of murine cd47 in porcine cells markedly inhibits macrophage - mediated phagocytosis of these cells in vitro and prolongs their survival in vivo . although a human sirp fusion protein was found to bind to porcine cd47 , porcine cd47 does not activate human sirp . as a result , porcine hematopoietic cells are rapidly phagocytosed by human macrophages , but expression of human cd47 in porcine cells results in attenuated phagocytosis by human macrophages . these findings indicate that transgenic pigs expressing human cd47 could be an important possibility to further help reducing the rejection of porcine cells in xenotransplantation . the finding that cd47 , by binding to sirp , could regulate rat alveolar macrophage fusion and formation of multinucleated giant cells in vitro suggested a function also in regulating formation of bone - resorbing osteoclasts and regulation of bone homeostasis . using functional blocking monoclonal antibodies against either cd47 or sirp , the formation of tartrate resistant acid phosphatase ( trap ) multinucleated osteoclasts is strongly inhibited in murine cell culture systems in vitro . this finding can also be confirmed in vivo , since cd47-deficient mice have reduced numbers of osteoclasts in bone and are protected from tumor metastasis and subsequent bone resorption . there are a few hypotheses presented to explain the mechanism whereby the interaction between cd47 and sirp promotes osteoclastogenesis . one suggests that binding of cd47 results in sirp tyrosine phosphorylation and subsequent recruitment of shp-1 to the phosphorylated itims , which next would mediate dephosphorylation of nonmuscle cell myosin iia to promote fusion and formation of osteoclasts . this hypothesis is contrasted by a study of sirp mutant mice , expressing a truncated nonsignaling sirp cytoplasmic domain , where cultures of bone marrow cells generated the same number of osteoclasts of the same size , as found in wild - type bone marrow cultures . the important observation made in the latter study was that osteoclasts generated in sirp mutant mice had an increased bone resorbing activity , suggesting that sirp negatively regulates this function in osteoclasts . the contradictory findings presented regarding a possible role of signaling through either cd47 or sirp during fusion of preosteoclasts and formation of multinucleated osteoclasts must also be put in context of the originally presented hypothesis that the interaction between cd47 and sirp may mainly facilitate cell fusion by promoting cell - cell adhesion and bringing the plasma membranes of two cells close enough to facilitate cell fusion . here it has also been highlighted that cd47 may interact with the shorter sirp isoform having only one extracellular igv domain , which would further reduce the distance between two opposing cellular membranes where cell fusion could take place . cd47 can regulate many important physiological cellular mechanisms by interacting with integrins , tsp-1 , or sirp. as outlined above , much knowledge has been collected over the past two decades , but much is probably still to be discovered regarding the functions of this protein . there are several areas where one can assume that further progress may result in new novel ways to interfere with biological mechanisms in pathological conditions . although cd47 is expressed by virtually all cells in the body , in healthy as well as pathological cells , the data so far indicates that cd47-induced apoptosis may still be a way to kill , for example , tumor cells , since it appears that tumor cells and activated cells are much more sensitive to this cell death mechanism than nonactivated nave cells or hematopoietic stem cells . the ability of cd47 to inhibit phagocytosis is clearly an important mechanism whereby innate cells as macrophages or dendritic cells can discriminate between self and non - self , and maintain tolerance to host cells . in the field of xenotransplantation , further development and research to create xenotransplants carrying human cd47 may prove important to obtain even better conditions where xenogenic cell , tissues , or organs can be tolerated when transplanted into humans . in the field of cancer research , where tumor cells may express higher levels of cd47 as a way of avoiding phagocytosis and clearance by phagocytic cells , and where cd47-blocking antibodies have been shown to promote clearance of and to reduce the dissemination of tumor cells , brings hope that careful development of reagents that can block the cd47/sirp interaction may indeed be useful to treat many forms of cancer without having too much of a negative side effect in terms of inducing clearance of host cells . also in the field of bone research , one may suggest that manipulation of the interaction between cd47 and sirp may be a way to modulate bone resorption and to prevent osteoporosis . however , data showing that these two proteins may also be involved in regulating bone formation indicate that manipulation of this signaling system in bone tissue may be more complicated .

interactions between cells and their surroundings are important for proper function and homeostasis in a multicellular organism . these interactions can either be established between the cells and molecules in their extracellular milieu , but also involve interactions between cells . in all these situations , proteins in the plasma membranes are critically involved to relay information obtained from the exterior of the cell . the cell surface glycoprotein cd47 ( integrin - associated protein ( iap ) ) was first identified as an important regulator of integrin function , but later also was shown to function in ways that do not necessarily involve integrins . ligation of cd47 can induce intracellular signaling resulting in cell activation or cell death depending on the exact context . by binding to another cell surface glycoprotein , signal regulatory protein alpha ( sirp ) , cd47 can regulate the function of cells in the monocyte / macrophage lineage . in this spotlight paper , several functions of cd47 will be reviewed , although some functions may be more briefly mentioned . focus will be on the ways cd47 regulates hematopoietic cells and functions such as cd47 signaling , induction of apoptosis , and regulation of phagocytosis or cell - cell fusion .

joubert syndrome ( js ) was initially described in 1968 in 4 siblings with agenesis of the cerebellar vermis presented with episodic hyperpnea , abnormal eye movements , ataxia , and intellectual disability1 ) . several years later , a pathognomonic midbrain - hindbrain malformation the molar tooth sign ( mts)was detected first in js2 ) , and then in several other conditions previously considered as distinct entities . this neuroradiological sign is now represented as a mandatory criterion for the diagnose of js . js can be assumed in all infants presented with hypotonia , altered respiratory pattern , abnormal eye movement , and developmental delay3 ) . in the newborn period , episodic hyperpnea , a subtle facial appearance , and abnormal eye movements nystagmus , colobomas , strabismus , ptosis , and retinal pigmentation are associated with eye abnormalities in neonates3 ) . concomitant symptoms of ptosis , anisocoria with unilateral miosis , and unilateral enophthalmos , which are compatible with horner syndrome ( hs ) , have not been documented in the children of js children thus far . we now describe a neonate of js presented with hypotonia and unique eye abnormalities of nystagmus , ptosis , anisocoria , and unilateral enophthalmos . to the best of our knowledge , a 2-month - old boy was referred to the pediatric neurology clinic due to bilateral nystagmus , bilateral ptosis with left - dominant , left - sided enophthalmos , and anisocoria since the second week after birth . the patient was the first baby of a healthy 28-year - old mother and a healthy 32-year - old father . the baby was a 3,050 g ( 10th25th percentile ) infant born at gestational age of 38 weeks by normal vaginal delivery after an uneventful pregnancy . his height was 48.5 cm ( 25th50th percentile ) , with a head circumference of 33 cm ( 10th25th percentile ) . after birth , the baby had laryngomalacia , hypotonia , and a systolic murmur best heard at the upper left sternal border . an electrocardiogram and echocardiogram revealed normal sinus rhythm , and a small sized secundum atrial septal defect . on physical examination , both pupils promptly reacted to light , but unilateral miosis and enophthalmos of the left eye and left - dominant bilateral ptosis were observed , which could be indicative of hs ( fig . other physical examination findings were unremarkable . in order to evaluate the symptoms of hs and horizontal nystagmus , magnetic resonance imaging ( mri ) of the orbit , orbit and brain mri showed hypoplastic cerebellar vermis and a narrowed anteroposterior diameter of the midbrain with a characteristic of molar tooth appearance ( fig . 2 ) , which were compatible with typical findings of js . neck mri showed no definite lesion or mass around the cervical sympathetic nerve chain , thymus , and mediastinum as the underlying cause of hs . laboratory examinations including renal and hepatic function tests and an abdominal ultrasonography showed normal findings . left - sided visual loss was found in ophthalmological examinations , and neither coloboma nor pigmentary retinal changes were observed . no abnormality was noted in an audiological test and follow - up electrocardiogram and echocardiogram . molecular genetic test was not performed . clinical development at the age of 10 months was global delayed , as explained by the following : he could not babble , follow with or turn his eyes on objects , control his head , sit independently , or hold his bottle . he underwent ophthalmologic surgery ( bilateral frontalis operation ) due to persistent visual disturbance by bilateral ptosis , and had distinct improvement of ptosis ; however , unilateral enophthalmos , and anisocoria remained ( fig . he is currently actively undergoing rehabilitation therapy , and will be regularly monitored through comprehensive developmental examination and follow - up imaging tests , including hepatorenal , cardiac , and ophthalmologic test . the gross photos of the figure were permitted by his parents through the informed consent . js is a rare autosomal - recessive disorder , which is characterized by midbrain - hindbrain malformation mainly in the form of agenesis or dysgenesis of cerebellar vermis3 ) . the incidence rate is 1 out of 800,000 to 100,000 per a year in the newborn5 ) . describe several diagnostic criteria ; ( 1 ) cranial mri findings demonstrating the mts on axial imaging with these three components : midline cerebellar vermis hypoplasia , deepened interpeduncular fossa , and thick , elongated superior cerebellar peduncles , ( 2 ) hypotonia in infancy , ( 3 ) global developmental delay / mental retardation , and ( 4 ) one or both of the following ( not absolutely required but supportive of the diagnosis ) : ( i ) irregular breathing pattern in infancy ( episodic tachypnea and/or apnea ) , and ( ii ) abnormal eye movement ( nystagmus , jerky eye movements , and difficulty with smooth visual pursuits ) . pathological findings of mts correspond to an image of severe hypo - dysplasia of the cerebellar vermis with midline clefting , fragmentation of cerebellar nuclei and heterotopias of purkinje - like neurons , along with dysplasia of the pontine and medullary structures such as the basis pontis , reticular formation , inferior olivary , dorsal column , and solitary tract nuclei . moreover , typical findings are expressed by the lack of decussation both of the superior cerebellar peduncles and of the corticospinal tracts at the medullary pyramids6 ) . a diagnosis of js should be assumed in all infants presented with hypotonia , abnormal eye movement and developmental delay . especially , a neonate of js shows the characteristic physical signs and symptoms ( table 1)3 ) . the infant may lie in a froglike position , and moderate to severe hypotonia can be observed3 ) . the most common presented feature in the newborn period is an abnormal breathing pattern , characterized by episodic hyperpnea23 ) . the distinctive short alternate episodes of apnea and hyperpnea or episodic hyperpnea lone , sometimes described as panting like a dog, may intensify with stress and progressively improve with age , usually disappearing around sixth months23 ) . the 7 children were diagnosed with js at younger than 1 year old of age78910 ) , and the case of neonatal onset was only one7 ) . the cardinal symptoms of them were unexplainable episodic tachypnea alternating with apnea during neonatal period , abnormal ocular movement , hypotonia , ataxia , and developmental delay . in rare cases most of the patients showed nystagmus , saccadic palsy , or congenital ocular motor apraxia as the abnormal ocular movement , however , one 7-month - old boy had a severe vision loss as leber 's congenital amaurosis8 ) . ocular and oculomotor involvement is common in js2411 ) , however , it has variable phenotypes among patients with js . colobomas , nystagmus , strabismus , ptosis , and abnormal retinal pigmentation are noted in neonates3 ) . genetype - phenytype correlations have stressed the predictive value for specific mutations of retinal involvement . retinal involvement is present in about 80% of patients with ahi1 and cep290 mutations12 ) . ocular misalignment is common in js411 ) , because the lesions of cerebellum lead to dysmetric and slow saccades . this patient showed unique eye abnormalities with transient nystagmus , anisocoria and enophthalmos , and eyelid ptosis . when both anisocoria and ptosis are coexistent , one must consider 2 main possible causes : a third nerve palsy ( parasympathetic pathway ) or a hs ( sympathetic pathway)13 ) . in most cases , a third nerve palsy can usually have disconjugate gaze with limitations of eye movements . hs is a problem of the disturbance of the oculosympathetic pathway13 ) , which is bilaterally originating in the hypothalamus , descending ipsilaterally in the reticular formation of the brainstem , eventually synapsing with the second - order neuron in the lower cervical or upper thoracic spinal cord . the second - order neuron enters the sympathetic chain , which then ascends within the soft tissues of the neck , outside of the spinal cord , where it is vulnerable to disruption at various locations . it finally synapses within the superior cervical ganglion to form the third - order neuron . this third - order neuron forms a plexus surrounding the carotid artery and into the cavernous sinus , finally innervating the dilator muscle of the iris and the mller muscle in the upper and lower eyelids . hs is characterized by a combination of ptosis due to superior tarsal muscle palsy , enophthalmos due to mller muscle palsy , rising of inferior eyelid due to inferior tarsal muscle palsy , miosis , and anhidrosis of the involved face when there is a defect of the sympathetic nerve route from hypothalamus to the orbit14 ) . typical causes include birth trauma , neuroblastoma , vascular anomalies of the large arteries , and chest surgery . if there is no history of birth trauma , an acquired hs in children should prompt evaluation for a tumor , particularly paraspinal neuroblastoma . imaging studies , such as mri of the head , neck , chest , and abdomen , as well as the measurement of urine catecholamines should be evaluated . these variable ocular phenotypes might be supported by a third nerve palsy and multiplex neuropathological findings of the cerebellar vermis and of several pontine and medullary structures . it may arise from the genetic heterogeneity of js and related disorders15 ) . in conclusion , we describe an infant of js with unique eye abnormalities , likely as symptoms of hs . it may suggest that a neonate or young infant with diverse eye abnormalities should be considered for early neuroimaging to evaluate the underlying etiology , although there may be no focal neurologic deficit or significant developmental delay . furthermore , once a diagnosis of js has been made , a further evaluation to assess the possibility of multiorgan involvement should be considered in children .

joubert syndrome ( js ) is characterized by the molar tooth sign ( mts ) with cerebellar vermis agenesis , episodic hyperpnea , abnormal eye movements , and hypotonia . ocular and oculomotor abnormalities have been observed ; however , horner syndrome ( hs ) has not been documented in children with js . we present the case of a 2-month - old boy having ocular abnormalities with bilateral nystagmus , left - dominant bilateral ptosis , and unilateral miosis and enophthalmos of the left eye , which were compatible with hs . brain magnetic resonance imaging ( mri ) revealed the presence of the mts . neck mri showed no definite lesion or mass around the cervical sympathetic chain . his global development was delayed . he underwent ophthalmologic surgery , and showed some improvement in his ptosis . to the best of our knowledge , the association of hs with js has not yet been described . we suggest that early neuroimaging should be considered for neonates or young infants with diverse eye abnormalities to evaluate the underlying etiology .

technetium-99 m ( tc-99 m ) in complex with methylene - diphosphonate ( mdp ) is a commonly used substrate in radionuclide bone imaging . bone scans utilizing this radiotracer are very sensitive and enable nuclear medicine physicians to screen for various osseous abnormalities and pathologies . localization of the radiotracer is accomplished through the exchange of tc-99 m mdp for phosphate compounds during active bone remodeling , and aggregation is subsequently detected using a gamma camera . even though the utilization of tc-99 m mdp bone scintigraphy is useful for identifying various physiologic abnormalities , its overall specificity is limited primarily by the lack of anatomic detail . it is therefore important to utilize the patient 's medical history , in conjunction with other forms of imaging , to aid in differentiating potential pathologies . here , we present a case in which tc-99 m mdp bone scintigraphy was utilized to raise concern for a serious abnormality despite relatively occult findings by conventional imaging modalities . a 70-year - old male presented with mild left hip pain and was found to have an elevated serum alkaline phosphatase ( alk ) level of 770 units / l . unfortunately , due to a language barrier , a complete medical history could not be obtained . in light of the patient 's elevated alk level , the planar whole body images [ figure 1a ] of the tc-99 m mdp bone scan demonstrated intense diffuse uptake extending from the left femoral head to the proximal shaft of the femur . initially , we felt this type of appearance could be compatible with early paget 's disease given the markedly elevated alk , despite the absence of apparent structural deformities . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . ( a ) the planar whole body images of the technetium-99 m methylene diphosphonate bone scan demonstrates intense diffuse uptake extending from the left femoral head to the proximal shaft of the femur ( double ended arrow ) . ( b ) the plain film of the left hip depicts three previously unreported fixation screws surrounded by osteopenic lesions ( arrow ) in the femoral neck and head . to confirm the initial diagnosis , the patient was sent directly for an x - ray [ figure 1b ] . however , the pattern of multifocal sclerosis ( cotton wool appearance ) that would be expected of paget 's disease was not present . instead , the radiograph demonstrated three previously unreported fixation screws , used to repair a femoral neck fracture ( later discovered to have occurred 1 year ago ) . surrounding the screws were poorly defined osteopenic , potentially lytic lesions in the femoral neck and femoral head [ figure 1b ] . correlation of the tc-99 m mdp bone scan with the x - ray changed the initial diagnosis from suspected paget 's disease to a differential of malignancy / metastasis , reparative phase of osteonecrosis , osteomyelitis , or reactive osteolysis to the fixation screws . though simple loosening of the fixation screws could have explained the patient 's mild hip pain , the activity demonstrated by the bone scan was much too diffuse and intense to support this diagnosis . to evaluate for metastatic disease , a computed tomography scan of the chest , abdomen , and pelvis was ordered the following day . evaluation of the scan of the left hip was limited due to streak artifact from the screws ; however , it was still possible to appreciate some mixed lytic / sclerotic changes in the native bone [ figure 2a and b ] . even though these changes raised the suspicion of osseous malignancy or metastasis , they were not pathognomonic , as hardware loosening , osteopenia , and degenerative changes could have a similar appearance . however these findings in combination with the bone scan findings , provided enough suspicion of a pathology sinister enough to refer the patient for surgical hardware revision with potential biopsy . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . ( a and b ) computed tomography of the pelvis reveal mixed lytic / sclerotic lesions ( arrows ) within the native bone , although evaluation of the left hip was limited due to streak artifact as seen on the two axial slices in figure 1 . surgical exploration of the left hip revealed a grossly abnormal appearance of the left proximal femur with areas of hemorrhagic bone surrounding lytic lesions . histologic study of the biopsy [ figure 3 ] demonstrated sarcomatous , spindle shaped cells with adjacent tumor osteoid production ; represented by eosinophilic , amorphous , fibrillary deposits between individual or small aggregates of tumor cells . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . histologic examination of the curettage ( using hematoxylin and eosin stain , 10 ) reveals sarcomatous , spindle shaped cells ( white arrows ) with adjacent tumor osteoid production ( black arrows ) ; represented by eosinophilic , amorphous , fibrillary deposits ( white arrows ) between individual or small aggregates of tumor cells ( black arrows ) . in context , the presence of osteosarcoma was highly unlikely as there have been no case reports of primary osteosarcomas occurring in a site of a fracture that was surgically repaired 1 year prior . additionally , the distal femur would be a far more likely site for a primary osteogenic sarcoma to occur . however , upon further interrogation of the patient 's past medical history , it was discovered that the patient had prostate cancer 11 years ago . given the standard of care at that time , one could surmise that he was treated with 60 - 70 gy of targeted external beam radiation to the prostate . the dose that he received and the time frame since receiving his radiation would be compatible with radiogenic osteosarcoma as there is a latency period of about 10 years . the repaired stress fracture 1 year prior was potentially pathologic from the malignancy , but was most likely missed due to the slightly atypical radiographic appearance on the plain film x - ray performed in the surgeon 's office . tc-99 m mdp bone scans are very sensitive tests for detecting a wide array of abnormalities within the bone . despite its comprehensive applicability , it is limited in identifying specific diseases and pathologies ; often relying on the addition of other investigative modalities to reach a final diagnosis . here , a tc-99 m mdp bone scan was used to identify the presence of abnormal bone remodeling , which led to further imaging work - up and confirmatory biopsy . osteosarcomas are particularly sensitive to tc-99 m mdp bone scans as their pathology involves active bone remodeling . however , its presence here was unexpected due to the atypical location on the bone scan and the limited available medical history ( due to a language barrier ) . when paired with the patient 's medical history of radiation treatment and recent fracture , the femur would have been exposed to scatter and bremsstrahlung radiation from its proximity to the treatment field . additionally , the 10-year latency period to developing malignancy is typical for post - external beam radiation therapy induced malignancies . the tc-99 m mdp bone scan demonstrated its value by raising the suspicion for a more sinister process in the left hip than was seen in radiographic appearance on plain film . this case demonstrated the value of tc-99 m mdp bone scintigraphy as an initial detector of bone abnormalities . its sensitivity prevents it from identifying specific pathologies without the aid other investigative methods as depicted in this case . this case also underscores the importance of considering the bone scan findings in conjunction with the patient 's clinical history and anatomic imaging . by using all this information in combination , we were able to sift through the differential and refer the patient for appropriate management .

we present a case of a 70-year - old male who was referred for a technetium-99 m methylene - diphosphonate bone scan for mild left hip pain and an elevated alkaline phosphatase level of 770 units / l . no additional information was provided and the patient 's history was limited due to a language barrier . we were able to ascertain that the patient had a remote history of prostate cancer , which had been treated with radiation . originally , we felt the bone scan was compatible with paget 's disease ; however , further work - up revealed the presence of osteosarcoma , which was potentially radiation - induced .

acute megakaryoblastic leukemia ( amkl ) with t(1;22)(p13;q13 ) represents < 1% of all acute myeloid leukemias ( aml ) , and is associated with the rbm15 ( ott)-mkl1 ( mal ) fusion gene . it occurs at higher incidence in the pediatric population and accounts for approximately 70% of infants with amkl 1,2 . in contrast to infants with 11q23-associated leukemia who frequently exhibit hyperleukocytosis , those with rbm15-mkl1 fusion - positive amkl usually present with decreased leukocyte count , severe anemia , and thrombocytopenia . the pathological findings show fibrosis along with the leukemic cells in the bone marrow and lymph node 3 . amkl with t(1;22)(p13;q13 ) is a rare disease that is often associated with massive organomegaly . neuron - specific enolase ( nse ) , the -subunit of enolase , is found not only in neuroendocrine cells but also in other cells including erythrocytes and subsets of lymphocytes 4,5 . increased levels of serum nse , we report on a neonate who presented with massive hepatomegaly and increased nse levels and was misdiagnosed with congenital neuroblastoma . the patient was the second daughter of healthy and non - consanguineous japanese parents , born at 38 weeks of gestation following an uncomplicated pregnancy , and her body weight was 3164 g. at birth , she was found to have abdominal distension . peripheral blood analysis demonstrated thrombocytopenia with a platelet count of 32 10/l . laboratory investigations revealed the following : total bilirubin of 8.8 mg / dl , direct bilirubin of 0.7 mg / dl , lactate dehydrogenase of 2942 iu / l , aspartate aminotransferase of 248 iu / l , alanine aminotransferase of 66 iu / l , -glutamyl transpeptidase of 222 iu / l , blood urea nitrogen of 4 mg / dl and creatinine of 0.3 mg / dl . serum nse level was extremely high ( 1050 ng / ml ; normal value < 25 ng / ml ) and -fetoprotein was 59,540 ng / ml ( within the normal range of age - matched controls ) . immunophenotyping and karyotyping of the bone marrow sample could not be examined because of the very small size of the sample . arrows indicate blast cells with large nuclei and basophilic cytoplasm ( may giemsa staining , original magnification 1000 ) . an initial diagnosis was made of neuroblastoma stage 4s with an unknown primary lesion , although urine vma and hva levels were normal . however , she died of pulmonary hemorrhage 1 day after therapy . abdominal mri imaging . reverse transcriptase - polymerase chain reaction ( rt - pcr ) assay for the detection of rbm15-mkl1 fusion transcripts was performed in a not bone marrow , but liver sample with forward primer rbm15-s ( 5-ttcctcagctgcatcagaca-3 ) located in rbm15 exon 1 ( genbank accession no . nm_022768.4 ) and reverse primers mkl1-as ( 5-tcattgaggtcatcggctag-3 ) and mkl1-as2 ( 5-tcattgaggtcatcggctag-3 ) located in mkl1 exon 7 ( genbank accession no . pcr amplifications were performed using the qiagen one - step rt - pcr kit ( qiagen gmbh , hilden , germany ) , in accordance with a previously published protocol 7 . sequence analysis was directly performed on the amplified rt - pcr product by using bigdye terminator cycle sequencing chemistry ( applied biosystems , foster city , ca ) on an automated sequencer abi prism 310 genetic analyser ( applied biosystems ) , in accordance with the manufacturer 's instructions . the patient was the second daughter of healthy and non - consanguineous japanese parents , born at 38 weeks of gestation following an uncomplicated pregnancy , and her body weight was 3164 g. at birth , she was found to have abdominal distension . peripheral blood analysis demonstrated thrombocytopenia with a platelet count of 32 10/l . laboratory investigations revealed the following : total bilirubin of 8.8 mg / dl , direct bilirubin of 0.7 mg / dl , lactate dehydrogenase of 2942 iu / l , aspartate aminotransferase of 248 iu / l , alanine aminotransferase of 66 iu / l , -glutamyl transpeptidase of 222 iu / l , blood urea nitrogen of 4 mg / dl and creatinine of 0.3 mg / dl . serum nse level was extremely high ( 1050 ng / ml ; normal value < 25 ng / ml ) and -fetoprotein was 59,540 ng / ml ( within the normal range of age - matched controls ) . immunophenotyping and karyotyping of the bone marrow sample could not be examined because of the very small size of the sample . arrows indicate blast cells with large nuclei and basophilic cytoplasm ( may giemsa staining , original magnification 1000 ) . an initial diagnosis was made of neuroblastoma stage 4s with an unknown primary lesion , although urine vma and hva levels were normal . however , she died of pulmonary hemorrhage 1 day after therapy . abdominal mri imaging . reverse transcriptase - polymerase chain reaction ( rt - pcr ) assay for the detection of rbm15-mkl1 fusion transcripts was performed in a not bone marrow , but liver sample with forward primer rbm15-s ( 5-ttcctcagctgcatcagaca-3 ) located in rbm15 exon 1 ( genbank accession no . nm_022768.4 ) and reverse primers mkl1-as ( 5-tcattgaggtcatcggctag-3 ) and mkl1-as2 ( 5-tcattgaggtcatcggctag-3 ) located in mkl1 exon 7 ( genbank accession no . pcr amplifications were performed using the qiagen one - step rt - pcr kit ( qiagen gmbh , hilden , germany ) , in accordance with a previously published protocol 7 . sequence analysis was directly performed on the amplified rt - pcr product by using bigdye terminator cycle sequencing chemistry ( applied biosystems , foster city , ca ) on an automated sequencer abi prism 310 genetic analyser ( applied biosystems ) , in accordance with the manufacturer 's instructions . , there were multiple nodules composed of diffuse proliferation of small round cells in the liver ( fig.3a ) . immunohistochemically , the proliferating cells were positive for cd41 , cd42b , cd43 , and nse , but negative for myeloperoxidase , tdt , cd7 , cd68 , cd163 , pgp9.5 , and tyrosine hydroxylase ( fig.3b ) . fluorescence in situ hybridization revealed chromosome 21 disomy , indicating that the patient did not have down syndrome . atypical cells are observed in the liver ( hematoxylin and eosin staining left panel : original magnification 200 ; right panel : original magnification 1000 ) . the lesion is positive for nse ( left panel ) and cd41 ( right panel ) ( original magnification 200 ) . lanes 1 , 2 and 4 indicate a 100-bp molecular marker , an internal control , and a negative control , respectively . ( b ) sequence analysis of the amplified rt - pcr product revealed an in - frame fusion between rbm15 exon 1 and mkl1 exon 5 . leukemia occurs much less frequently in the perinatal period than in later childhood . although not as common as neuroblastoma , leukemia is the leading cause of death due to neoplastic disease in neonates 8 . many cases of congenital neoplasms do not present with overwhelming signs and symptoms , and rapid and accurate diagnosis may be difficult . amkl is a rare variant of aml , and the leukemic blasts show characteristic morphologic and phenotypic features , indicating megakaryocytoid differentiation . a distinct entity characterized by t(1 ; 22)(p13;q13 ) , resulting in the rbm15-mkl1 fusion oncogene , has recently been identified . of the recognized subtypes of amkl , the disease associated with t(1;22 ) has clinicopathologic features that present in neonates and infants and is frequently associated with abdominal organomegaly 3,9 . the liver was involved as the primary site in some patients with congenital leukemia 9 . neonates with down syndrome sometimes present transient myeloproliferative disorder mimicking amkl 10 ; however , this patient was not diagnosed with down syndrome . at the initial evaluation , , the elevation of serum nse level and a few and absent blastic cells in the bone marrow and the peripheral blood might have led to a clinical misdiagnosis . nse is usually synthesized by neurons and neuroendocrine cells , and it is a useful marker for the diagnosis and monitoring of patients with neuroendocrine tumors such as neuroblastoma and small cell lung cancer 11 . however , nse is found not only in neuroendocrine cells but also in other cells including erythrocytes and subsets of lymphocytes 4,5 . some of the hematopoietic cell lines including t - cell leukemia and epstein barr virus - immortalized b lymphoblastoid cell lines produce nse . thus , nse is not exclusively expressed in neuroendocrine tumor cells 12 . in the present case , the fibrosis might have been caused by the production and secretion of transforming growth factor beta from the megakaryoblasts , which stimulates collagen synthesis in bone marrow fibroblasts 13 . there are very few documented cases of congenital leukemia in which the primary site of involvement was shown to be the liver , without initial bone marrow findings . a case of congenital anerythremic erythroleukemia was reported to present with hepatic failure and have a fetal outcome of multivisceral involvement 14 . an infant with amkl and a complex translocation demonstrated only 520% blasts on the initial bone marrow aspirate and core biopsy . therefore , myelodysplastic syndrome was initially diagnosed ; however , only later did blasts become apparent in the peripheral blood 15 . in the present case , amkl might have occurred in the fetal period because hematopoietic cells are mainly produced in the fetal liver . here , we report on a neonate who presented with massive hepatomegaly , increased serum nse levels , and was misdiagnosed with congenital neuroblastoma .

key clinical messagewe describe a neonate with abdominal distension , massive hepatomegaly , and high serum neuron - specific enolase level suggestive of congenital neuroblastoma . the patient died of pulmonary hemorrhage after therapy . autopsy revealed that the tumor cells in the liver indicated acute megakaryocytic leukemia with the rbm15-mkl1 fusion gene .

asthma severity and asthma control are often based on the presence and frequency of asthma symptoms , in particular respiratory symptoms such as exercise - induced asthma , limitation of physical activity , and frequency of night asthma symptoms . the revised version of the gina guidelines from 2006 recommends that disease classification can be changed from severity based on symptoms and airflow limitations to success of disease management . this is mainly based on frequency of symptoms and continues to include limitation of physical activity and night symptoms suggestive of level of airway inflammation and increasing need of inhaled corticosteroid ( ics ) . perception of respiratory symptoms may vary between individuals some might feel minor changes more than others . physical activity is an important release of asthma symptoms , revealing exercise - induced bronchoconstriction [ 3 , 4 ] , which is widely believed to be related to either airway inflammation or smooth muscle dysfunction [ 58 ] . the primary treatment strategy in airway inflammation should be inhaled steroid ; whereas smooth muscle dysfunction should be treated primarily with inhaled beta - agonist nighttime awakenings are probably related to increased airway inflammation [ 10 , 11 ] ; however , treatment strategies including lower dose of ics combined with long - acting beta2-agonist have been found to induce a more stable disease than a higher dose of ics alone , which is contradictory to increased inflammatory control . eia and night asthma might arise from airway inflammation , but other factors , such as smooth muscle dysfunction or differences in the patient 's perception / tolerance of symptoms , might play a role . the aim of the present study was to examine in a large population sample whether ahr is of importance for the perception of eia , that is , positive response to a questionnaire and nighttime symptoms . the hypothesis is that symptoms of eia in a general population are not usable for the diagnosis of astma . all asthmatic subjects ( n = 793 ) participating in two large asthma studies in our research unit were pooled in the present analysis [ 13 , 14 ] ; inclusion and exclusion criteria and the examinations performed were the same in all studies . subjects were aged 1444 years ( to minimize entry of patients with copd ) and all lived in copenhagen , denmark . exclusion criteria were respiratory illness other than asthma , for example , rhinitis as single disease , sarcoidosis , and cardiac illness . patients were excluded if they had withdrawn their consent after completing the questionnaires or if they had left the clinic without completing the clinical examination or diagnostic procedures . all participants received information in both oral and written form and gave their consent in writing before enrolment . subjects were asked about respiratory and allergic symptoms ( within the preceding four weeks and at any time ( ever asthma ) ) , use of medication , hospital referrals , and gp or specialist visits . the questions asked about asthma at the interview were adapted from studies by the american thoracic society , division of lung disease of the national heart , lung and blood institute . asthma was defined as asthma symptoms and signs of reversible airway disease , that is , either airway hyperresponsiveness ( ahr ) to inhaled methacholine with a pd20 4 mol , peak expiratory flow ( pef ) variability 20% , or at least a 15% increase in forced expiratory volume in one second ( fev1 ) after administration of a bronchodilator ( minimum 300 ml ) . asthma severity was classified according to the gina guidelines ( 1 ) mild intermittent : symptoms < once a week , nighttime symptoms < twice a month , fev1 > 80% predicted . ( 2 ) mild persistent : symptoms > once a week but < once a day , nighttime symptoms > twice a month , fev1 > 80% predicted . ( 3 ) moderate persistent : daily symptoms , nighttime symptoms > once a week or fev1 6080% predicted . ( 4 ) severe : continuous daytime symptoms , frequent nighttime symptoms or fev1 < 60% predicted . further , nighttime awakenings 0 ( never ) , nighttime awakenings ( nta ) 3 per month ( mild ) , nta 3 - 4 per month ( mild persistent ) , nta > 4 per months and fewer than 2 - 3 per week ( moderate ) , and nta > 4 per week ( severe ) . the severity of asthma ( mild , mild persistent , moderate , and severe ) was classified according to the 2004 gina guidelines , based on symptom frequency only , and asthma control was assessed according to the definition in the 2006 gina guidelines . details about education , socioeconomic aspects , and lifetime consumption of tobacco in pack years ( tobacco consumption [ g / day]/20 duration of smoking [ years ] ) were collected . never smokers were participants who have never smoked , whereas smokers included daily smokers and smokers with a more dispersed consumption . height and weight were measured and body mass index ( bmi ) was calculated as ( weight [ kg])/(height [ m ] ) . spirometry was performed on a 7-l dry wedge spirometer ( vitalograph ) in accordance with the ers and the percentage of predicted normal values of fev1 ( fev1%pred ) and fvc ( fvc%pred ) , and the fev1/fvc ratio was calculated . airway responsiveness to inhaled methacholine was measured in accordance with the method of yan et al . in all patients with fev1 the dose resulting in a 20% fall in fev1 ( pd20 ) was calculated , and the ratio dose response ( rdr ) was calculated as the decline in fev1 from inhaled saline divided by the highest dose of methacholine administered . a constant of four was added to all dose - response ratios to eliminate negative and zero values . measurement of fev1 was repeated 15 minutes after administration of 2 mg terbutaline in those with fev1 < 70% or 15 minutes after the last inhalation of methacholine in those with either symptoms or a significant decrease in fev1 ( i.e. , 20% ) . lastly , all subjects underwent an allergen skin prick test with ten inhalant allergens in accordance with the eaaci guidelines , and blood eosinophils were counted ( 10/l ) . all data were entered in the database by one person and 10% of all patient data were then proofread . when this was done , quality control was conducted by an experienced researcher on all outliers within the entire number of variables . incidence rates were calculated for the entire group and differences were tested by chi - squared analysis . further , differences in mean ( sd ) values between participants with and without respiratory symptoms were tested by parametric analysis ( student 's t - test ) . all included variables were examined in a univariate analysis with exercise - induced asthma or nighttime awakenings due to asthma as the dependent variable . in the event of a significant relationship , variables were entered in a linear regression analysis with backward elimination of all nonsignificant parameters after which a final regression analysis was performed . lastly , an odds ratio analysis was applied in the case of dichotomy variables . a p - value < .05 was considered significant . all 793 asthmatic subjects in the present survey were included based on respiratory symptoms and a positive test to methacholine provocation , inhaled beta - agonist or day - to - day variation in pef ( table 1 ) . the frequency of rhinitis was 71% among those with certain asthma , 62% had positive skin prick test , and 55% were never smokers . the frequency of exercise - induced asthma was as follows : never symptoms 9% ; 54% 2 times per week ; 22% 26 times per week ; 7% daily exercise symptoms ; 8% reported eia more than once a day . the night awakening due to asthma was 69% , 12% , 7% , 6% , and 6% , respectively . of the entire group , 29 asthmatic subjects ( 3.1% ) reported both severe eia and nighttime awakenings , and 55% reported neither symptom ( p < .001 ) . a univariate analysis including severity of eia ( table 2 ) and nighttime awakenings ( table 3 ) showed a significant association between airway inflammation , indicated by responsiveness to inhaled methacholine ( logrdr ) , and blood eosinophil count ( 10/l ) , or obstruction and severity of symptoms . the association between airway responsiveness and eia symptoms ( f = 2.5 , p < .05 , and rho = 0.1 , p = .01 ) was less close than that between airway responsiveness and nighttime awakenings due to asthma ( f = 3.5 , p < .01 and rho = 0.14 , p < .001 ) . further , no significant association was found between rhinitis and severe eia ( 15.4% versus 15.1% , ns ) and nighttime awakenings ( 11.9% versus 10.9% , ns ) ; atopic diseases was seldom seen in those with severe eia ( 10.0% versus 14.3% , p = .08 ) or among those with severe night symptoms ( 12.4% versus 21.0% , p < .01 ) . experience of severe eia was frequently found among female participants ( 21.0% versus 7.3% , p < .01 ) , and nighttime awakenings were found equally frequently among those who had severe eia ( 12.9% versus 8.5% , resp . , ns ) . lastly , those with severe eia also had many nighttime awakenings ( 56.8% versus 11.4% , p < .001 ) ; those with many nighttime awakenings also experienced many symptoms of eia ( 24.0% versus 4.8% , p < .001 ) . concerning eia , by including all variables in a multivariate analysis , logrdr was eliminated ; whereas fev1%pred ( .16 , p < .001 ) , smoking ( .098 , p < .05 ) , atopy ( .16 , p < .001 ) , sex ( .18 , p < .001 ) , and asthma treatment ( .17 , p < .01 ) were found to be of significant consequence for development of eia . women reported persistent exercise symptoms more frequently than did men ( 21% versus 7% , p < asthmatic subjects with persistent exercise symptoms had lower lung function than those without symptoms ( 88% versus 94% , p < .001 ) , and treatment with inhaled steroid was more frequently used among those with persistent exercise symptoms ( 41% versus 17% , p < .001 ) . including all variables in a multivariate analysis concerning nighttime awakenings showed that logrdr was eliminated as well ; whereas frequency of shortness of breath during daytime ( 0.263 , p < .001 ) , coughing ( 0.243 , p < .001 ) , and eia ( 0.102 , p = .066 ) were of significant consequence . furthermore , a higher level of eosinophils was associated with a higher level of nighttime awakenings ( 0.155 , p < .011 ) . these findings showed that among those with frequent night symptoms , 64% experienced daily coughing , 31% had daily dyspnoea , and the eosinophil count increased from 0.22 10/l ( no night symptoms ) to 0.35 10/l ( frequent night symptoms ) , ( normal cell count 0 - 0.4 10/l ) . the relative risk of having nighttime awakenings due to asthma was more than twofold higher among those with eia symptoms than among those without eia symptoms . in addition , the odds ratio ( ci95% ) was 2.77 ( 2.03.8 ) ( p < .001 ) of having nighttime awakenings due to asthma among those with eia symptoms . although many different surrogate variables representing airway inflammation such as bronchial responsiveness , low level of lung function , signs of airway obstruction ( low fev1/fvc ratio ) , positive skin prick test , high eosinophilic cell count , and many daily asthma symptoms were included in the analysis of this large uniform cohort of persons with asthma , the study showed great diversity . eia and nighttime awakenings due to asthma symptoms are symptoms of equal importance in the gina guidelines when evaluating the severity of asthma . according to the present guidelines , severity of asthma is based equally on the frequency of respiratory symptoms , such as exercise - induced asthma symptoms , and nighttime awakenings due to asthma symptoms . less controlled disease is usually an indicator of increased airway inflammation , and the guidelines therefore suggest initiation of inhaled steroid . from a theoretical viewpoint , exercise - induced bronchoconstriction could occur only in the event of airway inflammation , and the more severe the eia the more severe the airway inflammation that could probably be demonstrated [ 35 , 11 , 22 ] . symptoms of eia in elite athletes are thus not to be used as a predictor for asthma [ 23 , 24 ] as those symptoms are not related to either airway hyperresponsiveness to inhaled agents or to indirect provocation . it is thus unknown whether eia symptoms are closely associated with airway responsiveness and airway inflammation in a general population of asthmatic subjects who have demonstrated certain airway variability , because the majority of studies have been performed in groups with selected asthmatic subjects who report eia . however , the present study showed that factors important for the presence of eia symptoms and nighttime awakenings were outside the battery of inflammatory variables . these present findings indicate a close univariate association between eia symptoms and ahr , which is in agreement with earlier findings of a close association between degree of eia and ahr to inhaled agents [ 25 , 26 ] . however , in the final analysis many inflammatory variables were statistically eliminated , whereas eia symptoms continued to show a significant association with low level of lung function and positive skin prick test , which could suggest an association between markers of airway inflammation and eia in asthmatic subjects with a history of eia symptoms . one of which , the most important predictor of airway inflammation in the present study , may be having low level of lung function , as low level of lung function could be due to inflammatory swelling of the airway mucosa [ 2729 ] . the asthmatic women frequently reported persistent exercise - induced symptoms ; whereas nighttime awakenings in asthmatic participants in this large population were without sex association when interview by a specialist using a questionnaire - based interview with focus on the issue , as suggested by gina guidelines . it indicated that women may have a lower tolerance to eia or perhaps a different perception of eia compared with that of men . an explanation of women 's lower tolerance to eia could be a symptom of low fitness , and not astma or airway inflammation . women probably perform fewer physical activities than do men , especially among the oldest in this group ; consequently , when they exercise they develop more symptoms . another explanation could be the level of lung function , because although both women and men who reported severe eia generally had low level lung function , the women had a significantly larger reduction in fev1 percentage predicted than did the men ( data not shown ) , indicating that low ventilatory capacity could also be a satisfactory explanation of the present results of the eia symptoms . other studies indicate that asthmatic persons who are smokers have impaired effect of their asthma treatment , and compared with the asthmatic subjects who were non - smokers , the asthmatic smokers showed low level of lung function , less improvement in lung function , bhr , and symptoms [ 30 , 31 ] . these findings support our finding of more eia symptoms among those asthmatic subjects who smoked , although it is unexplained whether it is due to the smoking history or the low level of lung function , but it seems not to be inflammation . those asthmatic subjects with severe nighttime awakenings seem to have more severe illness than those with eia symptoms . those with night symptoms had a high eosinophilic count ; whereas the perception of exercise - induced symptoms had a cell count which was significantly lower . those with nighttime awakenings due to asthma had a more severe degree of airway responsiveness than did those with exercise symptoms ; moreover , those who had severe night awakening also had frequently signs of other asthma symptoms , such as dyspnoea and cough during daytime , all of which indicate that those with nighttime awakenings due to asthma might have severe airway inflammation and need treatment with inhaled steroid . these findings indicate that symptoms of eia may be less related to inflammation , whereas nighttime awakenings are more closely related to airway inflammation . the higher eosinophilic count found among those with many night awakenings is of significant importance as the prognosis of asthmatic subjects with high level of eosinophilic cell count ; many respiratory symptoms and severe ahr have been shown to be serious with a more severe and more variable asthmatic disease . nighttime awakenings due to asthma compared with eia symptoms without objective measurements are probably more likely to be based on airway inflammation , which indicates that these asthmatic subjects with nighttime awakenings , but not symptoms of eia , would probably benefit from increased treatment with inhaled steroid [ 3234 ] . however , we found a significantly increased risk of having nighttime awakenings among those with exercise - induced asthma symptoms . the risk was almost threefold increased among those with many eia symptoms and nighttime symptoms , indicating some association between the two different questions of asthma symptoms , and that although the nighttime awakenings seem to be more closely associated with airway inflammation , the presence of many exercise symptoms also has some relation to the presence of airway inflammation , but the majority of symptoms might be due to low level of fitness . based on these findings and earlier findings in elite athletes [ 23 , 24 ] the gina guidelines would most likely benefit from reducing the importance of eia symptoms as an initiator of increased treatment strategy of inhaled steroid without having an exercise test showing eib . these findings also emphasise the importance of objective measurement of asthma , including objective measurement of eia symptoms , before including eia as important for the level of asthma severity and the level of control . it could be speculated , however , whether some of these symptoms are based on a smooth airway dysfunction rather than inflammation or low level of fitness , indicating need of bronchodilator and not steroid , as all participants had proven asthma . in the present study a substantial number of asthmatic subjects were either untreated or undertreated ; this has been reported in earlier publications . these findings of undertreatment in the present population of asthmatic subjects highlight the importance of the present findings . the subjects were not contaminated with inhaled steroid , and even those who reported serious night awakening due to asthma were not receiving treatment according to the guidelines . in conclusion , in this large group of asthmatic subjects we found that eia symptoms and night symptoms were associated with ahr in a univariat analysis , but other factors seem of importance for the perception of eia and night symptoms . symptoms of eia in a general population are , like eia symptoms in elite athletes , not usable for the diagnosis of astma . night asthma is more closely associated with airway inflammation , which verifies what we had expected .

background . asthma experienced during exercise and during the night is based on the presence of airway hyperresponsiveness ( ahr ) . the aim of the present study was to examine whether ahr is a predictor of exercise - induced asthma ( eia ) and nighttime symptoms . material . we included 793 asthmatics subjects with symptoms and a positive asthma test . results . mean ( sd ) fev1 was 93% ( 15 ) , 71% had rhinitis , and 62% had atopy . both eia and nighttime symptoms were associated with ahr ; however , when including other factors of importance in a multivariate analysis , logrdr was eliminated , whereas fev1% pred ( p < .001 ) , smoking ( p < .05 ) , atopy ( p < .001 ) , sex ( p < .001 ) , and treatment ( p < .01 ) were associated with having eia while dyspnoea ( p < .001 ) , cough ( p < .001 ) , and eosinophils ( p < .01 ) were associated with frequent night symptoms . the risk of having nighttime awakenings due to asthma was more than twofold higher among those with eia symptoms than among those without symptoms ( or ( ci95% ) 2.77 ( 2.03.8 ) ( p < .001 ) ) . in conclusion . eia and night symptoms are associated with ahr , but other factors of importance eliminated this close association . night asthma is more closely associated with airway inflammation than ahr .

the use of regenerative procedures before implant - prosthetic oral rehabilitation is required to counteract vertical and horizontal bone loss and , mostly , to obtain an adequate bone quantity and quality to ensure primary stability at implant insertion . several bone grafting materials were proposed for bone regeneration , such as autogenous , allogenic and xenogenic biomaterials , or a combination of them and , as the biological response of host tissue can be related to biomaterial origin , attention was focused on the interactions occurring between grafts and host tissue . as already reported by mangano et al . , heterologous biomaterials were found to be as clinically efficient as autologous bone for their capacity of osteoconduction , even if little is known about their capability to be resorbed , and about the time they need to be fully replaced by newly formed bone tissue . in fact , a biomaterial showing a too rapid resorption rate may result unsuitable for bone augmentation procedures , because it could be completely resorbed before osteogenic cells have colonized the defect ; on the other hand , a biomaterial showing no resorption could cause problems to the regeneration of bone with a lower osteogenesis ability in respect to native autologous bone . in literature , only a few studies were reported about the use of an equine - derived bone substitute , which seems to be able to induce osteoblast differentiation , to be resorbed in vitro by osteoclasts and to be successfully used in mandibular ridge augmentation . moreover , the integration of a bone graft results in a remodeling process similar to the physiological bone healing event following a bone fracture . bone remodeling occurs through different steps , which start from a lesion of the vascular structure at the site of the injury reducing the supply of nutrients and determining an initial bone resorption . the hematoma generated activates signaling molecules and growth factors , which stimulate proliferation and differentiation of osteo - progenitor cells . among them , transforming growth factor- ( tgf- ) superfamily and angiogenic factors are included . in particular , tgf1 is essential for osteoblastic differentiation of mesenchymal precursors , it induces the synthesis of bone morphogenenetic proteins ( bmps ) and promotes osteoid and extracellular protein production such as collagen , osteopontin and osteonectin . moreover , tgf-1 is an important factor for osteoclasts and osteoblasts coupling : in fact , it not only promotes the recruitment of hematopoietic osteoclast precursors , but also shows an inhibitory effect on bone resorption and stimulates the production of osteoprotegerin ( opg ) . opg is expressed by osteoblasts and regulates bone homeostasis by inhibiting osteoclastogenesis and bone resorption . opg , binding to rankl on osteoblast / stromal cells , blocks the rankl / rank interaction between osteoblast / stromal cells and osteoclast precursors , inhibiting osteoclast precursor differentiation , reducing osteoclast production and regulating osteoclast - mediated resorption . moreover , in such processes a main role is also played by angiogenic factors , among which vascular endothelial growth factor ( vegf ) is included . vegf is produced by endothelial cells and osteoblasts and is involved in early bone remodeling phases since it controls osteoblast growth . the early phases of bone graft integration are followed by remodeling phenomena driven by molecules produced by osteoblasts , as bone sialoprotein ( bsp ) and secreted protein acidic and rich in cysteine ( sparc ) , leading to new extracellular matrix deposition and to its subsequent mineralization . bsp is a component of the extracellular matrix which plays important functions in the regulation of new bone apposition and remodeling . bsp expression is up - regulated by factors inducing osteoblast differentiation , released by active osteogenic cells at the site of new bone formation , even though it has also been shown to promote osteoclastic resorption of mineralized surfaces . sparc , also known as osteonectin , is a collagen - binding glycoprotein that appears to regulate cell growth through interactions with extracellular matrix ( ecm ) and cytokines . it is secreted by osteoblasts during bone formation , initiating a mineralization process and promoting mineral crystal formation . it is a modulator of the mineralizing process , essential for bone graft integration , such as the bone healing around dental implants . based on this knowledge , the aim of our work was to analyze and to compare the molecular events switched on by autologous or heterologous bone graft insertion , focusing our attention on tgf1 expression and opg / rankl ratio , to analyze resorption process , and to estimate bone graft vascularization , new bone formation and its mineralization , through vegf , bsp and sparc expression evaluation , respectively . by understanding the mechanism underlying graft integration and by comparing the results obtained with the use of the heterologous bone substitute to that obtained with the use of autologous bone it should be verified if equinederived bone substitute possesses characteristic , which may permit a good integration within the host bone tissue , thus clinically ensuring an adequate primary stability to the implant and predictability of the implant - prosthetic rehabilitation . twenty patients ( 13 males , 7 females ; age ranging 4558 ) with inadequate bone volume in the posterior maxilla , were scheduled for bone augmentation procedures followed by implant placement . the patients were divided into two groups ( n=10 ) , according to the severity and morphology of the bone defect , mirroring classes c and f of the chiapasco 's classification of the posterior maxilla ( table 1 ) . all patients received sufficient information about the inclusion in this study and gave written consent in accordance with italian legislation and with the code of ethical principles for medical research involving human subjects of the world medical association ( declaration of helsinki ) . ten patients ( 8 males , 2 females ; age ranging 4554 ) , having class c bone defects , underwent maxillary sinus augmentation procedure with a bone substitute of equine origin ( biobone osteoconductor mix , biosaf in s.r.l . , ancona , italy ) ( group 1 ) , and ten patients ( 5 males , 5 females ; age ranging 4658 ) , with class f bone defects , received an onlay bone graft and a maxillary sinus augmentation procedure with bone obtained from the parietal region of the calvaria ( group 2 ) . table 1chiapasco 's classification of the posterior maxilla.classheight of the residual ridgethickness of the residual ridgeinterarch distancea>5 mm < 8 mm6 mmnormalb>5 mm < 8 mm<6 mmnormalc<5 mm6 mmnormald<5 mm<6 mmnormale>5 mm < 8 mm6 mmincreasedf>5 mm < 8mm<6 mmincreasedg<5 mm6 mmincreasedh<5 mm<6 mmincreasedicompletely resorbed alveolar ridge with increased distance interarch and class iii skeletal relationships biobone osteoconductor mix is a reasorbable , collagen - deprived , and deantigenated osteoconductive biomaterial , obtained after a biological process of deantigenation at 37c in a humid atmosphere , made up of a corticocancellous mixture of equine origin ( particles width 0.51 mm ) . the autologous bone blocks from the parietal region of calvaria were taken from calvaria under general anesthesia by a piezoelectric instrument ( easy surgery , biosaf in s.r.l . ) , shaped according to the dimension of the defects , properly fitted in the recipient site , and fixed with lag screw to rebuild the alveolar ridge . all gaps between the bone blocks and the recipient sites and the maxillary sinus were packed with bone chips obtained from the same donor site , and the grafted areas covered with a resorbable barrier ( biobone collagen membrane , biosaf in s.r.l . ) post - operative healing was uneventful for all the patients , and therefore , after about 6 months they were scheduled for a second surgery for implant placement . contextually the intervention of implant insertion , bone samples were retrieved by a 3 mm - diameter and 8 mm - height trephine bur under sterile saline solution irrigation in the sites of implant placement , in order to obtain significant specimens of bone regenerated with heterologous bone substitute in group 1 patients , while in group 2 patients bone samples were obtained from the lateral maxillary wall between the sites of implant placement . samples of bone tissues were fixed in 10% phosphate - buffered formalin for 24 h , and decalcified in 10% tetrahydrated edta , according to data sheet ( mielodec kit , bio - optica , milan , italy ) . subsequently , they were dehydrated through ascending alcohols and xylene , and then paraffin embedded . samples were dewaxed ( xylene and alcohol at progressively decreasing concentrations ) , sliced 5 m thick and processed for hematoxylin - eosin staining and for immunohistochemical analysis . in order to detect tgf1 , opg , rankl , vegf , bsp , and sparc proteins , immunohistochemistry was performed on 5 m - thick sections by means of ultravision lp detection system hrp polymer & dab plus chromogen ( lab vision thermo , ca , usa ) . to reduce non - specific background staining due to endogenous peroxidase slides were then incubated in the presence of mouse anti - tgf1 , anti - rankl and anti - sparc monoclonal antibodies , and rabbit anti - vegf polyclonal antibody ( santa cruz biotechnology , santa cruz , ca , usa ) , mouse anti - opg monoclonal antibody ( acris antibodies , herford , germany ) , and mouse anti - bsp monoclonal antibody ( calbiochem , darmstadt , germany ) . peroxidase was developed using diaminobenzidin chromogen ( dab ) , and nuclei were hematoxylin counterstained . randomly selected slides belonging to each sample were then observed by means of leica dm 4000 light microscopy ( leica cambridge ltd , cambridge , uk ) , equipped with a leica dfc 320 camera ( leica cambridge ltd ) for computerized images . after digitizing the images obtained from the immunohistochemical stained sections , qwin plus 3.5 software ( leica cambridge ltd ) was used to evaluate tgf1 , opg , rankl , vegf , bsp and sparc expression . image analysis of protein expression was performed through the quantification of immunohistochemical brown chromogen , expressed as percentage of positive area respect to total area of the field , as an average value per ten fields , randomly chosen , for each sample at light microscope observation . moreover , intensity of staining ( is ) was graded on a scale of 04 , according to the following assesssments : 0 , no detectable staining ; 1 , weak staining ; 2 , moderate staining ; 3 , strong staining ; 4 , very strong staining , as previously reported . the positive immunolabeling for tgf1 , opg , rankl , and vegf was cytoplasmic ; the positive immunolabeling for bsp and sparc was in the pericellular space . quantification of immunohistochemical brown chromogen was performed at 20 magnification by three different researchers , and the final result was a mean value of the three separate evaluations . cohen 's kappa coefficient was applied to measure the agreement between the three observers and averaged over all three to evaluate overall agreement using the following grading : 00.2 ( slight ) , 0.210.40 ( fair ) , 0.410.60 ( moderate ) , 0.610.80 ( substantial ) , and 0.811.0 ( almost perfect ) negative control images were randomly chosen . the statistical significance of the results was evaluated by the wilcoxon , mann - whitney test , using r software , ver . after collecting results , the mean data were reported and showed in an histogram using excel 2010 for microsoft windows . twenty patients ( 13 males , 7 females ; age ranging 4558 ) with inadequate bone volume in the posterior maxilla , were scheduled for bone augmentation procedures followed by implant placement . the patients were divided into two groups ( n=10 ) , according to the severity and morphology of the bone defect , mirroring classes c and f of the chiapasco 's classification of the posterior maxilla ( table 1 ) . all patients received sufficient information about the inclusion in this study and gave written consent in accordance with italian legislation and with the code of ethical principles for medical research involving human subjects of the world medical association ( declaration of helsinki ) . ten patients ( 8 males , 2 females ; age ranging 4554 ) , having class c bone defects , underwent maxillary sinus augmentation procedure with a bone substitute of equine origin ( biobone osteoconductor mix , biosaf in s.r.l . , ancona , italy ) ( group 1 ) , and ten patients ( 5 males , 5 females ; age ranging 4658 ) , with class f bone defects , received an onlay bone graft and a maxillary sinus augmentation procedure with bone obtained from the parietal region of the calvaria ( group 2 ) . table 1chiapasco 's classification of the posterior maxilla.classheight of the residual ridgethickness of the residual ridgeinterarch distancea>5 mm < 8 mm6 mmnormalb>5 mm < 8 mm<6 mmnormalc<5 mm6 mmnormald<5 mm<6 mmnormale>5 mm < 8 mm6 mmincreasedf>5 mm < 8mm<6 mmincreasedg<5 mm6 mmincreasedh<5 mm<6 mmincreasedicompletely resorbed alveolar ridge with increased distance interarch and class iii skeletal relationships biobone osteoconductor mix is a reasorbable , collagen - deprived , and deantigenated osteoconductive biomaterial , obtained after a biological process of deantigenation at 37c in a humid atmosphere , made up of a corticocancellous mixture of equine origin ( particles width 0.51 mm ) . the autologous bone blocks from the parietal region of calvaria were taken from calvaria under general anesthesia by a piezoelectric instrument ( easy surgery , biosaf in s.r.l . ) , shaped according to the dimension of the defects , properly fitted in the recipient site , and fixed with lag screw to rebuild the alveolar ridge . all gaps between the bone blocks and the recipient sites and the maxillary sinus were packed with bone chips obtained from the same donor site , and the grafted areas covered with a resorbable barrier ( biobone collagen membrane , biosaf in s.r.l . ) post - operative healing was uneventful for all the patients , and therefore , after about 6 months they were scheduled for a second surgery for implant placement . contextually the intervention of implant insertion , bone samples were retrieved by a 3 mm - diameter and 8 mm - height trephine bur under sterile saline solution irrigation in the sites of implant placement , in order to obtain significant specimens of bone regenerated with heterologous bone substitute in group 1 patients , while in group 2 patients bone samples were obtained from the lateral maxillary wall between the sites of implant placement . samples of bone tissues were fixed in 10% phosphate - buffered formalin for 24 h , and decalcified in 10% tetrahydrated edta , according to data sheet ( mielodec kit , bio - optica , milan , italy ) . subsequently , they were dehydrated through ascending alcohols and xylene , and then paraffin embedded . samples were dewaxed ( xylene and alcohol at progressively decreasing concentrations ) , sliced 5 m thick and processed for hematoxylin - eosin staining and for immunohistochemical analysis . in order to detect tgf1 , opg , rankl , vegf , bsp , and sparc proteins , immunohistochemistry was performed on 5 m - thick sections by means of ultravision lp detection system hrp polymer & dab plus chromogen ( lab vision thermo , ca , usa ) . to reduce non - specific background staining due to endogenous peroxidase slides were then incubated in the presence of mouse anti - tgf1 , anti - rankl and anti - sparc monoclonal antibodies , and rabbit anti - vegf polyclonal antibody ( santa cruz biotechnology , santa cruz , ca , usa ) , mouse anti - opg monoclonal antibody ( acris antibodies , herford , germany ) , and mouse anti - bsp monoclonal antibody ( calbiochem , darmstadt , germany ) . peroxidase was developed using diaminobenzidin chromogen ( dab ) , and nuclei were hematoxylin counterstained . negative controls were performed by omitting the primary antibody . randomly selected slides belonging to each sample were then observed by means of leica dm 4000 light microscopy ( leica cambridge ltd , cambridge , uk ) , equipped with a leica dfc 320 camera ( leica cambridge ltd ) for computerized images . after digitizing the images obtained from the immunohistochemical stained sections , qwin plus 3.5 software ( leica cambridge ltd ) was used to evaluate tgf1 , opg , rankl , vegf , bsp and sparc expression . image analysis of protein expression was performed through the quantification of immunohistochemical brown chromogen , expressed as percentage of positive area respect to total area of the field , as an average value per ten fields , randomly chosen , for each sample at light microscope observation . moreover , intensity of staining ( is ) was graded on a scale of 04 , according to the following assesssments : 0 , no detectable staining ; 1 , weak staining ; 2 , moderate staining ; 3 , strong staining ; 4 , very strong staining , as previously reported . the positive immunolabeling for tgf1 , opg , rankl , and vegf was cytoplasmic ; the positive immunolabeling for bsp and sparc was in the pericellular space . quantification of immunohistochemical brown chromogen was performed at 20 magnification by three different researchers , and the final result was a mean value of the three separate evaluations . cohen 's kappa coefficient was applied to measure the agreement between the three observers and averaged over all three to evaluate overall agreement using the following grading : 00.2 ( slight ) , 0.210.40 ( fair ) , 0.410.60 ( moderate ) , 0.610.80 ( substantial ) , and 0.811.0 ( almost perfect ) negative control images were randomly chosen . the statistical significance of the results was evaluated by the wilcoxon , mann - whitney test , using r software , ver . after collecting results , the mean data were reported and showed in an histogram using excel 2010 for microsoft windows . morphological analysis was performed at light microscope after hematoxylin - eosin staining ( figure 1 ) . in group 1 specimens the native bone tissue and connective tissue fibers could be identified . moreover , the grafted biomaterial particles are easily distinguished ( purple areas ) due to their lack of tissue structure , as better shown by the inset . group 2 sample shows large mineralized areas close to dense areas of connective tissue , within which newly formed bone is recognizable because of osteocyte lacunae lack and absence of lamellar organization ( inset ) . molecular modifications occurring after bone substitutes placement , concerning their ability to be integrated and to be clinically suitable , tgf-1 expression , essential for osteoblastic differentiation and osteoid and extracellular proteins production , do not show any statistically significant difference between the two experimental groups ( figure 2 ) ; in fact , moderate tgf-1 immunolabeling is seen both in group 1 and group 2 . moreover , since tgf-1 stimulates opg production , inhibiting the rankl / rank interaction , opg and rankl expressions were checked . moderate opg immunolabeling is found in group 1 and weak opg immunolabeling in group 2 , whereas weak rankl immunolabeling is seen both in group 1 and group 2 . however , group 1 samples show a mean ratio of about four fold higher than that observed in group 2 specimens ( 2.4 vs 1.1 ) , due to the concomitant increase of opg and a significant decrease of rankl expression in samples from sites regenerated with the equinederived bone substitute ( figure 3 ) . when the expression of vegf , an angiogenic factor involved in early bone remodeling phases , was evaluated a very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 magnification 20. group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area . inset ( 40x ) shows , in group 1 specimens , grafted biomaterial particles , in group 2 specimens , large mineralized areas within which newly formed bone can be recognized because of osteocyte lacunae lack and absence of lamellar organization ; xb , xenogenous bone ; ob , old bone ; nb , new bone . magnification 20. group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area . inset ( 40x ) shows , in group 1 specimens , grafted biomaterial particles , in group 2 specimens , large mineralized areas within which newly formed bone can be recognized because of osteocyte lacunae lack and absence of lamellar organization ; xb , xenogenous bone ; ob , old bone ; nb , new bone . figure 2a ) immunohistochemical analysis of tgf1 expression in group 1 and group 2 specimens . magnification 20. group 1 : bone tissue specimens obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . moderate tgf1 immunolabeling of both group 1 and group 2 bone tissue ; no tgf1 immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of tgf1 positive area sd , determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification . magnification 20. group 1 : bone tissue specimens obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , moderate tgf1 immunolabeling of both group 1 and group 2 bone tissue ; no tgf1 immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of tgf1 positive area sd , determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification . figure 3a ) immunohistochemical analysis of opg expression in group 1 and group 2 specimens . moderate opg immunolabeling in group 1 and weak opg immunolabeling in group 2 bone tissue ; no opg immunostaining is seen in negative control . b ) immunohistochemical analysis of rankl expression , in group 1 and group 2 specimens , respectively . group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . weak rankl immunolabeling in both group 1 and group 2 bone tissue ; no rankl immunostaining is seen in negative control . moderate opg immunolabeling in group 1 and weak opg immunolabeling in group 2 bone tissue ; no opg immunostaining is seen in negative control . b ) immunohistochemical analysis of rankl expression , in group 1 and group 2 specimens , respectively . group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . weak rankl immunolabeling in both group 1 and group 2 bone tissue ; no rankl immunostaining is seen in negative control . figure 4a ) immunohistochemical analysis of vegf expression in group 1 and group 2 specimens . magnification 20. group 1 : bone tissue samples obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 bone tissue ; no vegf immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of vegf positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.05 ) . magnification 20. group 1 : bone tissue samples obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 bone tissue ; no vegf immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of vegf positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.05 ) . finally , new bone formation and bone mineralizing processes were assessed taking into consideration bsp and sparc expression levels , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 is seen , whereas weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 is evidenced ( p<0.001 ) ( figure 5 ) . for all densitometric evaluations , interobserver agreement , measured using the kappa coefficient , was 0.90 ( almost perfect ) . figure 5a ) immunohistochemical analysis of bsp expression in group 1 and group 2 specimens , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 bone tissue ; no bsp immunostaining is seen in negative control . b ) immunohistochemical analysis of sparc expression , in group 1 and group 2 specimens , respectively . magnification 20. group 1 : bone tissue specimenss obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 bone tissue ; no sparc immunostaining is seen in negative control . c ) graphic representation of densitometric analysis of bsp and sparc positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.001 ; * * p<0.001 ) . a ) immunohistochemical analysis of bsp expression in group 1 and group 2 specimens , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 bone tissue ; no bsp immunostaining is seen in negative control . b ) immunohistochemical analysis of sparc expression , in group 1 and group 2 specimens , respectively . magnification 20. group 1 : bone tissue specimenss obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 bone tissue ; no sparc immunostaining is seen in negative control . c ) graphic representation of densitometric analysis of bsp and sparc positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.001 ; * * p<0.001 ) . morphological analysis was performed at light microscope after hematoxylin - eosin staining ( figure 1 ) . in group 1 specimens the native bone tissue and connective tissue fibers could be identified . moreover , the grafted biomaterial particles are easily distinguished ( purple areas ) due to their lack of tissue structure , as better shown by the inset . group 2 sample shows large mineralized areas close to dense areas of connective tissue , within which newly formed bone is recognizable because of osteocyte lacunae lack and absence of lamellar organization ( inset ) . molecular modifications occurring after bone substitutes placement , concerning their ability to be integrated and to be clinically suitable , tgf-1 expression , essential for osteoblastic differentiation and osteoid and extracellular proteins production , do not show any statistically significant difference between the two experimental groups ( figure 2 ) ; in fact , moderate tgf-1 immunolabeling is seen both in group 1 and group 2 . moreover , since tgf-1 stimulates opg production , inhibiting the rankl / rank interaction , opg and rankl expressions were checked . moderate opg immunolabeling is found in group 1 and weak opg immunolabeling in group 2 , whereas weak rankl immunolabeling is seen both in group 1 and group 2 . however , group 1 samples show a mean ratio of about four fold higher than that observed in group 2 specimens ( 2.4 vs 1.1 ) , due to the concomitant increase of opg and a significant decrease of rankl expression in samples from sites regenerated with the equinederived bone substitute ( figure 3 ) . when the expression of vegf , an angiogenic factor involved in early bone remodeling phases , was evaluated a very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 magnification 20. group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area . inset ( 40x ) shows , in group 1 specimens , grafted biomaterial particles , in group 2 specimens , large mineralized areas within which newly formed bone can be recognized because of osteocyte lacunae lack and absence of lamellar organization ; xb , xenogenous bone ; ob , old bone ; nb , new bone . magnification 20. group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area . inset ( 40x ) shows , in group 1 specimens , grafted biomaterial particles , in group 2 specimens , large mineralized areas within which newly formed bone can be recognized because of osteocyte lacunae lack and absence of lamellar organization ; xb , xenogenous bone ; ob , old bone ; nb , new bone . figure 2a ) immunohistochemical analysis of tgf1 expression in group 1 and group 2 specimens . magnification 20. group 1 : bone tissue specimens obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . moderate tgf1 immunolabeling of both group 1 and group 2 bone tissue ; no tgf1 immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of tgf1 positive area sd , determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification . magnification 20. group 1 : bone tissue specimens obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , moderate tgf1 immunolabeling of both group 1 and group 2 bone tissue ; no tgf1 immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of tgf1 positive area sd , determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification . figure 3a ) immunohistochemical analysis of opg expression in group 1 and group 2 specimens . moderate opg immunolabeling in group 1 and weak opg immunolabeling in group 2 bone tissue ; no opg immunostaining is seen in negative control . b ) immunohistochemical analysis of rankl expression , in group 1 and group 2 specimens , respectively . group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . weak rankl immunolabeling in both group 1 and group 2 bone tissue ; no rankl immunostaining is seen in negative control . moderate opg immunolabeling in group 1 and weak opg immunolabeling in group 2 bone tissue ; no opg immunostaining is seen in negative control . b ) immunohistochemical analysis of rankl expression , in group 1 and group 2 specimens , respectively . group 1 : bone tissue specimens obtained from equine - derived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . weak rankl immunolabeling in both group 1 and group 2 bone tissue ; no rankl immunostaining is seen in negative control . figure 4a ) immunohistochemical analysis of vegf expression in group 1 and group 2 specimens . magnification 20. group 1 : bone tissue samples obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 bone tissue ; no vegf immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of vegf positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.05 ) . magnification 20. group 1 : bone tissue samples obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , negative control . very strong vegf immunolabeling in group 1 and strong vegf immunolabeling in group 2 bone tissue ; no vegf immunostaining is seen in negative control . b ) graphic representation of densitometric analysis of vegf positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.05 ) . finally , new bone formation and bone mineralizing processes were assessed taking into consideration bsp and sparc expression levels , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 is seen , whereas weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 is evidenced ( p<0.001 ) ( figure 5 ) . for all densitometric evaluations , interobserver agreement , measured using the kappa coefficient , was 0.90 ( almost perfect ) . figure 5a ) immunohistochemical analysis of bsp expression in group 1 and group 2 specimens , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 bone tissue ; no bsp immunostaining is seen in negative control . b ) immunohistochemical analysis of sparc expression , in group 1 and group 2 specimens , respectively . magnification 20. group 1 : bone tissue specimenss obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 bone tissue ; no sparc immunostaining is seen in negative control . c ) graphic representation of densitometric analysis of bsp and sparc positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.001 ; * * p<0.001 ) . a ) immunohistochemical analysis of bsp expression in group 1 and group 2 specimens , respectively . weak bsp immunolabeling in group 1 and moderate bsp immunolabeling in group 2 bone tissue ; no bsp immunostaining is seen in negative control . b ) immunohistochemical analysis of sparc expression , in group 1 and group 2 specimens , respectively . magnification 20. group 1 : bone tissue specimenss obtained from equinederived bone substitute grafted area ; group 2 : bone tissue specimens obtained from calvaria bone grafted area ; c(- ) , weak sparc immunolabeling in group 1 and moderate sparc immunolabeling in group 2 bone tissue ; no sparc immunostaining is seen in negative control . c ) graphic representation of densitometric analysis of bsp and sparc positive area sd determined by direct visual counting of ten fields ( mean values ) for each of five slides per specimens at 20 magnification ( * p<0.001 ; * * p<0.001 ) . regenerative procedures employing autologous , homologous or heterologous bone grafts lead to bone defect repair through different biological mechanisms . the very good results obtained with the use of autologous bone grafts were widely demonstrated . histological and clinical studies showed the possibility to have predictable results with the use of calvaria bone grafts , with no inflammatory phenomena and a minimum resorption rate during the healing period , along with a high rate of clinical success for the subsequent implant rehabilitation . clinicians are constantly searching for a heterologous bone substitute that combines the osteo - regenerative features of autologous bone eliminating the limits imposed by the need for a second surgery . most of the currently used heterologous grafts do not show ideal characteristics for bone regeneration such as osteogenic , osteoinductive and angiogenic potentials , biological safety , no size restrictions , long shelf life and reasonable cost . the present study aimed to evaluate , by morphological and immunohistochemical analyses , an equine bone substitute and an autologous bone graft , 6 months after the graft placement , in terms of ability of integration with the host tissue . complete healing is obtained during the remodeling phase , when osteoblasts and osteoclasts cooperate to convert the callus of a fracture in a definitive functional bone structure . for this reason , the first step of our study was to evaluate tgf1 expression , as its role in osteoblast / osteoclast coupling was demonstrated . both group 1 and group 2 show a discrete tgf1 expression , suggesting that bone remodeling phenomena occurred in all the grafted areas . to better elucidate the host tissue response to the different bone substitutes , attention was focused on the opg / rankl ratio , often used as a bone resorption index . in fact , opg , by inhibiting the rankl / rank binding , protects osteoclastsmediated bone resorption acting on osteoclasts differentiation from precursors . recent studies showed an increasing expression and activity of rankl in bone pathologies characterized by bone resorption such as osteoporosis and osteoarthritis , and a compensatory increase of opg in bone diseases , such as paget 's syndrome , in which an anomalous higher bone formation takes place . the balance between rankl and opg controls the osteoclast activity and undergoes an endocrine regulation . in fact , it is usually preferable to evaluate opg / rankl ratio rather than opg and rankl absolute values . thus an opg / rankl ratio < 1 suggests a rankl predominant activity , and , as a consequence , bone resorption events are predominant ; on the other hand an opg / rankl ratio > 1 reveals opg greater activity and predominant in new bone formation processes . the specimens of both groups show an opg / rankl ratio > 1 , suggesting that the bone protection process was predominant on rankl / rank cascade activation . however , the lower values of group 2 specimen led us to hypothesize that the integration between host bone and autologous graft could be at a more advanced stage in respect to group 1 specimens , since the occurrence of bone resorption and new bone apposition phenomena was nearer to balance in group 2 . this evidence is also confirmed by morphological analysis , that reveals a stronger integration between host tissue and bone graft in group 2 specimens while , on the contrary , in group 1 specimens some of the equine - derived biomaterial particles are still evident and few newly formed mineralized tissue areas can be observed . in order to support these morphological aspects , angiogenesis , new bone formation and mineralization processes angiogenesis has a crucial role in the regulation of bone remodeling and repair .. new blood vessel formation is essential to allow circulating osteoclast and osteoblast precursors to move towards the site of remodeling . moreover , vegf supports osteoblast growth in the initial phase of bone graft integration . in our study , vegf expression is higher in group 1 specimens indicating that intense neo - angiogenic phenomena were occurring in heterologous bone grafted sites , six months after the maxillary sinus augmentation procedure . this evidence could point to the fact that in autologous bone grafts this phase was already ended . in previous reports from our laboratory , in fact , extra - oral autologous bone grafts , showing an intense angiogenesis phenomena four months after grafting , with vegf values significantly higher than in native bone tissue were found . furthermore , the expression of molecules , such as bsp and sparc , index of graft consolidation involved in new bone formation and mineralizing activity , indicate that sites treated with calvaria bone graft seemed to reach earlier a higher stage of mineralization compared to the equine bone grafted specimens . such results led us to conclude that host bone tissue , undergoing regenerative phenomena , positively reacted to the placement of both biomaterials . in particular , the equine - derived biomaterial shows good characteristics , in terms of both clinical and microscopic integration . however , at the same experimental time , sites treated with autologous bone clearly show a better organization , which could ensure a better primary stability to the implant and a higher predictability of the implant - prosthetic rehabilitation . in addition , in sites treated with autologous bone , a balance between matrix deposition and resorption is observed , suggesting that the regenerative process shows a higher rate of progression , than in sites treated with the equine bone graft in which this process seems to be slower .

autologous bone is considered the gold standard for bone regeneration , even if different heterologous bone substitutes have been proposed to overcome the limits related to its use . the aim of this study was to analyze and to compare the molecular events switched on by autologous or heterologous bone graft insertion , focusing on tgf1 expression and opg / rankl ratio , to analyze resorption process , and estimating graft vascularization , new bone tissue deposition and its mineralization , through vegf , bsp and sparc expression evaluation , respectively . patients needing pre - prosthetic rehabilitation of the posterior maxilla were treated using an equine - derived biomaterial ( group 1 ) or calvaria autologous bone ( group 2 ) , according to the morphology of the bone defect . bone graft integration was evaluated on bone samples obtained from the treated areas at the moment of dental implant insertion , by morphological and immunohistochemical analyses for tgf1 , opg , rankl , vegf , bsp , and sparc expression . morpho - logical analysis shows the presence of biomaterial residual granules in group 1 , in parallel to a good integration between graft and host tissue . moderate tgf1 expression is seen in both group 1 and group 2 . opg / rankl ratio appears higher in group 1 ; vegf expression appears very strong in group 1 and strong in group 2 , while bsp and sparc expression results weak in group 1 and moderate in group 2 . our results reveal the good integration between both types of graft and the host tissue , even though autologous graft seems to produce a faster regenerative process , as evidenced by the different expression of the investigated molecules . according to these observations , the clinical use of heterologous particulate equine - derived biomaterial may ensure long - term predictability of implantprosthetic rehabilitation , comparable to that obtained with autologous bone graft .

cherubism is an inherited , fibro - osseous condition characterized by firm , painless swelling of the jaws , and was first described by jones in 1933 . clinically cherubism is characterized by fullness of the cheeks and jaw bones that results in a round face with retraction of the lower eyelids and exposure of the sclera below the irises ; the heaven look produced is suggestive of a cherub and gave rise to the name of this condition . the radiographical appearance is characterized by bilateral , multilocular radiolucent lesions , which often begin near the angle of the mandible and spread to the ramus and body of the mandible . the presence of eosinophilic , collagenous material around small capillaries in histopatholgical sections is of value in the diagnosis of cherubism . a 25-year - old male patient of non - consanguineous parents reported to our department with a complaint of bilateral painless swelling of both jaws . later he noticed progressive increase in the swelling in his jaws which continued till it attained the present size . the size of the swelling had been stable for the past year and had not regressed or increased . on extra - oral examination , the swellings appeared ovoid in shape , with well - defined borders . the swelling was more prominent in the parasymphysis and body of the mandible and molar regions . eyes were upturned with sclera visible below the iris giving an eye raised to heaven appearance . there was a difference in the ocular levels , right eye being at a slightly higher level [ figure 1 ] . obliteration of buccal sulcus of mandible was noticed in the molar regions . in the maxilla multiple swellings were seen , which were hard , sessile , non - tender , and covered with pink mucosa . extra - orally the swellings appeared ovoid in shape , with well - defined borders . the orthopantamograph revealed multiple impacted teeth in the mandible , with multiloculated osteolytic lesions involving the entire body as well as the ramus of the mandible sparing the condyles [ figure 2 ] . a 3-dimensional computed tomography showed multiloculated cystic lesions affecting the body and rami of the mandible and also the maxilla . the lesion caused marked expansion of the bones , with a multifocal cortical breakthrough [ figure 3 ] . the orthopantamograph revealed multiple impacted teeth in the mandilble ( red arrows ) with multiloculated osteolytic lesions involving mandible and maxilla ( blue arrows ) . 3-dimensional computed tomography scan showing multiloculated cystic lesions affecting the body and rami of the mandible ( blue arrows ) and maxilla with raised orbital floor ( red arrows ) . biopsy to obtain specimen from both the jaws in the alveolar region was performed under local anesthesia.microscopy revealed highly cellular stroma with many multinucleated giant cells . the cellular stroma showed spindle cell fibroblasts with vesicular nuclei arranged in fascicles , whorled pattern and irregularly in few areas . the giant cells were unequally distributed and were of different sizes with varied number of nuclei resembling osteoclasts . there were few blood vessels and areas of extravasated blood and minimal amount of collagen was seen . microscopy revealed highly cellular stroma consisting of plenty of multinucleated giant cells ( black arrow ) . the cellular stroma consists of spindle cell fibroblasts with vesicular nuclei arranged in fascicles ( blue arrow ) . the grotesque appearance and the parent 's insistence on esthetic improvement compelled the surgeon to attempt cosmetic surgical recontouring of the jaws under general anaesthesia . the entire mandible and maxilla was degloved intraorally . the exposed lesion tissue consisted of multiple locules with reddish hue and semi - hard consistency . buccal surfaces of maxilla and mandible were decorticated and curetted as much as possible to provide optimal cosmetic improvement . impacted and loose teeth were removed . patient had no recurrence of the lesion during the 2-year follow - up period [ figure 6 ] . the orthopantamograph revealed multiple impacted teeth in the mandible , with multiloculated osteolytic lesions involving the entire body as well as the ramus of the mandible sparing the condyles [ figure 2 ] . a 3-dimensional computed tomography showed multiloculated cystic lesions affecting the body and rami of the mandible and also the maxilla . the lesion caused marked expansion of the bones , with a multifocal cortical breakthrough [ figure 3 ] . the orthopantamograph revealed multiple impacted teeth in the mandilble ( red arrows ) with multiloculated osteolytic lesions involving mandible and maxilla ( blue arrows ) . 3-dimensional computed tomography scan showing multiloculated cystic lesions affecting the body and rami of the mandible ( blue arrows ) and maxilla with raised orbital floor ( red arrows ) . biopsy to obtain specimen from both the jaws in the alveolar region was performed under local anesthesia.microscopy revealed highly cellular stroma with many multinucleated giant cells . the cellular stroma showed spindle cell fibroblasts with vesicular nuclei arranged in fascicles , whorled pattern and irregularly in few areas . the giant cells were unequally distributed and were of different sizes with varied number of nuclei resembling osteoclasts . there were few blood vessels and areas of extravasated blood and minimal amount of collagen was seen . microscopy revealed highly cellular stroma consisting of plenty of multinucleated giant cells ( black arrow ) . the cellular stroma consists of spindle cell fibroblasts with vesicular nuclei arranged in fascicles ( blue arrow ) . the grotesque appearance and the parent 's insistence on esthetic improvement compelled the surgeon to attempt cosmetic surgical recontouring of the jaws under general anaesthesia . the entire mandible and maxilla was degloved intraorally . the exposed lesion tissue consisted of multiple locules with reddish hue and semi - hard consistency . buccal surfaces of maxilla and mandible were decorticated and curetted as much as possible to provide optimal cosmetic improvement . impacted and loose teeth were removed . patient had no recurrence of the lesion during the 2-year follow - up period [ figure 6 ] . no cause and effect relationship with trauma , infection , or hemorrhage has ever been verified . anderson suggested that a genetically induced biochemical abnormality stimulates the giant cell lesions characteristic of cherubism . a molecular pathogenesis of cherubism has been proposed ; sh3bp2 gene mutations cause dysregulation of the msx-1 gene , which is involved in regulating mesenchymal interaction in craniofacial morphogenesis . clinically the lesion of cherubism produces painless , typically slow expansion of the affected area of jaw . the facial expansion maybe quite mild or may result in considerable deformity . more extensive involvement may result in enlargement and widening of the alveolar bone . progressive involvement of the anterior maxillary segment bone stretches the skin and the lower eyelid beneath the orbit , exposing the lower sclera . the developing permanent teeth in the areas of involvement may be displaced , malformed , or absent . history , clinical course , and the findings in our case corresponds to the classic features of cherubism . irregular , multilocular , well - defined , translucent , cystic spaces causing expansion of bone and sparing only a thin layer of cortex are usually seen . the teeth may be displaced , unerupted , or appear to be floating in the cyst - like spaces . in the mandible , the inferior alveolar canal maybe displaced and lesion may involve the alveolar process , the angle and ramus . it may advance toward the incisors and obliterate the sigmoid notch , but the condyles may be spared . periosteal new bone formation is never present . in the maxilla the lesion is similar and the maxillary antra may be completely obliterated only to become normally pneumatized as the lesion regress . seward and hankey have proposed a grading system based on the radiographical location of the lesions in the jaws . it is as follows : grade 1 : involvement of bilateral mandibular molar regions and ascending rami , mandible body , or mentis . grade 2 : involvement of bilateral maxillary tuberosities ( in addition to grade 1 lesions ) and diffuse mandibular involvement . grade 3 : massive involvement of the entire maxilla and mandible , except the condyles . grade 4 : involvement of both jaws , including the condyles . according to ramon and engelberg grade 4 lesions our case could possibly be classified as above grade 3 cherubism as there was huge deformity of the mandible , especially the bilateral posterolaterosuperior extension of maxillary lesions with slight elevation of orbital floor . the lesions of cherubism are not distinctive histologically and are difficult to differentiate from other giant cells containing fibro - osseous lesions , making diagnosis dependent on the clinical findings impossible . hamner histochemically demonstrated this perivascular cuffing to be collagen protein and felt its presence was of value in the diagnosis of cherubism . surgical treatment appears to be unnecessary for grade 1 and 2 cases , in the absence of secondary disturbances . curettage appears to be necessary in more aggressive cases ( grade 3 ) , to reduce maxillofacial deformity that occurs after puberty . dukart et al . , found that surgical curettage and recontouring performed during a period of rapid growth of cherubism lesions not only offer a favorable immediate result but also arrests active growth of remnant lesions while stimulating bone regeneration . the rationale of calcitonin administration is that it inhibits the osteoclastic activity of the giant cells . radiation therapy is ineffective and contraindicated in view of the risk of osteoradionecrosis , interference with dentofacial growth and development , and the effect on future surgical procedure . in our case , the patient was an adolescent affected psychologically by his grotesque appearance , which was also a concern for his parents . therefore , a conservative surgical curettage and recontouring of the jaws was performed under general anesthesia to address his esthetic concern . two years of postoperative follow - up showed a sustained esthetic improvement achieved by surgery of the grade 3 lesion .

cherubism is a rare non - neoplastic hereditary disease related to genetic mutations characterized by symmetrically swollen cheeks , particularly over the angles of the mandible , and an upward turning of the eyes . the affected mandible and maxilla begin to swell in early childhood , and gradually increase until the age of puberty . apparently , surgical intervention is unnecessary unless significant functional , esthetic , or emotional disturbances develop . in the present paper , we report a case of cherubism , with classic features that was classified as grade 3 , managed with surgery and followed up for 2-years after treatment .

we analyzed culture - positive cases of mdr tb reported to the california tb registry from january 1 , 2004 , through december 31 , 2007 . california state law ( health and safety code title 17 2505 ) requires reporting of all verified cases of tb , submission of all m. tuberculosis isolates to local public health laboratories , and submission of all mdr m. tuberculosis isolates to the california department of public health microbial diseases laboratory . testing for first- and second - line drug susceptibilities was performed at local laboratories or at the microbial diseases laboratory by using bactec 460 ( becton dickinson diagnostic instruments , sparks , md , usa ) , mgit 960 ( becton dickinson ) , or the agar proportion method . some isolates were forwarded to the centers for disease control and prevention ( cdc ; atlanta , ga , usa ) for additional second - line drug testing or for confirmation of drug resistance . patients were included in the study if mdr m. tuberculosis was identified on > 1 isolate . demographic and clinical information for all patients with mdr tb was abstracted from state tb surveillance forms ( report of a verified case of tuberculosis ) , which have high validity ( 18 ) . all protocols were approved by the california committee for the protection of human subjects and university of california , san francisco , committee for the protection of human subjects . for each isolate , genomic dna was extracted from solid media ( lowenstein - jensen slants , middlebrook 7h10 or 7h11 agar ) , liquid media ( bactec 12 b or mgit [ becton dickinson ] ) , or smear - positive sputum sediments . a real - time pcr assay with 6 molecular beacon probes was performed by using an iq5 icycler instrument ( bio - rad , hercules , ca , usa ) to screen for mutations associated with isoniazid and rifampin resistance ( 19 ) . two molecular beacons that targeted katg ( codon 311317 ) and the inha promoter were used to detect isoniazid resistance conferring mutations , and 4 molecular beacons that targeted the core region of rpob were used to detect rifampin resistance conferring mutations . isolates with mutations in katg detected by the wild - type probe were further tested with another molecular beacon that specifically targeted katg s315 t ( agc acc ) . when molecular beacon analysis did not show katg s315 t or -c15 t inha promoter mutations , the entire fura - katg locus ( h37rv : 21536262156657 , 3,031 bp ) was sequenced as described ( 6 ) . sequence data were generated by using abi bigdye v3.1 dye terminator sequencing chemistry and the abi prism 3730xl capillary dna analyzer ( applied biosystems , foster city , ca , usa ) at the genomic core facility , university of california , san francisco ( www.genomics.ucsf.edu/sequencing/index.aspx ) , and were analyzed with clustalw ( www.ebi.ac.uk/tools/clustalw/index.html ) . spacer oligonucleotide typing ( spoligotyping ) and mycobacterial interspersed repetitive unit ( miru ) typing were performed in accordance with the centers for disease control and prevention universal genotyping program procedures ( 20 ) . spoligotyping was performed by using luminex - based methods to detect 43 known spacer sequences in the direct repeat locus ( 21 ) . a capillary sequencer , ceq 8000 ( beckman , fullerton , ca , usa ) , was used to analyze the number of repeated sequences at each of the 12 loci . is6110-based restriction fragment length polymorphism ( rflp ) genotyping was performed following standardized methods ( 23 ) . rflp patterns were compared by using bioimage whole band analyzer software version 4.2.1 ( bioimage corp . , ann arbor , mi , usa ) ( 24 ) . rflp patterns with < 20 identical bands , or > 20 bands and differing by no more than a single band , were considered matched . is6110 rflp band assignment was edited by 2 independent readers , and the cluster assignment was confirmed visually spoligotype families h , lam , and t , x , s were considered to be of euro - american lineage ; beijing of east - asian lineage ; eai of indo - oceanic lineage ; and cas of east african - indian lineage ( 5 ) . cases were defined as clustered if > 2 isolates from cases reported during the study period shared the same miru and spoligotype , matched is6110 rflp , and had specific drug resistance conferring mutations for isoniazid and rifampin . clustering was assumed to represent both transmission of m. tuberculosis and progression to active disease , leading to secondary case generation within the period of the study . cases not in a cluster were considered to be the result of reactivation of latent infection . patients with the earliest case report date within a cluster were regarded as index cases . the proportion of clustered mdr tb cases was analyzed as the number of clustered cases divided by the total number of culture - positive cases that occurred during the study period . because of limited sample size , the independent effects of katg s315 t and phylogeographic lineage on clustering were estimated by using exact logistic regression methods . refugee status and refugee resettlement during the study period could bias our results in that overrepresentation of ethnic groups or geographic locations with a high prevalence of particular strain - specific factors ( phylogeographic lineage or drug - resistance mutations ) could confound the association under study . to examine this influence and potential clustering of tb cases within households or communities related to refugee resettlement , sensitivity analysis was conducted by reestimation of study results after excluding 1 ) patients immigrating from refugee settings within the past 3 years and 2 ) the single largest patient cluster , which accounted for 40% of all clustered cases . in a separate analysis , standard logistic regression was used to estimate the effect of katg s315 t and phylogeographic lineage on sputum smear positivity , again controlling for refugee status . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated to measure associations of interest . categorical data ( e.g. , sex , foreign vs. us birth , homelessness ) were compared by using fisher exact tests . the wilcoxon rank - sum test was used to determine differences in the distribution of continuous variables ( e.g. , age , time from entry into the united states to tb diagnosis ) . interaction between the independent variables was assessed separately for each factor by stratification and statistical testing ( breslow - day with the tarone correction and zelen test ) , with p<0.2 assumed to indicate the presence of interaction . analyses were performed by using stata 10 ( statacorp . , college station , tx , usa ) and statexact 8 ( cytel software corp . , cambridge , ma , usa ) . for each isolate , genomic dna was extracted from solid media ( lowenstein - jensen slants , middlebrook 7h10 or 7h11 agar ) , liquid media ( bactec 12 b or mgit [ becton dickinson ] ) , or smear - positive sputum sediments . a real - time pcr assay with 6 molecular beacon probes was performed by using an iq5 icycler instrument ( bio - rad , hercules , ca , usa ) to screen for mutations associated with isoniazid and rifampin resistance ( 19 ) . two molecular beacons that targeted katg ( codon 311317 ) and the inha promoter were used to detect isoniazid resistance conferring mutations , and 4 molecular beacons that targeted the core region of rpob were used to detect rifampin resistance conferring mutations . isolates with mutations in katg detected by the wild - type probe were further tested with another molecular beacon that specifically targeted katg s315 t ( agc acc ) . when molecular beacon analysis did not show katg s315 t or -c15 t inha promoter mutations , the entire fura - katg locus ( h37rv : 21536262156657 , 3,031 bp ) was sequenced as described ( 6 ) . sequence data were generated by using abi bigdye v3.1 dye terminator sequencing chemistry and the abi prism 3730xl capillary dna analyzer ( applied biosystems , foster city , ca , usa ) at the genomic core facility , university of california , san francisco ( www.genomics.ucsf.edu/sequencing/index.aspx ) , and were analyzed with clustalw ( www.ebi.ac.uk/tools/clustalw/index.html ) . spacer oligonucleotide typing ( spoligotyping ) and mycobacterial interspersed repetitive unit ( miru ) typing were performed in accordance with the centers for disease control and prevention universal genotyping program procedures ( 20 ) . spoligotyping was performed by using luminex - based methods to detect 43 known spacer sequences in the direct repeat locus ( 21 ) . a capillary sequencer , ceq 8000 ( beckman , fullerton , ca , usa ) , was used to analyze the number of repeated sequences at each of the 12 loci . is6110-based restriction fragment length polymorphism ( rflp ) genotyping was performed following standardized methods ( 23 ) . rflp patterns were compared by using bioimage whole band analyzer software version 4.2.1 ( bioimage corp . , ann arbor , mi , usa ) ( 24 ) . rflp patterns with < 20 identical bands , or > 20 bands and differing by no more than a single band , were considered matched . is6110 rflp band assignment was edited by 2 independent readers , and the cluster assignment was confirmed visually . the phylogeographic lineage of strains was determined from spoligotyping results . spoligotype families h , lam , and t , x , s were considered to be of euro - american lineage ; beijing of east - asian lineage ; eai of indo - oceanic lineage ; and cas of east african - indian lineage ( 5 ) . cases were defined as clustered if > 2 isolates from cases reported during the study period shared the same miru and spoligotype , matched is6110 rflp , and had specific drug resistance conferring mutations for isoniazid and rifampin . clustering was assumed to represent both transmission of m. tuberculosis and progression to active disease , leading to secondary case generation within the period of the study . cases not in a cluster were considered to be the result of reactivation of latent infection . patients with the earliest case report date within a cluster were regarded as index cases . the proportion of clustered mdr tb cases was analyzed as the number of clustered cases divided by the total number of culture - positive cases that occurred during the study period . because of limited sample size , the independent effects of katg s315 t and phylogeographic lineage on clustering were estimated by using exact logistic regression methods . refugee status and refugee resettlement during the study period could bias our results in that overrepresentation of ethnic groups or geographic locations with a high prevalence of particular strain - specific factors ( phylogeographic lineage or drug - resistance mutations ) could confound the association under study . to examine this influence and potential clustering of tb cases within households or communities related to refugee resettlement , sensitivity analysis was conducted by reestimation of study results after excluding 1 ) patients immigrating from refugee settings within the past 3 years and 2 ) the single largest patient cluster , which accounted for 40% of all clustered cases . in a separate analysis , standard logistic regression was used to estimate the effect of katg s315 t and phylogeographic lineage on sputum smear positivity , again controlling for refugee status . odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated to measure associations of interest . categorical data ( e.g. , sex , foreign vs. us birth , homelessness ) were compared by using fisher exact tests . the wilcoxon rank - sum test was used to determine differences in the distribution of continuous variables ( e.g. , age , time from entry into the united states to tb diagnosis ) . interaction between the independent variables was assessed separately for each factor by stratification and statistical testing ( breslow - day with the tarone correction and zelen test ) , with p<0.2 assumed to indicate the presence of interaction . analyses were performed by using stata 10 ( statacorp . , college station , tx , usa ) and statexact 8 ( cytel software corp . , cambridge , ma , usa ) . during the study period , 11,395 cases of tb were reported in california , of which 9,037 ( 79% ) had positive cultures . of these , of the 141 ( 2% ) incident mdr tb cases , 123 ( 87% ) had isolates available for miru , spoligotyping , and is6110 rflp analysis . isolates unavailable for genotyping ( n = 18 ) were more often from los angeles county ; other demographic and clinical characteristics were similar to those of analyzed cases . twenty - five mdr m. tuberculosis isolates were aggregated in 8 clusters ( 1 cluster of 10 cases , 1 cluster of 3 cases , and 6 clusters of 2 cases ) for an overall cluster proportion of 20% ( 25/123 ) . excluding the 8 index cases , 14% ( 17/123 ) of all cases were considered to have resulted from recent transmission and rapid progression to disease ( secondary cases ) . within clusters , a median of 3 months elapsed between successive secondary cases ( range 020 months ) . of the 123 total cases , 113 ( 92% ) occurred among foreign - born patients , with more than half ( 56% ) occurring among immigrants from mexico , philippines , the people s republic of china , or vietnam ( table 1 ) . seven of 8 index cases occurred either in mexican immigrants ( 3/8 ) or recent refugees from thailand , lao people s democratic republic , or india ( 4/8 ) . median time from patient arrival in the united states to tb diagnosis was approximately twice as long for clustered as for nonclustered cases ( 4.3 years vs. 2.4 years ) and 3 times as long for cases in mexican - born persons as for cases in persons from other countries ( 7.3 years vs. 2.3 years ) . * mdr tb , multidrug - resistant tuberculosis ; or , odds ratios ; ci , confidence interval ; iqr , interquartile range ; afb , acid - fast bacilli . ors describe the association between the characteristic of interest and mdr tb case - clustered status . afghanistan ( 1 ) , burma ( 1 ) , cambodia ( 5 ) , ethiopia ( 1 ) , indonesia ( 1 ) , mongolia ( 1 ) , nepal ( 1 ) , peru ( 2 ) , south korea ( 5 ) , and ukraine ( 1 ) . # nonclustered cases : cervical lymph node ( 5 ) , bone ( 1 ) , other ( 1 ) ; clustered cases : pleural ( 1 ) . * * culture positive after > 8 months of treatment ; limited to pulmonary tb patients who were alive at diagnosis and had an initial positive sputum culture . younger persons were more likely than older persons to harbor strains involved in mdr tb clusters ( table 1 ) . hiv infection was unusual in this patient population ; only 3 ( 4% ) of 75 patients with known hiv status were hiv - infected . twenty - eight percent ( 35/123 ) of patients reported a history of active tb ; this proportion did not vary between clustered and nonclustered cases ( p = 0.75 ) . time to culture negativity ( 2.4 vs. 2.8 months , p = 0.95 ) , treatment failure , or death did not differ between clustered and nonclustered cases ( p = 0.57 ) among 105 ( 85% ) of 123 cases for which data were available . seventy - five percent of mdr m. tuberculosis strains harbored the isoniazid resistance conferring mutation katg s315 t , including all ( 100% ) clustered strains ( table 2 ) . when we controlled for strain lineage and refugee status , katg s315 t was inversely associated ( or 0.28 , 95% ci 0.090.89 , p = 0.03 ) with sputum smear positivity ( table 3 ) . most rifampin resistance conferring mutations were found between positions 529 and 534 of the rpob gene , likely indicative of the serine to leucine substitution at position 531 ( s531l ) of the rpob gene ( 27 ) . the association between this mutation and clustering did not reach statistical significance ( or 2.2 , 95% ci 0.87.4 ; p = 0.16 ) . * mdr tb , multidrug - resistant tuberculosis ; s315 t , serine - to - threonine substitution at position 315 . novel mutations detected : y413stop , t314 t ( silent ) , w161 g , d61e ( fur a ) , r145p , p325l , and v633f . inha promoter mutation was concomitant with 4/91 ( 4% ) isolates harboring the katg s315 t and 2/8 ( 25% ) isolates with katg mutations other than s315 t . no mutations detected by molecular beacons ; sequencing was not possible for these isolates because of degraded dna . rifampin resistance conferring mutations were not detected by the molecular beacon assay for 4 isolates . * mdr tb , multidrug - resistant tuberculosis ; s315 t , serine - to - threonine substitution at position 315 ; or , odds ratio ; ci , confidence interval . mdr m. tuberculosis isolates were distributed among east - asian ( 47% ) , euro - american ( 30% ) , indo - oceanic ( 20% ) , and east african indian ( 3% ) phylogeographic lineages . lineage could not be established for 11 cases . on univariate analysis , east - asian strain lineage was associated with clustering ( table 4 ) , but not with adverse outcome ( death or treatment failure ) . mdr , multidrug - resistant tuberculosis ; or , odds ratio ; ci , confidence interval . clustering was independently associated with katg s315 t ( or 11.2 , 95% ci 2.2- ; p = 0.004 ) and refugee status ( or 6.0 , 95% ci 1.236.2 ; p = 0.03 ) in exact multivariate analyses in which strain lineage and sputum smear positivity were controlled for ( table 5 ) . the estimated association between katg s315 t and case clustering did not appreciably change in sensitivity analyses that excluded either all recently arrived refugees or the single largest patient cluster . * mdr tb , multidrug - resistant tuberculosis ; or , odds ratio ; ci , confidence interval . in this 4-year population - based molecular epidemiologic study , transmission followed by secondary case generation contributed to 14% , or 1 of every 7 , mdr tb cases in california . clustered cases occurred more often among younger persons and persons who had emigrated from mexico and refugee settings in southeast asia . in addition , pathogen - specific factors were associated with clustering of mdr tb cases , independent of traditional clinical and demographic risk factors . the proportion of mdr tb cases attributed to transmission in california was higher than that reported by other investigators in most low incidence settings ( 2832 ) . the largest clusters in our study resulted from mdr tb outbreaks in california after resettlement of hmong refugees in 20052006 ( 33 ) and resettlement of tibetan refugees in 20012006 . the associations between pathogen - specific factors and case clustering could be due to regional differences in strain prevalence and preferential migration of persons with specific strains to california . we attempted to control for these factors by using highly stringent criteria to define clustered cases and by adjusting for refugee status in our multivariate model . however , without detailed contact information and contact tracing , we can not be certain of the extent to which transmission or progression to active disease occurred within or outside california . given that most clustered cases occurred among persons residing in the united states for > 3 years and that us - born persons were involved in 3 of 8 clusters , at least some proportion of mdr tb transmission is likely to have occurred in california . this observation suggests that although most mdr tb cases in the united states are related to the migration of persons already infected with drug - resistant m. tuberculosis , a small but notable proportion may be due to ongoing transmission . heterogeneity in the reproductive success of drug - resistant m. tuberculosis is now well established ( 34,35 ) . in this study , the katg s315 t mutation was the only isoniazid resistance conferring mutation found among clustered mdr tb cases . the high prevalence of katg s315 t among mdr strains ( 12,14,3638 ) and the association of this mutation with increased secondary case generation among isoniazid - monoresistant strains ( 6,7 ) have been documented . we report that the katg s315 t isoniazid resistance conferring mutation retains an independent effect on clustering of mdr tb cases despite the presence of mutations that confer resistance to additional drugs . in particular , secondary case generation did not significantly vary according to site of rpob mutations that confer rifampin resistance , which supports the hypothesis that these are no - cost mutations or that compensatory mutations commonly exist ( 39 ) . the katg s315 t mutation is thought to preserve fitness through the relative preservation of catalase - peroxidase production ( 9 ) , although whether this mutation is associated with different clinical phenotypes is unknown . in this study , we noted an inverse association between presence of the katg s315 t mutation and sputum smear positivity . in addition , the katg s315 t mutation was associated with case clustering , independent of sputum smear status . these findings suggest that the katg s315 t mutation may preserve the ability of m. tuberculosis to transmit and cause secondary cases through mechanisms unrelated to conventional indices of disease severity , such as the presence of abundant acid - fast bacilli in sputum . first , mdr tb in california is occurring predominantly among patients who were not born in the united states , with some cases from recent transmission and rapid progression to disease . our study suggests that in california , younger persons and persons who have emigrated from mexico and from refugee settings may be at higher risk for transmitting mdr m. tuberculosis . likewise , our findings reinforce the need for giving priority to screening and prevention activities in immigrant communities and us investment in international tb control ( 40 ) . second , if our results are verified in other settings , tb - control programs should consider pathogen - specific factors such as isoniazid resistance conferring mutations when planning the intensity of contact investigation and secondary case - finding activities . first , our estimates of case clustering are imprecise because of the limited number of mdr tb cases observed during the study period . however , these estimates are the best currently available , given that the data make up the largest population - based tb registry in the united states . second , although our definition of genotypic clustering was highly rigorous , the lack of detailed epidemiologic information precluded confirmation of transmission within california . third , because our study did not include pan - susceptible or isoniazid - monoresistant strains , we can not comment directly on mdr m. tuberculosis pathogenicity relative to these groups . lastly , our analyses implicitly assume independence of outcome events , and household or community - level factors potentially associated with clustering were not available . future studies should be designed so that statistical methods can be used that are able to accommodate the possible effects of within - household clustering . we found a substantial proportion of mdr tb cases and case clustering in california among non us - born persons , and the katg s315 t mutation was independently associated with clustering . validation of these findings in larger cohorts and in different population settings may have crucial public health consequences .

toc summary : type of isoniazid resistance conferring mutation may be a determinant of genotypic clustering .

historically , presence of heterotopic gastric mucosa in the intestinal tract has never aroused particular interest among clinicians . apart from its occurrence in the meckel s diverticulum , the condition is regarded as clinically insignificant . gastric metaplasia in the duodenum has been described by several authors , however , the significance of its occurrence remains unknown . extensive use of gastroduodenal fibroscopy enabled the authors to obtain biopsies of the duodenal mucosa . this paper will describe the incidence of gastric metaplasia in duodenum in various gastroduodenal diseases and discuss the pathogenetic role of gastric metaplasia in peptic ulcer . the authors obtained two or three pieces of random biopsy specimens from apparently normal mucosa in the anterior wall of duodenal bulb using gastroduodenal fiberscope on 65 subjects . all biopsies were immediately fixed in 10% formalin solution , embedded in paraffin , and 5 mm thick sections were stained with haematoxylin and eosine and pas , as described by mark and drysdale for sharp distinction of gastric metaplasia . the authors attempted to locate the fundic gland in all specimens but failed . consequently , the possibility of gastric heterotopia was excluded . the surface epithelium of gastric mucosa stained strongly with pas reaction contrasting sharply against the duodenal epithelium , where only goblet cells and the brush border disclosed a positive reaction ( fig . duodenal ulcer and prepyloric ulcers are grouped together since they have the same pathogenetic mechanism as proposed by johnson . the gastric ulcer group consisted solely of patients with ulcers located at the body , angle , or proximal antrum : two of the subjects in this group were additionally diagnosed with duodenitis . the incidence of gastric metaplasia according to its underlying diseases gastric metaplasia was not observed in normal subjects with exception to one ( 5.2% ) . in patients with duodenal and/or prepyloric ulcer ( s ) , its incidence was 72.2% , which was higher than in normal controls ( 5.2% ) and those with gastric ulcer ( 36.4% ) , nevertheless the difference lacked statistical significance ( p<.25 ) . three of 7 patients with duodenitis ( 42.8% ) , and one of 8 patients with gastric erosions ( 12.5% ) , had gastric metaplasia . the incidence of gastric metaplasia in the patients with gastic ulcer seemed to be varied according to the location of ulcer . although the number of cases was too low to evaluate it more thoroughly , the incidence of metaplasia was higher ( 66.7% ) in the patients with ulcers located in lower portion of the stomach ( prepyloric and pyloric ) than ulcers located in lower portion of the stomach ( prepyloric and pyloric ) than ulcers located in upper portion of the stomach ( 57.1% in those with ulcers located in angle and proximal antrum : 0% in those with body ulcer ) . the gastric mucosa in the duodenum can be classified into congenital heterotopic gastric mucosa and acquired gastric metaplasia as shown in table 3 . until now , many authors have made the mistake of combining metaplasia and true heterotopic gastric mucosa . since taylor reported earlier ( in 1927 ) two cases of gastric heterotopia in the duodenum , many authors described heterotopic gastric mucosa in duodenum as slightly raised patches consisting of chief cells and parietal cells . as true gastric heterotopia is frequently seen in meckel s diverticulum , and occasionally in rectum and small intestine , it is usually regarded as congenital in origin . the gastric metaplasia , a term which was first suggested by lessels , is an incomplete form of gastric heterotopia , and is composed of foveolar and pyloric gland : it can be identified only with microscopy . the incidence of true gastric heterotopia is suggested to be present in approximately 2% of the population . however , microscopic gastric metaplasia is far more frequently seen than true heterotopia and it is especially common in duodenal ulcer patients . the incidence of gastric metaplasia in duodenal ulcer patients was reported to be 51.9% by johansen , 74.3% by urakami and 72.2% by these authors series , which was quite similar to previous reports . it is unknown whether the gastric metaplasia has a protective role or a harmful effect in the formation of duodenal ulcer . the presence of gastric epithelium in the duodenum could indicate a simple protective response to excessive acid secretion . urakami reported that the gastric metaplasia is less frequent in the active stage of duodenal ulcer , and its incidence increases in the ulcer s healing stage . jochi found that gastric metaplasia was frequently seen in the margin of the ulcer and appeared to occur with the healing process of the ulcer . suzuki divided the gastric metaplasia into three types : foveolar cell metaplasia , parietal cell metaplasia , fully developed fundic gland metaplasia : he reported that each incidence had been 79.2% , 10.4% . and this study was intended only to reveal the incidence of gastric metaplasia in various gastroduodenal disease patients . the study could be expanded to clarify possible relations between acid secretion , stage of peptic ulcer , and stomach cancer .

to investigate the incidence of duodenal gastric metaplasia and its underlying gastric or duodenal diseases , the authors obtained endoscopic biopsy specimens from the duodenal bulb at random sites during endoscopy from 19 normal subjects , 11 patients with gastric ulcer , 18 with duodenal and/or prepyloric ulcer ( s ) , 7 with duodenitis and 8 with gastric erosions . the biopsy specimens were assessed with pas staining to confirm gastric metaplasia.the incidence of duodenal gastric metaplasia was 72.2% in patients with duodenal and/or prepyloric ulcer ( s ) , which contrasted with the patients with gastric ulcers ( 36.4% ) , duodenitis ( 42.9% ) , gastric erosions ( 12.5% ) , and normal subjects ( 5.3%).in conclusion , the results suggest that gastric metaplasia seen predominantly in patients with duodenal ulcer , seems to be related to hyperacidity and it plays some role in the pathogenesis of peptic ulcer in duodenum .

periodontal diseases are polymicrobial immune - inflammatory infectious diseases that can lead to the destruction of periodontal ligaments and adjacent supportive alveolar bone . the subgingival plaque contains more than 700 bacterial species , and some of these microorganisms have been shown to be responsible for initiation / progression of periodontal diseases [ 1 , 2 ] . the red complex , which includes porphyromonas gingivalis , treponema denticola , and tannerella forsythia ( formerly bacteroides forsythus ) , encompasses the most important pathogens in adult periodontal disease . additionally , fusobacterium nucleatum , prevotella species , eikenella corrodens , peptostreptococcus micros , and campylobacter rectus are increased in deep periodontal pockets and are implicated as possible periodontopathogens [ 14 ] . these bacteria are not usually found alone , but in combination in the periodontal pockets , suggesting that some bacteria may cause destruction of the periodontal tissue in a cooperative manner . studies using animal models have reported the synergistic pathogenicity of mixed infections with p. gingivalis - t . furthermore , coaggregation , nutrient effects , and modulation of virulence factors by periodontopathogens or by interspecies interactions between periodontopathogenic and nonpathogenic organisms have been reported to contribute to oral microbial pathogenesis . this paper focuses on interspecies pathogenic interactions within the red complex , in particular the combinations of p. gingivalis - t . potential therapies using normal inhabitants of the oral microbiota that have an antagonistic relationship with the red complex are discussed . p. gingivalis possesses many virulence factors , such as fimbriae , lipopolysaccharides , and proteases [ 1315 ] . the arg - gingipain ( rgp ) and lys - gingipain ( kgp ) cysteine proteinases are important for the virulence of p. gingivalis as they elicit dysfunction of inflammatory and immune responses and can degrade various connective tissue proteins [ 16 , 17 ] . rgp is encoded by two separate genes ( rgpa and rgpb ) , whereas kgp is encoded by a single gene ( kgp ) . in a murine abscess model , rgpa and rgpb double and kgp single - mutants induced smaller abscesses than did the wild type , and the rgpa - rgpb - kgp triple ( gingipain - null ) mutant showed negligible lesion formation . these findings indicate that gingipains play an important role in abscess formation in mice . compared to the abscess formation induced by monoinfection with each bacterium in the abovedescribed murine model , mixed infection with wild - type p. gingivalis atcc 33277 and t. forsythia atcc 43037 showed a synergistic effect on abscess formation . on the other hand , mixed infection with p. gingivalis mutants devoid of gingipain ( rgpa rgpb , kgp , and rgpa rgpb kgp ) with t. forsythia showed only just an additive effect on abscess formation . these findings suggest that the gingipains of p. gingivalis play an important role in the pathological synergism between p. gingivalis and t. forsythia . a combination of t. forsythia strains isolated from periodontitis patients and p. gingivalis also showed synergistic pathogenesis in a rabbit abscess model . the researchers found 100% abscess formation in rabbits with mixed infection using p. gingivalis and t. forsythia but 0% in rabbits with monoinfection with each bacterium . recently , verma et al . injected p. gingivalis fdc 381 and t. forsythia atcc 43037 into the oral cavity of rats and evaluated the synergistic effect of mixed infection on periodontal disease . mixed colonization by p. gingivalis and t. forsythia in the rat oral cavity was confirmed by polymerase chain reaction ( pcr ) . the induction of moderate periodontal inflammation and pronounced apical migration of junctional epithelium , generation of a specific immunoglobulin g ( igg ) antibody response , and stimulation of both th1- and th2-like immune responses as reflected by the serum igg subclass profiles were evident . mixed infection with p. gingivalis and t. forsythia resulted in a significant increase in interproximal alveolar bone resorption compared to that in rats infected with a single bacterial strain and controls , but there was no synergy between the bacteria . the researchers indicated that the virulence of p. gingivalis and t. forsythia mixed infection resulted from the immune - inflammatory responses and the lack of humoral immune protection during periodontitis in rats . mice that were first infected with a wild - type strain and subsequently reinfected with the same wild - type strain showed significantly smaller lesions than control mice that were mock - infected with medium only and then re - infected with the wild - type strain . assessed the serum igg antibody response in a murine abscess model after mixed infection with various levels of p. gingivalis atcc 33277 or t. forsythia atcc 43037 whole bacteria cell antigens . after mixed infection , igg antibody responses to p. gingivalis increased in proportion to the level of t. forsythia injected . in contrast , igg antibody responses to t. forsythia did not correlate with the level of p. gingivalis injected . reasons for this difference may be that p. gingivalis and t. forsythia induced different antibody responses after mixed infection in mice ; alternatively , these bacteria may exhibit different interactions in terms of growth at the injected sites . t. forsythia , which is a fastidious anaerobic gram - negative rod , is frequently isolated together with p. gingivalis , especially from the active state of periodontitis [ 2427 ] . it is well known that the growth of t. forsythia is accelerated on blood agar when cocultivated with p. gingivalis or f. nucleatum , suggesting that a form of symbiosis occurs with respect to nutrition . sonicated cell extracts of t. forsythia atcc 43037 stimulate growth of p. gingivalis atcc 33277 in nutrition - decreased medium in a dose - dependent manner , whereas the cell extract of t. forsythia had no stimulatory effect on the growth of the rgpa rgpb kgp triple ( gingipain - null ) mutant of p. gingivalis . these results suggest that gingipains of p. gingivalis play an important role in the digestion or uptake of the growth - promoting factor derived from t. forsythia . the growth - promoting interaction between p. gingivalis and t. forsythia may be partly related to synergistic virulence in a murine abscess model . t. forsythia is a member of the polymicrobial flora that invades buccal epithelial cells taken directly from the mouth . epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses and for forming reservoirs important in recurrent infections . t. forsythia possesses some putative virulence factors , such as a trypsin - like protease , a sialidase , hemagglutinin , components of the bacterial s - layer , and a cell surface - associated and secreted protein ( bspa ) . bspa has been recognized as a virulence factor important for alveolar bone loss in mice . inagaki et al . investigated the epithelial cell adherence and invasion abilities of t. forsythia and reported that these are dependent on bspa . additionally , they found that p. gingivalis fdc 381 or its outer membrane vesicles enhance the attachment and invasion of t. forsythia atcc 43037 to epithelial cells . t. denticola , a small oral spirochete , is frequently found with p. gingivalis in progressing periodontitis lesions [ 1 , 3335 ] . t. denticola is located within the surface layers of the subgingival plaque , whereas p. gingivalis is observed predominantly beneath the spirochete layer ; a symbiotic nutrient utilization relationship between these two periodontopathogens has been shown in vitro . the growth - stimulating factors produced by p. gingivalis atcc 33277 and t. denticola atcc 35405 have been identified as isobutyric acid and succinic acid , respectively . coculture of p. gingivalis fdc 381 and t. denticola atcc 35405 induced synergistic biofilm formation and coaggregation . confocal microscopy demonstrated that p. gingivalis attaches to the substratum first as the primary colonizer followed by coaggregation with t. denticola to form a mixed biofilm . the t. denticola flagellar mutant and cytoplasmic filament mutant exhibit significantly reduced biofilm formation with p. gingivalis . similarly , the p. gingivalis gingipain mutant and major fimbriae mutant exhibited significantly reduced biofilm formation with t. denticola . using two - dimensional electrophoresis followed by a ligand overlay assay with p. gingivalis fimbriae , hashimoto et al . determined that dentilisin , a chymotrypsin - like proteinase of t. denticola , was the p. gingivalis fimbriae - binding protein . these results support the hypothesis that these two organisms assist each other 's survival in subgingival sulcus and explain why they are frequently isolated together from subgingival plaque . synergistic virulence of mixed p. gingivalis and t. denticola infection has been assessed in several animal lesion models . using a murine abscess model , one group reported that high doses of p. gingivalis w50 ( 1 - 2 10 cells per dose ) together with t. denticola atcc 35404 ( 1 10 or 1 10 cells per dose ) had no effect on the formation and size of the spreading lesion caused by p. gingivalis . however , at low p. gingivalis doses ( 1 - 2 10 cells ) , addition of t. denticola ( 1 10 cells ) significantly enhanced the virulence of p. gingivalis compared with monoinfection . investigation of the synergistic virulence of p. gingivalis and t. denticola using a murine experimental model of periodontitis found that a 1 : 1 ratio of p. gingivalis w50 and t. denticola atcc 35405 coinoculum at 5 10 or 1 10 total bacterial cells induced the same level of bone loss as four doses of 1 10 p. gingivalis . coinoculation induced strong p. gingivalis - specific t cell proliferative and interferon- ( ifn- ) cytokine responses and induced a strong t. denticola - specific ifn- cytokine response . another study using a rat model of periodontal disease reported that a mixed p. gingivalis fdc 381 ( 5 10 cells ) and t. denticola atcc 35404 ( 5 10 cells ) infection produced significantly more interproximal and horizontal alveolar bone loss compared to monoinfections ( 1 10 cells ) ; however , there was no synergy between p. gingivalis and t. denticola . furthermore , colonization of these bacteria was observed in the rat oral cavity during 7 weeks of periodontal disease , resulting in the generation of a specific serum igg antibody response that reflected oral infection and the induction of an inflammatory response consistent with the established characteristics of periodontitis . these results suggest that p. gingivalis and t. denticola act synergistically ( with no synergy between the bacteria ) to stimulate the host immune response and to induce alveolar bone loss in a rat model of periodontitis . epithelial cells and macrophages play a major role in the host response to periodontopathogens , and secretion of inflammatory mediators and matrix metalloproteinases ( mmps ) by these host cells contribute to periodontal tissue destruction . investigated the inflammatory response of an in vitro macrophage / epithelial cell coculture model following mono- or mixed infections with whole bacterial cells of the red - complex and their lipopolysaccharide ( lps ) . mono- or mixed infections of the coculture model induced the secretion of interleukin-1 beta ( il-1 ) , il-6 , il-8 , prostaglandin e2 ( pge2 ) , and mmp-9 . all lps mono- or mixed infections induced an increase in chemokine , mmp-9 , and pge2 production . compared to mono - infections with individual bacterial species , no synergistic effects on cytokine , pge2 , or mmp-9 production by the bacterial mixtures tested were observed . p. gingivalis and t. forsythia induced the secretion of rantes ( regulated and normal t cell expressed and secreted ) , whereas t. denticola alone or in combination with p. gingivalis resulted in a significant decrease in rantes levels . rantes degradation by mono- or mixed infections with red - complex bacteria resulted in massive proteolytic degradation of rantes by t. denticola . the ability of periodontopathogens to degrade cytokines and chemokines in vivo may play an important role in their pathogenicity by disrupting the host inflammatory response . recently , polymicrobial infection with p. gingivalis , t. denticola , and t. forsythia in a rat model of periodontal disease was investigated . a 1 10 cell mixture ( 1 ml ) containing an equal number of cells of each bacterium was injected into the rat oral cavity . pcr of the bacterial dna in the oral sample revealed that polymicrobial infection enhanced colonization by p. gingivalis , t. denticola , and t. forsythia compared to their levels in monomicrobial infections . oral infection of rats with a polymicrobial consortium comprising p. gingivalis , t. denticola , and t. forsythia induced significant increases in maxillary and mandibular alveolar bone resorption compared to those resulting from any of the monomicrobial infections ( p < 0.001 ) . the levels of serum igg against all of the bacteria in the polymicrobial infection were lower than the respective levels induced by monomicrobial infections . this suggested that the host response to the polymicrobial infection was altered , resulting in enhanced evasion of protective immune responses by the bacterial consortium . most therapeutic modalities for treatment of periodontitis aim to remove pathogens and kill all bacteria in the periodontal pocket . understanding the effect of interbacterial interactions on the pathogenesis of periodontitis may facilitate development of novel treatment modalities , such as the inhibition of adherence using antagonists , passive immunization , replacement therapy , regulation of levels of nonpathogenic bacteria to modulate virulence , probiotics , and interference with signaling mechanisms . the disruption of the harmonic relationship between the host and commensal microorganisms is considered to be an important factor for the development of oral pathologies . , we discuss the significance of interbacterial antagonism for maintenance and recovery of a healthy oral microbiota . moreover , antagonistic bacteria have the potential for probiotic action , which may protect against periodontitis . staphylococcus aureus and streptococcus mutans isolates inhibited the growth of t. denticola and p. gingivalis . s. aureus strains produced a bacteriocin - like inhibitory substance , whereas the inhibitory effect of s. mutans was related to the production of lactic acid . s. sanguinis , s. cristatus , s. salivarius , s. mitis , actinomyces naeslundii , and haemophilus parainfluenzae inhibited the adhesion of standard p. gingivalis strains in vitro . s. cristatus arginine deiminase repressed fima , a major subunit protein of the long fimbriae , and inhibited biofilm formation by p. gingivalis . an analysis of the ability of clinical isolates from healthy and periodontitis patients to inhibit the growth of periodontopathogens showed that the number of isolates from healthy volunteers that inhibited either p. gingivalis or p. intermedia was significantly higher than that from diseased patients . these isolated growth - inhibiting strains included some viridans group streptococcus , actinomyces , and bifidobacterium strains . compared to these isolates , commercial dietary probiotics showed stronger inhibition of the periodontopathogens . in a study that compared oral lactobacilli from patients with chronic periodontitis and periodontally healthy subjects , the most prevalent species in healthy subjects were lactobacillus gasseri and l. fermentum , whereas the most prevalent species in subjects with periodontitis was l. plantarum . furthermore , the greatest antimicrobial activities were associated with l. paracasei , l. plantarum , l. rhamnosus , and l. salivarius . the international guidelines for the evaluation of probiotics confirm that these four organisms exhibit both high antimicrobial activity and high tolerance to environmental stress . some oral bacteria possess the potential for antagonism towards periodontopathogens , which highlights the therapeutic potential of stimulation of oral health using beneficial effector strains . most evidence indicates that probiotics in the gut do not populate the gastrointestinal microbiota permanently , and they disappear from feces soon after cessation of probiotic ingestion . probiotic bacteria used in the human oral cavity include bifidobacterium and lactobacillus species , and most of them were not derived from the oral cavity . l. reuteri and l. salivarius colonized the oral cavity of patients ; however , the study was of only 2 weeks duration [ 5759 ] . another study reported that oral administration of l. salivarius decreased the proportion of l. salivarius in saliva during the 4- and 8-week intervention periods , although the sampling and analysis methods differed . organisms antagonistic to periodontopathogens that are derived from typical representatives of the oral microbiota and have probiotic potential may overcome the weaknesses associated with exogenous probiotic bacteria .

the red complex , which includes porphyromonas gingivalis , treponema denticola , and tannerella forsythia ( formerly bacteroides forsythus ) , are recognized as the most important pathogens in adult periodontal disease . these bacteria are usually found together in periodontal pockets , suggesting that they may cause destruction of the periodontal tissue in a cooperative manner . this article discusses the interspecies pathogenic interactions within the red complex .

technological advances in three - dimensional ( 3d ) imaging such as cone - beam computed tomography ( cbct ) appear to offer significant advantages in both quality and quantity of data representing true anatomy , . the increased use of computed tomography in dentistry has also spurred the improvement of existing software designed primarily for dental use , as well as the development of new software . however , it is necessary to select protocols and to assess the reliability and accuracy of landmark identification in 3d images . development of 3d imaging into a practical and usable cephalometric method was hindered for many years by technologic limitations , high costs , and high radiation doses . advances in cbct technology make its use feasible for specific indications , such as impacted teeth , craniofacial anomalies , airway and temporomandibular joint disorders . in these cases , cbct images could be used for routine cephalometric purposes beyond the diagnostic purposes for which they were primarily taken . this has been extensively done for cephalometric measurements , but to a much lesser extent for maxillofacial computed tomography imaging . whether cbct should be used routinely in all orthodontic patients the voxel is the smallest unit of ct images and its size has an influence on image spatial resolution . by decreasing the voxel size the image spatial resolution increases as there is a decrease in partial volume averaging . on the other hand , by decreasing the voxel size , the scan time increases , as does the probability of patient movement . this is especially of concern in the case of younger individuals , who constitute the overwhelming majority of orthodontic patients . this issue of increased radiation dose to children , and specifically for orthodontic purposes , has caused widespread concern and has been the source of much discussion even in the lay literature . in order to verify the clinical reliability of measurements on multiplanar sections and 3d reconstructions derived from cbct , it is also necessary to evaluate the measurements using anatomic landmarks without the use of metallic markers . the most recent studies involving cbct scans have shown reproducibility and accuracy of cephalometric measurements performed on lateral cephalograms reformatted from cbct images or in 3d - cbct images . however , most of the studies used radiopaque markers and these might have an influence on the accuracy and reproducibility of the measurements . the presence of metallic markers eliminates errors from landmark identification because metallic landmarks can be easily identified and located with a high degree of accuracy and precision . the aim of this study was to verify the influence of voxel size on the accuracy and reproducibility of linear measurements of the mandible performed without metallic markers on 3d cbct images , comparing multiplanar sections and 3d rendering images . approval for this study was received from the ethics committee at the university of so paulo , bauru , brazil . ten dry adult human mandibles , selected from the collection of dry skulls of the department of anatomy from the bauru school of dentistry were scanned . to fit the inclusion criteria , the mandibles had to be well preserved with no large metallic restorations on the teeth , in order to avoid scattering and artifacts . the sample size of each group was calculated based on an alpha significance level of 0.05 and beta of 0.2 to achieve 80% of power . to provide soft - tissue equivalent attenuation , the mandibles were positioned with the mandibular plane parallel and the midsagittal plane perpendicular to the floor , in accordance with the manufacturer s instructions for performing the scans in the clinical setting . the cbct images were acquired using the i - cat classic 3d dental imaging system ( i - cat classic , imaging sciences international , hatfield , pa , usa ) . each mandible was scanned twice : once with a 0.20 voxel resolution ( 36.12 mas , 40 sec , fov of 8 cm , 120 kv ) and once with a 0.40 voxel resolution ( 18.45 mas , 20 sec , fov of 8 cm , 120 kv ) . cbct data were exported in dicom ( digital imaging and communication in medicine ) multi - file format and imported into dolphin11.5 software ( dolphin imaging & management solutions , patterson technology , chatsworth , ca , usa ) . a computer a505-s6975 ( satellite , toshiba , tokyo , japan ) with a dedicated 512-mb video card on a 17-in crystalbrite lcd flat - panel color screen with a maximum resolution of 1440x900 pixels measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d . the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image . it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups , each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) . the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated . figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points figure 2points . reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images figure 3definitions of mandibular linear measurements for 3d rendering images , the landmarks were identified directly on the surface for 3d images . the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) . the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies . in this initial study , we selected landmarks that are relatively easy to identify linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) . the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) . intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements . all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc . measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d . the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image . it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups , each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) . the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated . figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points figure 2points . reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images figure 3definitions of mandibular linear measurements for 3d rendering images , the landmarks were identified directly on the surface for 3d images . the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) . the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies . in this initial study , we selected landmarks that are relatively easy to identify . linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) . the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) . intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements . all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc . , chicago , il , usa ) . the measurements for investigator 1 showed excellent reliability with intraclass correlation coefficients ( icc ) ranging from 0.93 to 1 ( icc average g2:0.97 ; g3:0.97 ; g4:0.98 and g5:0.98 ) . additionally , the mean absolute differences between the first and second measurements were less than 0.40 mm ( mean average difference g2:0.11 mm and g3:0.02 mm ) for multiplanar images and less than 0.64 mm ( mean average difference g4:0.14 mm and g5:0.03 mm ) for 3d model images . for investigator 2 , the results also showed excellent reliability , with icc coefficients ranging from 0.88 to 0.99 ( icc average - g2:0.96 ; g3:0.95 ; g4:0.97 and g5:0.97 ) . table 1intraobserver and interobserver variation for each group and analysis methods [ intraclass correlation coefficient ( icc ) and bland - altman ] multiplanar images3d model imagesvariable g2g3g4g5 0.2 voxel ( n=10)0.4 voxel ( n=10)0.2 voxel ( n=10)0.4 voxel ( n=10)intraobserver obser veradsd95% ciiccadsd95% ciiccadsd95% ciiccadsd95% ciiccc c 10.010.36 - 0.69 ; 0.711 - 0.010.15 - 0.31 ; 0.2910.120.67 - 1.20 ; 1.440.990.180.77 - 1.34 ; 1.700.98 2 - 0.410.75 - 1.89 ; 1.070.98 - 0.680.75 - 2.16 ; 0.800.970.170.87 - 1.54 ; 1.880.980.460.73 - 0.98 ; 1.900.98mf mf 1 - 0.130.46 - 1.03 ; 0.770.99 - 0.190.49 - 1.14 ; 0.760.980.170.72 - 1.25 ; 1.590.96 - 0.210.49 - 1.17 ; 0.750.98 2 - 0.301.18 - 2.16 ; 2.010.93 - 0.080.68 - 1.41 ; 1.250.97 - 0.210.82 - 1.82 ; 1.400.96 - 0.031.33 - 2.63 ; 2.570.92c mf 1 - 0.041.09 - 2.18 ; 2.100.970.310.67 - 0.99 ; 1.610.980.640.78 - 0.90 ; 2.180.980.260.81 - 1.32 ; 1.840.98 2 - 0.581.21 - 2.95 ; 1.790.95 - 0.920.97 - 2.81 ; 0.970.960.480.92 - 1.33 ; 2.290.970.420.75 - 1.04 ; 1.880.98c mf 10.190.84 - 1.46 ; 1.840.990.240.90 - 1.53 ; 2.010.970.390.67 - 0.93 ; 1.710.980.100.70 - 1.28 ; 1.480.99 2 - 0.660.81 - 2.24 ; 0.920.97 - 0.811.27 - 3.30 ; 1.680.940.100.73 - 1.34 ; 1.540.990.630.85 - 1.03 ; 2.290.97c mf 10.401.22 - 2.0 ; 2.800.950.051.40 - 2.68 ; 2.780.930.041.00 - 1.93 ; 2.010.960.261.06 - 1.83 ; 2.350.95 2 - 1.050.90 - 2.82 ; 0.720.91 - 0.291.01 - 2.27 ; 1.690.95 - 0.090.61 - 1.28 ; 1.100.990.410.68 - 0.92 ; 1.740.97c mf 10.121.40 - 2.62 ; 2.860.94 - 0.041.13 - 2.27 ; 2.190.950.090.90 - 1.67 ; 1.850.970.090.73 - 1.35 ; 1.530.98 2 - 0.650.88 - 2.36 ; 1.060.96 - 0.690.72 - 2.10 ; 0.720.96 - 0.020.99 - 1.96 ; 1.920.970.061.01 - 1.93 ; 2.050.97mco lco ( right)1 - 0.030.39 - 0.80 ; 0.740.98 - 0.070.40 - 0.84 ; 0.700.980.140.28 - 0.42 ; 0.700.99 - 0.030.45 - 0.91 ; 0.850.98 20.260.47 - 0.66 ; 1.180.970.180.71 - 1.22 ; 1.580.950.770.54 - 0.29 ; 1.830.900.680.55 - 0.40 ; 1.760.92mco lco ( left)10.390.44 - 0.47 ; 1.250.94 - 0.380.53 - 1.42 ; 0.660.930.010.35 - 0.67 ; 0.690.98 - 0.050.53 - 1.10 ; 1.000.95 2 - 0.050.53 - 1.08 ; 0.990.950.300.81 - 1.29 ; 1.890.88 - 0.080.43 - 0.93 ; 0.770.980.140.33 - 0.50 ; 0.780.98lco lco 10.300.38 - 0.34 ; 0.940.99 - 0.220.50 - 1.22 ; 0.780.99 - 0.030.31 - 0.65 ; 0.591 - 0.150.70 - 1.52 ; 1.220.99 2 - 0.300.70 - 1.09 ; 1.690.980.150.67 - 1.15 ; 1.450.990.770.53 - 0.28 ; 1.820.970.780.53 - 0.27 ; 1.830.97mco mco 1 - 0.070.35 - 0.76 ; 0.620.990.080.54 - 0.98 ; 1.140.98 - 0.220.35 - 0.91 ; 0.470.99 - 0.120.50 - 1.11 ; 0.870.99 2 - 0.330.51 - 1.34 ; 0.680.98 - 0.620.35 - 1.31 ; 0.070.97 - 0.080.35 - 0.78 ; 0.620.99 - 0.051.29 - 0.62 ; 0.520.99total10.11 0.970.02 0.970.14 0.980.03 0.98 2 - 0.41 0.96 - 0.35 0.950.18 0.970.35 0.97interobserverc c 1 x 2 - 0.540.98 - 2.46 ; 1.380.960.000.49 - 0.95 ; 0.960.99 - 0.110.88 - 1.84 ; 1.630.98 - 0.150.56 - 1.25 ; 0.950.99mf mf 1 x 21.000.61 - 0.20 ; 2.190.910.940.54 - 0.12 ; 1.990.923.591.490.66 ; 6.510.263.251.450.38 ; 6.090.45c mf 1 x 2 - 1.201.18 - 3.51 ; 1.150.93 - 1.491.09 - 3.63 ; 0.640.90 - 2.420.80 - 3.99 ; -0.840.84 - 2.560.66 - 3.86 ; -1.260.82c mf 1 x 20.631.05 - 1.45 ; 2.700.970.060.80 - 1.51 ; 1.640.980.071.20 - 2.30 ; 2.430.96 - 0.340.57 - 1.46 ; 0.790.99c mf 1 x 2 - 1.091.34 - 3.72 ; 1.530.89 - 1.070.94 - 2.91 ; 0.790.92 - 2.421.38 - 5.13 ; 0.300.70 - 2.471.06 - 4.56 ; -0.370.69c mf 1 x 20.400.60 - 0.78 ; 1.590.98 - 0.790.81 - 0.79 ; 2.380.950.070.68 - 1.28 ; 1.410.980.100.58 - 1.05 ; 1.240.99mco lco ( right)1 x 2 - 1.770.54 - 2.83 ; -0.710.69 - 1.840.50 - 2.83 ; -0.860.67 - 0.180.84 - 1.83 ; 1.460.92 - 0.490.46 - 1.40 ; 0.410.95mco lco ( left)1 x 2 - 1.430.54 - 2.49 ; -0.360.63 - 1.390.63 - 2.63 ; -0.140.63 - 1.130.84 - 2.79 ; 0.520.72 - 0.730.56 - 1.85 ; 0.380.86lco lco 1 x 2 - 1.420.55 - 2.50 ; -0.340.92 - 1.450.45 - 2.34 ; -0.550.93 - 0.091.16 - 2.35 ; 2.190.96 - 0.150.93 - 1.96 ; 1.670.97mco mco 1 x 21.990.461.08 ; 2.890.762.120.431.27 ; 2.960.761.340.610.14 ; 2.530.861.170.480.21 ; 2.130.90total -0.34 0.86 - 0.49 0.87 - 0.13 0.82 - 0.24 0.86ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient cbct multiplanar reconstruction measurements showed good to high interobserver reliability with icc ranging from 0.63 to 0.99 ( mean average g2=0.86 and g3=0.87 ) with the absolute differences between the first and second measurements ranging for 0 to 2.12 mm ( mean average difference g2:-0.34 mm and g3:-0.49 mm , table 1 ) . for 3d reconstruction measurements , independently of the voxel size ( g4 or g5 ) , most of the variables presented good to high interobserver reliability , with the exception of inter - mental foramen width ( mf mf ) that had fair reliability on 0.2 mm voxel images ( g4=0.26 ) and moderate reliability for 0.4 voxel size ( g5=0.45 ) . the mean values and standard deviations of the physical measurements and of the cbct measurements for all groups are presented in table 2 . two variables ( mf mf and c mf ) at groups g2 and g3 were significantly smaller than physical measurements ( g1 ) . for 3d rendering images ( g4 and g5 ) , only two variables out of 10 were not significantly smaller than the physical measurements . no significant difference was found between the voxel size both for 3d rendering and multiplanar sections . table 2mean ( mm ) and standard deviation ( sd ) of linear measurements for dry mandibles compared to cone - beam computed tomography ( cbct ) images with different voxel sizes [ analysis of variance ( anova ) for repeated measures and dunnett s tests ] physical measurementmultiplanar images3d model images variableg1g2g3g4g5p 0.2 voxel0.4 voxel0.2 voxel0.4 voxel ( n=10)(n=10)(n=10)(n=10)(n=10 ) meansdmeansdmdmeansdmdmeansdmdmeansdmd c c 101.033.98101.154.150.11100.974.01 - 0,06101.124.060,09101.184.260.140.697mf mf 49.17 3.0347.91 2.98 - 1.2647.91 2.81 - 1,2647.16 2.64 - 2,0147.76 3.01 - 1.420.000*c mf 76.43 4.7176.50 4.420.0776.24 4.17 - 0,1875.59 4.60 - 0,8475.51 4.49 - 0.920.000*c mf 104.41 4.63103.82 4.79 - 0.58103.56 4.26 - 0,85102.76 4.38 - 1,64103.10 4.47 - 1.310.000*c mf 75.16 3.9975.66 3.640.5075.69 3.580,5374.48 3.46 - 0,6874.25 3.30 - 0.910.000*c mf 102.65 3.92102.44 4.02 - 0.21102.33 3.76 - 0,32101.29 3.90 - 1,36101.38 3.81 - 1.270.000*mco lco 21.42 2.4321.68 2.280.2621.69 2.280,2619.61 2.08 - 1,8120.07 2.19 - 1.350.000*mco lco 21.37 2.0521.28 1.67 - 0.0921.02 1.68 - 0,3419.31 1.82 - 2,0619.14 1.74 - 2.230.000*lco lco 124.25 4.05124.00 4.03 - 0.25123.78 4.10 - 0,47122.73 4.21 - 1,53122.74 4.04 - 1.510.000*mco mco 83.873.0984.072.860.2084.132.970,2684.072.810,283.762.98 - 0.110.251sd , standard deviation ; md , mean difference * statistically significantdifferent letters represent statistically significant differences ( dunnett s test ) sd , standard deviation ; md , mean difference statistically significant different letters represent statistically significant differences ( dunnett s test ) the availability of this technology is undoubtedly expanding the use and application of 3d imaging in the field of orthodontics . however , while cbct provides many advantages , patient radiation dose is still higher than conventional cephalometric and panoramic digital imaging modalities . this study was performed to evaluate the reliability and accuracy of linear measurements between common landmarks of the mandible taken from cbct multiplanar sections and 3d volumetric renderings with different voxel sizes . in addition , larger voxel sizes are consistent with the alara principle , keeping the dose as low as reasonably achievable for the intended purpose of the scan . most of the previous studies performed to validate cbct for 3d cephalometrics measured linear distances between anatomical landmarks in dry skulls in combination with radiopaque markers . cephalometric analyses are subject to the influence of the examiner , landmark identification and type of image analyzed ( 2d sections or 3d image ) . the major question of this study was if the difficulty in identifying the landmarks in 3d images can influence the reliability and accuracy of the measurements , and for this reason , metallic markers were not used . this method is close to the real clinical procedures and is more clinically relevant than making measurements between well - defined metallic landmarks . there was excellent intraobserver reliability for both examiners with the mean icc for all groups above 0.95 and mean average differences less than 0.41 mm , independently of the protocol analyzed ( table 1 ) . these differences are not clinically significant for orthodontic / surgical diagnosis and treatment planning , and acceptable for skeletal measurements . additionally , good to high interobserver precision was identified for all groups , with exception of measurement mf the main explanations for the good reliability found in this study are the use of landmarks which are easily identifiable , the calibration between observers and the high spatial definition of cbct images . the differences between multiplanar sections and 3d volume rendering images were probably because landmarks on multiplanar images were identified on the orthogonal slices simultaneously instead of being identified directly on the surface of 3d reformatted images . our study also evaluated the accuracy of measurements performed in cbct images ( table 2 ) . two variables at g2 and g3 and 8 variables at g4 and g5 were significantly smaller than physical measurements ( g1 ) . ( 2009 ) , who showed a tendency for the cbct measurements to slightly underestimate the gold standard . the consistency of the differences we found suggests a systematic error in the 3d model measurement method . one possible explanation is that this error might have been introduced by the measurement software , mainly building line . although few software systems currently have a 3d cephalometric module , most of them have not been tested or validated . the results of the validation measurements for a specific machine or software can not be extrapolated to the entire cbct machine and software . most of them are designed differently and software updates are constant and frequent . in addition , the difference ranged from 0.06 to 2.23 mm . in agreement with previous reports , the average difference in our study is below clinical significance . according to grauer , et al . ( 2009 ) , generating measurements in 3d volumetric images rather than simultaneously in sets of 2d multiplanar images introduces error because of the difficulty involved in locating landmarks in 3d space and the inaccuracies of the user entered threshold used for the construction of 3d virtual surface models . rendered for this reason , greater caution is suggested for linear measurements performed on 3d models . the spatial resolution of the image has an influence in the accuracy of the measurements . most studies on cbct with a large field of view used 0.4 mm voxel size , whereas others with a small field of view used a 0.2 mm or smaller voxel sizes . none of the measurements showed significant difference between the voxel sizes , independently of the method of measurement ( table 2 ) . ( 2010 ) , who have suggested that 0.4 mm voxel resolution is adequate for performing measurements of craniofacial structures . while small voxel sizes increase the image resolution , they also may increase the image noise . the benefits of a shorter scanning time , with its reduced likelihood of patient movement and especially the lower exposure dose compensates for the poorer resolution . besides , large measurements are less influenced by small differences in spatial resolution . on the other hand , the diagnostic ability of cbct images for evaluating small structures appears to be influenced by voxel size . the two most common voxel sizes used in orthodontics 0.3 and 0.4 mm provide lower spatial resolution than smaller voxel sizes . orthodontic scans with resolutions of 0.3 and 0.4 voxels are ideal for general treatment planning but should be used with caution if the goal is to assess small variations in bone thickness . ( 2009 ) investigated the usefulness of cbct for identifying artificial external root resorption in images with voxel sizes of 0.4 , 0.3 , and 0.2 mm . they concluded that , even though the results were the same for the different voxel sizes , diagnosis was easier at smaller voxel sizes of 0.3 and 0.2 mm . although the benefits of a shorter scanning time satisfy the as low as reasonably achievable principle , the risks of misdiagnosis and treatment complications must also be weighed . consequently , a scanning protocol with a 0.40 mm voxel size might not be suitable for every patient . voxel size may vary according to the size of the structures to be analyzed as well as to the level of detail desired . in this study , we measured distances that are in excess of the resolutions 0.2 and 0.4 mm of the scan , so that voxel size would not very likely influence the measurements . unlike in conventional cephalometrics , where all the landmarks are identified in one image , namely , the lateral cephalogram , with cbct each landmark must be identified in three different images ( axial , coronal and sagittal ) , making the process of performing the measurements in cbct images more time - consuming for the orthodontist . further , cbct demands a higher radiation dose than traditional cephalometric images . for these reasons , its use should be limited to specific indications , for example , patients with impacted teeth , or those with facial asymmetries or craniofacial anomalies where cbct is better able to quantify the differences between the right and the left side of craniofacial structures . evidence - based will tell whether future advances , especially in terms of dose reduction , will make cbct appropriate for routine use in all orthodontic patients . linear measurements obtained on multiplanar 2d cbct images with 0.2 and 0.4 voxel sizes using i - cat scanner and dolphin software are reliable and accurate for clinical diagnosis and treatment planning . caution should be taken in linear measurements on 3d rendering images , because the measurements were reliable , but not accurate . reducing the voxel size from 0.4 to 0.2 mm does not influence the accuracy and reliability of measurements of large craniofacial structures on cbct-3d images .

objective the purpose of this study was to determine the accuracy and reliability of two methods of measurements of linear distances ( multiplanar 2d and tridimensional reconstruction 3d ) obtained from cone - beam computed tomography ( cbct ) with different voxel sizes.material and methods ten dry human mandibles were scanned at voxel sizes of 0.2 and 0.4 mm . craniometric anatomical landmarks were identified twice by two independent operators on the multiplanar reconstructed and on volume rendering images that were generated by the software dolphin. subsequently , physical measurements were performed using a digital caliper . analysis of variance ( anova ) , intraclass correlation coefficient ( icc ) and bland - altman were used for evaluating accuracy and reliability ( p<0.05).results excellent intraobserver reliability and good to high precision interobserver reliability values were found for linear measurements from cbct 3d and multiplanar images . measurements performed on multiplanar reconstructed images were more accurate than measurements in volume rendering compared with the gold standard . no statistically significant difference was found between voxel protocols , independently of the measurement method.conclusions linear measurements on multiplanar images of 0.2 and 0.4 voxel are reliable and accurate when compared with direct caliper measurements . caution should be taken in the volume rendering measurements , because the measurements were reliable , but not accurate for all variables . an increased voxel resolution did not result in greater accuracy of mandible measurements and would potentially provide increased patient radiation exposure .

the following terms were used to describe the different kinds of genetic maps used in this study , as suggested by hudson et al . ( 2012 ) : 1 ) sex - averaged map : a consensus map for both parents of a pedigree ; 2 ) consensus map : an integrated map based on segregation data from individual component maps ; 3 ) composite map : an integrated map of different individual component maps built by a marker - merging method ; and 4 ) component map : each of the maps used in the construction of a composite map . the graphics and the representations of genetic maps were produced with r 3.1.0 ( r core team 2014 ) . we used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . the first six maps were obtained from three controlled crosses ( pedigrees # 1 , # 2 , and # 3 in supplementary fig . s1 , supplementary material online ) between three different genotypes : corsica landes ( cl ) , morocco landes ( ml ) , and corsica morocco ( cm ) . in total , 106 , 117 , and 94 full - sibs the regression mapping algorithm of joinmap 4.1 ( van ooijen 2011 ) was used to produce two maps for each parental genotype ( one per cross ) , according to a two - way pseudotestcross mapping strategy ( grattapaglia and sederoff 1994 ) , using testcross markers ( i.e. , segregating in a 1:1 mendelian ratio ) only . the genetic maps were then combined into sex - averaged maps ( corsica , landes and morocco , see supplementary fig . s1 , supplementary material online ) with the function combine groups for map integration of joinmap 4.1 . more details on the construction of the maps can be found in lagraulet ( 2015 ) . the first population was a three - generation inbred pedigree consisting of an f2 population ( # 4 in supplementary fig . s1 , supplementary material online ) resulting from the selfing of an interprovenance tree ( landes corsica ) . the second population was a three - generation outbred pedigree ( g2 , # 5 ) resulting from a controlled cross of two intraprovenance hybrid trees ( landes landes ) . for the f2 population , two different sets of individuals were used to generate two maps ( f2_o and f2_n ) with the record algorithm ( van os et al . for the g2 population , one map for each parent ( g2 m and g2f ) was produced with the regression mapping algorithm of joinmap 4.1 ( van ooijen , 2011 ) . the f2_o , g2 m , and g2f maps included different marker types : aflp , single sequence repeat ( ssr ) , expressed sequence tag ( est ) , and snps from different arrays ( chancerel et al . 2011 , 2013 ) , whereas the f2_n map contained only snps from the 9k snp - array ( plomion et al . we made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map ( referred to as f2c by plomion et al . the last four maps were generated from two different f1 crosses : c14c15 ( # 6 in supplementary fig . s1 , supplementary material online ) and gal1056oria6 ( # 7 ) . from the initial parental maps of the c14c15 mapping population described by de miguel ( 2012 ) , we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . ( 2011 ) and 273 snps from a 1,536 snp - array ( sez - laguna et al . for both pedigrees , we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . the four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers ( ssrs , ests , sampls , and snps ) . for all maps , genetic distances in centimorgans ( cm ) were calculated with the kosambi mapping function ( kosambi 1943 ) . we carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family , for which sequence information was publicly available . eckert et al . ( 2010 ) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda ( accession number : the white spruce pedigree was an f1 full - sib family , whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . lind et al . ( 2014 ) provided the most saturated and gene - rich map to date for pic . this map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . a detailed list of mapping features for each component map included in this study is available in table 1 . a high - density linkage map for c. japonica was incorporated into this study , as a representative species from the cupressaceae family ( moriguchi et al . this map was constructed from an f1 full - sib family ( table 1 ) , with the regression mapping algorithm implemented in joinmap v 3.0 . the 17 maps described above were mostly constructed with snp markers ( 100% of the markers for picea sp . , 98% for p. pinaster , and 90% for p. taeda ) . the flanking sequences of each snp marker were compared with the most recent unigene sets available for each species to obtain the sequence of unigenes containing the mapped snps : canales et al . this comparison was carried out with the blastn tool ( the blast 1 step in fig . unigenes with a percentage identity exceeding 95% with mapped snp flanking sequences were retained for the next step . ( 2014 ) , pine v3 , was then used as the reference for the identification of homologous unigenes within pinaceae species . a second sequence comparison ( r - blast 2 in fig . 1a ) was performed , between the mapped unigenes of each species and the unigene sequences of pine v3 . for this interspecific comparison , only sequences with a reciprocal best hit with a percentage identity exceeding 85% , an e value below e , and an alignment of more than 200 bp were retained as homologous unigenes . homologous unigenes between different species were considered as orthologs if they were positioned in the same lg ( i.e. , syntenic unigenes ) . identified orthologous unigenes were used as anchor markers to construct a composite linkage map for each genus ( pinus and picea ) , as a preliminary step in the construction of a composite map for the pinaceae family including both genera . we used the r package lpmerge ( endelman and plomion 2014 ) to integrate component linkage maps into a composite map without the use of segregation data . lpmerge assessed the goodness of fit of the composite map by calculating a root mean squared error ( rmse ) per lg , by comparing the position ( in cm ) of all markers on the composite map with that on the component maps . we calculated this metric for different maximum interval sizes ( parameter k in the algorithm ) , ranging from 1 to 10 . the value of k minimizing the mean rmse per lg was selected for construction of the composite map . this method was used for the construction of all the composite species maps reported here . further details about the production of each composite map are described below . before integrating the 14 base genetic linkage maps into a single composite map , we established consensus maps ( supplementary fig . s1 , supplementary material online ) based on markers common to different accessions across pedigrees ( corsica , landes , morocco genotypes for pedigrees # 1 , # 2 , and # 3 , respectively ) , or accessions within pedigrees ( coca and gxo for pedigrees # 6 and # 7 , respectively ) , or based on the merging of different data sets of the same pedigree ( f2 for pedigree # 4 ) . this process , designed to increase the number of markers common to component maps , was facilitated by the use of the same 12k ( chancerel et al . 2013 ) and 9k ( plomion et al . 2015 ) snp - arrays for some pedigrees . the snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . this step , which was essential for the use of lpmerge ( i.e. , same marker name for orthologous markers ) , also made it possible to check the collinearity between maps . thus , nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps ( supplementary table s2 , supplementary material online ) . finally , lpmerge was used to create the composite map for p. pinaster . given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps , each component map was assigned the same weight in lpmerge . the snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster ( canales et al . we established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . this process was designed to increase the number of markers common to the component maps for each genus ( pinus and picea ) . lpmerge was used to build these two composite maps , following the same procedure as described for p. pinaster . we discarded nonsyntenic unigenes , except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map , from the construction of composite linkage maps . noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . abies ( lind et al . 2014 ) , when the map for this species was compared with that for pic . abies lg7 ( renamed lg2 after comparison with the p. pinaster reference map ) was reconstructed from genotyping data provided as supplementary material by lind et al . ( 2014 ) , using the same parameters described in lind s article and the same mapping software ( joinmap v4.1 ) . two markers with a log10 ( p ) > 1 that produced a large number of double recombinants were excluded from this lg map . we were thus able to map 16 additional markers , and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . the available map for c. japonica consisted of 77% of snp markers ( moriguchi et al . sequences of unigenes containing the mapped snps were retrieved from the unigene set developed by ueno et al . 2014 ) mapped in the pinaceae composite map using tblastx ( blast 3 , fig . different e - value cutoff for homologous unigenes identification between pinaceae and c. japonica was tested : lower than 1e and 1e . selected homologs from the pinaceae and c. japonica linkage maps were used for comparative mapping . we established orthologous blocks within lgs where several homologous unigenes were shared between both families . different thresholds were also tested to consider an orthologous block within an lg : blocks with at least four and six shared unigenes . the most parsimonious evolutionary model between pinaceae and cupressaceae considering the existence of the identified cars was proposed . circular genetic maps used in interfamily comparative mapping were drawn with circos software ( krzywinski et al . the following terms were used to describe the different kinds of genetic maps used in this study , as suggested by hudson et al . ( 2012 ) : 1 ) sex - averaged map : a consensus map for both parents of a pedigree ; 2 ) consensus map : an integrated map based on segregation data from individual component maps ; 3 ) composite map : an integrated map of different individual component maps built by a marker - merging method ; and 4 ) component map : each of the maps used in the construction of a composite map . the graphics and the representations of genetic maps were produced with r 3.1.0 ( r core team 2014 ) . we used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . the first six maps were obtained from three controlled crosses ( pedigrees # 1 , # 2 , and # 3 in supplementary fig . s1 , supplementary material online ) between three different genotypes : corsica landes ( cl ) , morocco landes ( ml ) , and corsica morocco ( cm ) . in total , 106 , 117 , and 94 full - sibs the regression mapping algorithm of joinmap 4.1 ( van ooijen 2011 ) was used to produce two maps for each parental genotype ( one per cross ) , according to a two - way pseudotestcross mapping strategy ( grattapaglia and sederoff 1994 ) , using testcross markers ( i.e. , segregating in a 1:1 mendelian ratio ) only . the genetic maps were then combined into sex - averaged maps ( corsica , landes and morocco , see supplementary fig . s1 , supplementary material online ) with the function combine groups for map integration of joinmap 4.1 . more details on the construction of the maps can be found in lagraulet ( 2015 ) . the first population was a three - generation inbred pedigree consisting of an f2 population ( # 4 in supplementary fig . s1 , supplementary material online ) resulting from the selfing of an interprovenance tree ( landes corsica ) . the second population was a three - generation outbred pedigree ( g2 , # 5 ) resulting from a controlled cross of two intraprovenance hybrid trees ( landes landes ) . for the f2 population , two different sets of individuals were used to generate two maps ( f2_o and f2_n ) with the record algorithm ( van os et al . for the g2 population , one map for each parent ( g2 m and g2f ) was produced with the regression mapping algorithm of joinmap 4.1 ( van ooijen , 2011 ) . the f2_o , g2 m , and g2f maps included different marker types : aflp , single sequence repeat ( ssr ) , expressed sequence tag ( est ) , and snps from different arrays ( chancerel et al . 2011 , 2013 ) , whereas the f2_n map contained only snps from the 9k snp - array ( plomion et al . we made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map ( referred to as f2c by plomion et al . the last four maps were generated from two different f1 crosses : c14c15 ( # 6 in supplementary fig . s1 , supplementary material online ) and gal1056oria6 ( # 7 ) . from the initial parental maps of the c14c15 mapping population described by de miguel ( 2012 ) , we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . ( 2011 ) and 273 snps from a 1,536 snp - array ( sez - laguna et al . for both pedigrees , we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . the four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers ( ssrs , ests , sampls , and snps ) . for all maps , genetic distances in centimorgans ( cm ) were calculated with the kosambi mapping function ( kosambi 1943 ) . we carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family , for which sequence information was publicly available . eckert et al . ( 2010 ) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda ( accession number : the white spruce pedigree was an f1 full - sib family , whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . lind et al . ( 2014 ) provided the most saturated and gene - rich map to date for pic . this map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . a detailed list of mapping features for each component map included in this study is available in table 1 . a high - density linkage map for c. japonica was incorporated into this study , as a representative species from the cupressaceae family ( moriguchi et al . this map was constructed from an f1 full - sib family ( table 1 ) , with the regression mapping algorithm implemented in joinmap v 3.0 . we used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . the first six maps were obtained from three controlled crosses ( pedigrees # 1 , # 2 , and # 3 in supplementary fig . s1 , supplementary material online ) between three different genotypes : corsica landes ( cl ) , morocco landes ( ml ) , and corsica morocco ( cm ) . in total , 106 , 117 , and 94 full - sibs the regression mapping algorithm of joinmap 4.1 ( van ooijen 2011 ) was used to produce two maps for each parental genotype ( one per cross ) , according to a two - way pseudotestcross mapping strategy ( grattapaglia and sederoff 1994 ) , using testcross markers ( i.e. , segregating in a 1:1 mendelian ratio ) only . the genetic maps were then combined into sex - averaged maps ( corsica , landes and morocco , see supplementary fig . s1 , supplementary material online ) with the function combine groups for map integration of joinmap 4.1 . more details on the construction of the maps can be found in lagraulet ( 2015 ) . the first population was a three - generation inbred pedigree consisting of an f2 population ( # 4 in supplementary fig . s1 , supplementary material online ) resulting from the selfing of an interprovenance tree ( landes corsica ) . the second population was a three - generation outbred pedigree ( g2 , # 5 ) resulting from a controlled cross of two intraprovenance hybrid trees ( landes landes ) . for the f2 population , two different sets of individuals were used to generate two maps ( f2_o and f2_n ) with the record algorithm ( van os et al . for the g2 population , one map for each parent ( g2 m and g2f ) was produced with the regression mapping algorithm of joinmap 4.1 ( van ooijen , 2011 ) . the f2_o , g2 m , and g2f maps included different marker types : aflp , single sequence repeat ( ssr ) , expressed sequence tag ( est ) , and snps from different arrays ( chancerel et al . 2011 , 2013 ) , whereas the f2_n map contained only snps from the 9k snp - array ( plomion et al . we made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map ( referred to as f2c by plomion et al . the last four maps were generated from two different f1 crosses : c14c15 ( # 6 in supplementary fig . s1 , supplementary material online ) and gal1056oria6 ( # 7 ) . from the initial parental maps of the c14c15 mapping population described by de miguel ( 2012 ) , we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . ( 2011 ) and 273 snps from a 1,536 snp - array ( sez - laguna et al . for both pedigrees , we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . the four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers ( ssrs , ests , sampls , and snps ) . for all maps , genetic distances in centimorgans ( cm ) were calculated with the kosambi mapping function ( kosambi 1943 ) . we carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family , for which sequence information was publicly available . eckert et al . ( 2010 ) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda ( accession number : tg091 , http://dendrome.ucdavis.edu/cmap/ , last accessed may 21 , 2015 ) . the map provided by pavy et al . the white spruce pedigree was an f1 full - sib family , whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . lind et al . ( 2014 ) provided the most saturated and gene - rich map to date for pic . this map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . a detailed list of mapping features for each component map included in this study is available in table 1 . a high - density linkage map for c. japonica was incorporated into this study , as a representative species from the cupressaceae family ( moriguchi et al . this map was constructed from an f1 full - sib family ( table 1 ) , with the regression mapping algorithm implemented in joinmap v 3.0 . the 17 maps described above were mostly constructed with snp markers ( 100% of the markers for picea the flanking sequences of each snp marker were compared with the most recent unigene sets available for each species to obtain the sequence of unigenes containing the mapped snps : canales et al . this comparison was carried out with the blastn tool ( the blast 1 step in fig . unigenes with a percentage identity exceeding 95% with mapped snp flanking sequences were retained for the next step . ( 2014 ) , pine v3 , was then used as the reference for the identification of homologous unigenes within pinaceae species . a second sequence comparison ( r - blast 2 in fig . 1a ) was performed , between the mapped unigenes of each species and the unigene sequences of pine v3 . for this interspecific comparison , only sequences with a reciprocal best hit with a percentage identity exceeding 85% , an e value below e , and an alignment of more than 200 bp were retained as homologous unigenes . homologous unigenes between different species were considered as orthologs if they were positioned in the same lg ( i.e. , syntenic unigenes ) . identified orthologous unigenes were used as anchor markers to construct a composite linkage map for each genus ( pinus and picea ) , as a preliminary step in the construction of a composite map for the pinaceae family including both genera . we used the r package lpmerge ( endelman and plomion 2014 ) to integrate component linkage maps into a composite map without the use of segregation data . lpmerge assessed the goodness of fit of the composite map by calculating a root mean squared error ( rmse ) per lg , by comparing the position ( in cm ) of all markers on the composite map with that on the component maps . we calculated this metric for different maximum interval sizes ( parameter k in the algorithm ) , ranging from 1 to 10 . the value of k minimizing the mean rmse per lg was selected for construction of the composite map . this method was used for the construction of all the composite species maps reported here . further details about the production of each composite map are described below . before integrating the 14 base genetic linkage maps into a single composite map , we established consensus maps ( supplementary fig . s1 , supplementary material online ) based on markers common to different accessions across pedigrees ( corsica , landes , morocco genotypes for pedigrees # 1 , # 2 , and # 3 , respectively ) , or accessions within pedigrees ( coca and gxo for pedigrees # 6 and # 7 , respectively ) , or based on the merging of different data sets of the same pedigree ( f2 for pedigree # 4 ) . this process , designed to increase the number of markers common to component maps , was facilitated by the use of the same 12k ( chancerel et al . 2013 ) and 9k ( plomion et al . 2015 ) snp - arrays for some pedigrees . the snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . this step , which was essential for the use of lpmerge ( i.e. , same marker name for orthologous markers ) , also made it possible to check the collinearity between maps . thus , nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps ( supplementary table s2 , supplementary material online ) . given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps , each component map was assigned the same weight in lpmerge . the snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster ( canales et al . we established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . this process was designed to increase the number of markers common to the component maps for each genus ( pinus and picea ) . lpmerge was used to build these two composite maps , following the same procedure as described for p. pinaster . we discarded nonsyntenic unigenes , except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map , from the construction of composite linkage maps . noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . abies ( lind et al . 2014 ) , when the map for this species was compared with that for pic . abies lg7 ( renamed lg2 after comparison with the p. pinaster reference map ) was reconstructed from genotyping data provided as supplementary material by lind et al . ( 2014 ) , using the same parameters described in lind s article and the same mapping software ( joinmap v4.1 ) . two markers with a log10 ( p ) > 1 that produced a large number of double recombinants were excluded from this lg map . we were thus able to map 16 additional markers , and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . before integrating the 14 base genetic linkage maps into a single composite map , we established consensus maps ( supplementary fig . s1 , supplementary material online ) based on markers common to different accessions across pedigrees ( corsica , landes , morocco genotypes for pedigrees # 1 , # 2 , and # 3 , respectively ) , or accessions within pedigrees ( coca and gxo for pedigrees # 6 and # 7 , respectively ) , or based on the merging of different data sets of the same pedigree ( f2 for pedigree # 4 ) . this process , designed to increase the number of markers common to component maps , was facilitated by the use of the same 12k ( chancerel et al . 2013 ) and 9k ( plomion et al . 2015 ) snp - arrays for some pedigrees . the snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . this step , which was essential for the use of lpmerge ( i.e. , same marker name for orthologous markers ) , also made it possible to check the collinearity between maps . thus , nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps ( supplementary table s2 , supplementary material online ) . given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps , each component map was assigned the same weight in lpmerge . the snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster ( canales et al . we established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . this process was designed to increase the number of markers common to the component maps for each genus ( pinus and picea ) . lpmerge was used to build these two composite maps , following the same procedure as described for p. pinaster . we discarded nonsyntenic unigenes , except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map , from the construction of composite linkage maps . noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . abies ( lind et al . 2014 ) , when the map for this species was compared with that for pic . abies lg7 ( renamed lg2 after comparison with the p. pinaster reference map ) was reconstructed from genotyping data provided as supplementary material by lind et al . ( 2014 ) , using the same parameters described in lind s article and the same mapping software ( joinmap v4.1 ) . two markers with a log10 ( p ) > 1 that produced a large number of double recombinants were excluded from this lg map . we were thus able to map 16 additional markers , and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . the available map for c. japonica consisted of 77% of snp markers ( moriguchi et al . 2012 ) . sequences of unigenes containing the mapped snps were retrieved from the unigene set developed by ueno et al . 2014 ) mapped in the pinaceae composite map using tblastx ( blast 3 , fig . different e - value cutoff for homologous unigenes identification between pinaceae and c. japonica was tested : lower than 1e and 1e . selected homologs from the pinaceae and c. japonica linkage maps were used for comparative mapping . we established orthologous blocks within lgs where several homologous unigenes were shared between both families . different thresholds were also tested to consider an orthologous block within an lg : blocks with at least four and six shared unigenes . the most parsimonious evolutionary model between pinaceae and cupressaceae considering the existence of the identified cars was proposed . circular genetic maps used in interfamily comparative mapping were drawn with circos software ( krzywinski et al . all the linkage maps described here are available from the pinus portal ( a european genetic and genomic resource for pinus ) through the pinusmap application ( https://w3.pierroton.inra.fr/pinusportal/index.php ) . accession numbers for marker sequences used in this study are available in supplementary table s3 , supplementary material online . supplementary figures s1s5 and tables s1s3 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .

although recent advances have been gained on genome evolution in angiosperm lineages , virtually nothing is known about karyotype evolution in the other group of seed plants , the gymnosperms . here , we used high - density gene - based linkage mapping to compare the karyotype structure of two families of conifers ( the most abundant group of gymnosperms ) separated around 290 ma : pinaceae and cupressaceae . we propose for the first time a model based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of pinaceae ( with n = 12 ) and cupressaceae ( with n = 11 ) . the considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the pinaceae . it also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes .

bluetongue viruses , pathogenic only to domestic cattle and wild ruminants ( barratt - boyes and maclachlan , 1995 ; tsuboi and imada , 1997 ) , are non - pathogenic to humans ( xiao et al . , 2004 ; hu et al . , 2008 ) and thus , humans do not have pre - existing antibodies to btvs ( jeggo et al . , 1983 ; barratt - boyes and maclachlan , 1995 ; dong et al . , 1998 ) not only can btv be grown easily in vitro ( jameson and grossberg , 1981 ; du , 1985 ; samal et al . , 1985 ; sundin and mecham , 1989 ; dong et al . , 1998 ; wechsler and mcholland , 1998 ; chen et al . , 1999 ; prasad et al . , 1999 ; xue , 2001 ; lei et al . , 2004 ; xiao et al . , 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) , it has powerful oncolytic activity against many different in vitro cultured cancer cell lines ( chen et al . 2008 ) , such as a498 human kidney cells ( atcc : htb-4 ) , hep - g2 human liver cells ( atcc : hb-8065 ) , a549 human lung epithelial cells ( atcc : ccl-185 ) , baby hamster kidney cells ( bhk-21 ) , monkey kidney ( vero ) cells ( ramig et al . , 1989 ) , mouse fibroblast cells ( nih 3t3 ) , spc - a-1 cells ( lei et al . , 2004 ) , ma 782 cells ( liang et al . , 2006 ) , and in mouse models . no normal human cells such as the primary human embryo lung fibroblast ( hel ) and primary murine embryos fibroblast ( mef ) have ever been successfully infected by btv ( dong et al . , 1998 ; chen et al . , 1999 ; , 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) and thus , humans are normally sero - negative . madin darby canine kidney ( mdck ) which is sensitive to reovirus apoptosis also proved resistant to btv infection except in high moi ( unpublished data ) . btvs can also infect cultured insect cells ( king and alders , 1985 ; samal et al . , 1987 ; guirakhoo et al . , 1995 ; mullens et al . , 1995 ; mertens et al . , 1996 ; xu et al . , 1997 ; tan et al . , 2001 ) . in the last decade , our lab and collaborators have used both cell culture systems ( dong et al . , 1998 ; xiao et al . , 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) and mice ( manuscript in preparation ) to investigate and determine the oncolytic activities and capacities of btvs and their selective degradation of human cancer cells as potential treatment of human cancers . human cancer cell lines such as hep-3b and hep - g2 , a549 , a498 , spc - a-1 , and ma 782 cells infected with btv 10 , 11 , 13 , 17 , and hbc3 at various mois exhibited the following easily detected characteristics 36 h post - infection ( pi ) but not in hel and mef cells in multiple determinations ( xiao et al . , 2004 ; hu et al . , 2008 ) . briefly : between 70 and 90% cytopathic effects ( cpe ) were readily observed and detected along with cellular apoptosis and necrosis in many human cancer cell lines . various degrees of morphological and cellular damage could be visualized by either light , confocal , or electron microscopy ( em ) . cellular dilated endoplasmic reticulum , nuclear chromatin condensation , and cytoplasmic shrinking were readily detected and easily observed in em . btv viral genomic ds - rna fragments and viral mrnas could be found in btv-10 , 13 , 17 , and hbc3 infected human cancer cells , but not in normal hel or mef cells by gel electrophoresis , elisa , rip , western blots , pcr , and qrt - pcr ( tiwari et al . , 2000 ) . the survival rate of several human cancer cell lines was low if high moi was used . primary hel and mef cells survived very well for 69 days independent of the moi used for testing . human cancer cells infected with btvs had more efficient viral replication and provided higher viral progeny yields . selective cell cycle arrest at sub - g1 peak in several human cancer cells infected with btv-10 ( hu et al . , 2008 ) and hbc3 similar to the reovirus - induced g2/m cell cycle arrest ( poggioli et al . , 2000 after tumor cells were grown in culture and then injected as xenografts into mice ( eight mice / sample ) , tumors developed and all these mice died in less than 8 days ( preliminary data ) . when 0.1 ml of btvs with different mois in sterile phosphate - buffered saline ( pbs ) was injected at three different sites of each xenograft , the sizes of tumors in mice were reduced 6085% . most mice with the reduced tumors survived more than 35 days before they were sacrificed for tissue and organ samples ( kidney , lung , liver , heart . etc . thus , btvs selective cytotoxic effects to human tumor cells in vitro and partially in vivo have been clearly indicated and demonstrated . further in vivo research and clinical studies are now in progress to document and investigate this unique oncolyitc potential of btvs which will provide us better and safer application in humans either in monotherapy or combination therapy in the near future . bluetongue viruses administration has been shown to cause cell death and apoptosis in several cell lines as well as in several human tumor cell lines ( wechsler and mcholland , 1998 ; xiao et al . , 2004 ; liang et al . , 2006 ; hu et al . , 2008 ) . however , in experiments with cultured human cancer cells , the cells detached and died progressively throughout btv infection . all btv genes are transcribed and btv proteins are expressed during infection . in the analysis of secreted analyte expression , cytokine and chemokine levels from mock- and btv-17 infected cells were determined using multiplex immunoassay kits and reagents supplied from quansys biosciences ( logan , ut , usa ) , according to the supplier 's protocol . twenty - five analytes were tested and these included il-1a , il-1b , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12p40 , il-12p70 , il-13 , ifn , tnf , tnf , tgf - b1 , mcp-1 , rantes , ang2 , hgf , timp , tpo , vegf , pdgf , fgf - basic , and crp ( manuscript in preparation ) . of the 25 analytes tested for during infection , only seven were found to be expressed from five cell lines : a498 human kidney cells , hep - g2 human liver cells , a549 human lung epithelial cells ( atcc : htb-4 , hb-8065 , ccl-185 , respectively ) , bhk-21 cells , or vero cells . of these seven analytes , five were found to be consistently elevated upon btv infection , with the expression pattern consistent among cell lines . these are il-6 , il-8 , mcp-1 , rantes , and fgf. the levels of vegf and timp were similar in control and btv - infected cells . however , a498 kidney cells responded to infection with the greatest secretion of cytokines , followed by a549 lung cells while hep - g2 liver cells secreted the least amount of cytokines . polyinosine : polycytosine ( poly i : c ) is a synthetic ds - rna that has been used to assess the role of ds - rna segments of many ds - rna viruses in vitro since it can induce differential interferon and cytokine production ( der et al . , 1998 ; , 2003 ; chiang et al . , 2006 ) . however , poly i : c alone did not cause cell death or have a significant impact on cytokine or chemokine expression in btv - infected human cancer cell lines . only il-6 and il-8 were found to be significantly elevated during poly i : c treatment . increases in il-6 and il-8 were observed only in kidney cells ( a498 and vero ) and were most prevalent in samples treated with 1 g / ml the other cytokines mcp-1 , rantes , and fgf were all at expression levels comparable to the placebo treated cells . with the identification of some of the antigenic epitopes of various btv proteins ( wang et al . gould et al . , 1988a , 1994 ; grieder and schultz , 1989 ; geysen , 1990 ; li and yang , 1990 , 1992 ; marshall and roy , 1990 ; mecham and jochim , 1990 ; eaton et al . , 1991 ; rossitto and maclachlan , 1992 ; du plessis et al . , 1994 , 1995 ; , 1997 ; nagesha et al . , 2001 ) , btv immunity has only been studied partially in the last two decades because of lack of supporting funding and reliable immune assays ( jeggo and wardley , 1982a , b ; gibbs et al . , 1983 ; jeggo et al . , 1983 ; campbell and grubman , 1985 ; stott et al . , 1985 ; fukusho et al . , 1987a ; marshall and roy , 1990 ; martyn et al . , 1991 ; ; lin and zhou , 1996 ; wade - evans et al . , 1996 ; odeon et al . , 1999 ; prasad and minakshi , 1999 ; demaula et al . , 2000 ; roy , 2003 ) . with the recent development of more reliable immune assays and microarrays , we have found that btv can induce inflammatory cytokine expression in immortalized human kidney , lung , and liver cells and subsequently kill the infected cell with cell line - dependent severity . analysis of results from the cytokine profiles and expression levels in these five cell lines indicated that human tumor cells and tumor reduction might be a result of the combined effects of direct viral induced cell death and recruitment of t - lymphocytes ( jeggo and wardley , 1982a , b ; jones et al . , 1996 , 1997 ) to the tumor through elevated cytokine / chemokine production ( shmulevitz et al . , 2005 , 2010 ) . very high levels of pro - inflammatory cytokines were secreted from btv - infected cells and cytokine levels secreted from each of the seven cell types were directly proportional to the measured death of infected cells . thus , we hypothesize that the cell death and cytokine expression are related and may be caused by shared signaling pathways or events . if these same events occurred in human tumors , btv would be expected to reduce tumor size in infected patients by directly causing infected human cancer cell death and by inducing cytokine expression from the tumor that would lead to lymphocyte recruitment to the site of the tumor . thus , btv has the therapeutic potential in cancer because of its selective ability to cause preferential apoptosis of various human cancer cell lines and tumors . ideal oncolytic viruses should have essential survival data , systemic effect , resist immune system clearance , targeted delivery , and distribution , be highly mobile for intravenous spread , travel to and kill distant metastases , have a high rate of replication to stay ahead of immune system clearance , tolerable toxicity , and side effects , etc . systemic delivery makes sense in cancer where primary tumors and metastases are dispersed . however , systemic approach in cancer is a big problem because we do not have a good target specific delivery device that would facilitate uptake specifically for certain cells . if this can be achieved , then it would be better than the current and conventional therapies . however , it is very difficult to get enough control patients in clinical trials and to get biopsies from different tissues , organs and tumors after the oncolytic virus treatments to determine the efficacy or survival of most oncolytic viruses . oncovirologists consider oncolytic virus as a dark horse treatment but not a dead one since they know that many anti - cancer agents appear to be actively effective in lab cultures and animal models but failed in human trials . many oncolytic viruses ( norman and lee , 2005 ; alain et al . , 2006 , 2007 ; kim et al . , 2006 ; shen and nemunaitis , 2006 ; burroughs et al . , 2007 ; qi et al . , 2007 , 2009 ; ou and yen , 2010 ) that are currently in phase i / ii / iii human clinical trial status include adenovirus , vaccinia virus , coxsackie virus , reovirus , newcastle disease virus , herpes simplex virus 1 , measles virus , and seneca valley virus ( burroughs et al . , 2007 ) . in 2007 , the only oncolytic virus approved for phase iii clinical trials in china was the adenovirus ( h101 ) with e1b deletion produced jointly by the sunway biotech of china and onyx pharmaceuticals of emeryville , ca , usa ( privilege communication ) . since mid-2005 to the end of 2010 , several other oncolytic viruses have also been approved for phase iii clinical trials primarily outside us as briefly described below . thus , cancer therapy with oncolytic viruses has survived and revived , and this unique approach is poised for a comeback ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al . the results of the last 1015 years of investigation generated by researchers have increased interest in oncolytic viruses . investigators have generated and obtained some genetically modified viruses with greater oncolytic potentials ( aghi and martuza , 2005 ; norman and lee , 2005 ; alain et al . a significant effort is underway to understand the genetic and mechanistic basis of cancers , selective degradation of cancer cells by these oncolytic viruses , and their effects on downstream cellular events and pathways ( strong et al . vorburger et al . , 2004 ; aghi and martuza , 2005 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; liu , 2006 ; kim et al . , 2007 ; liu et al . , 2007 ; 2009 ; hill and lee , 2010 ; ou and yen , 2010 ; thirukkumaran et al . , 2010 ) . increasing attention is being paid to protein families like proteases , caspases , and kinases which play critical roles in cancer . current therapeutic approaches for human cancer include the regimen of selective surgery , high dose but cytotoxic chemotherapy with two to three drugs and radiation . these approaches are not very effective to combat the increasing numbers of human cancers . using oncolytic viruses generates a paradigm shift from current cancer treatment using chemotherapy and radiation which typically destroy both normal and cancer tumor cells . as we learn more about cancer biology and oncolytic viruses , new strategies for treating cancers are rapidly evolving . uses of oncolytic virus alone or with synergistic anti - cancer drugs or irradiation in human clinical trials are currently under consideration or in progress . reovirus leads the field of oncolytic viruses and positive clinical trials are very encouraging ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al . 2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) . other companies and institutes are not far behind . in mid - may 2010 , researchers of the university of helsinki and oncos therapeutics have treated 200 patients with their modified gmcsf armed oncolytic adenovirus which exhibited anti - tumor immunity that recruited natural killer cells and induced tumor - specific cytotoxic t - cells with strong efficacy and safety . the media report indicated that its clinical benefit is about 45% according to the recist criteria with no grade four to five side effects detected . this represents an extension of their initial investigation in 2008 . with these successful applications and those from other oncolytic viruses , we can now add the simple but oncolytic btv as a key component to the armamentarium of oncologists to combat and destroy the insidious cancer cells since btvs have similar characteristics and additional subtle advantages over reovirus and other oncolytic viruses . bluetongue viruses and reovirus are more advantageous than the genetically modified oncolytic dna viruses since the integration of viral ds - rna genome into the host cell genome have not been shown or reported . since humans are sero - negative to btvs , potential future use of btv to degrade human cancer by direct intratumoral injection to degrade the injected tumors will not be inhibited by pre - existing immune components within human cancer cells . . the current mechanisms of oncolytic reovirus toward human cancer cells have recently been summarized ( strong et al . , 1998 ; norman et al . , 2004 ; norman and lee , 2005 ; shmulevitz et al . , 2005 , 2010 ; alain et al . , 2006 ; kim et al . , 2009 ; hill and lee , 2010 ; thirukkumaran et al . , 2010 ) . genetically modified reovirus serotype 3 , but not types 1 and 2 , is a proprietary product called reolysin produced by reolytics biotech , inc . data for cancer treatment with this agent from the last decade in animal models and lately in phase i / ii human clinical trials have shown encouraging indications of its potential oncolytic activities in cancer cells bearing an activated ras pathway . phase iii human clinical trials have been approved and initiated in 2009 . however , the pre - existing anti - reovirus sera in humans worldwide can trigger greater immune responses against this virus , reducing its efficacy and effectively shutting down its potential oncolytic treatment . uses of oncolytic btv have greater advantages over reovirus since no anti - btv serum is detected in most people globally . even though btv injected into tumors can activate dendritic cells for early detection of viral infection , it will take 1014 days before adaptive immune responses can be generated against btvs which can eradicate xenografted tumor cells in mice in less than 10 days ( preliminary data ) . oncolytics biotech at calgary , ab , canada showed the uses of reovirus ( reolysin ) plus cisplatin , a standard chemotherapy agent in both cultured melanoma cells and a mouse model . the combined uses of reolysin and cisplatin were much better than either of these two agents alone since they appeared to work synergistically ( abstract of the 4th international symposium on the treatment of cancers with oncolytic viruses at scottsdale , arizona march 1517 , 2007 ) . furthermore , they had also showed that simultaneous and combined uses of reolysin and gemcitabine could completely eradicate the transplanted colon cancer in four of the five mice ( abstract of the american association for cancer research ( aacr ) annual meeting in april 2007 ) . since mid - april , 2007 , oncolytics biotech has demonstrated some success in multiple phase ii trials with several medical centers in the uk and us , administering intravenous reolysin to patients with sarcomas that have metastasized to the lung . multiple clinical trials for other tumors with the us national cancer institute ( nci ) were conducted in late 2007 . this will help to determine whether reolysin can migrate systemically to metastasized tumor sites and exhibit its oncolytic activity . oncolytics biotech has expended more than 65 million dollars and a decade of genetic modifications of reolysin before human clinical trials were approved . similar and greater expenditures have also been made by other companies and institutes in the last 1215 years to develop different oncolytic viruses with tremendous personnel efforts before some clinical trials are approved and conducted . we strongly believe that btv is the front runner of the next generation oncolytic viruses since it needs no genetic modifications and attenuation , and carries no payloads . it can grow fast and spread easily within many human cancer cells . since there is no pre - existing neutralizing antibody against btv in humans , they should be undetected nor immunologically inhibited in the initial injection alone or with anti - cancer agents . in addition to the comparison of the oncolysis of human cancer cells by reovirus and btv as shown in table 2 , btv has additional advantages . briefly : since btv does not infect normal human cells , we do not have to suppress the immune system before use btv is very stable since it can be stored at ph 8.28.4 at 4c for over 20 years with only 510% loss of infectivity ( unpublished data ) . thus , it can be shipped by air - mail globally without significant loss of its infectivity and oncolytic activities ( unpublished data ) . 2-d gel electrophoresis and the 3-d assay ( lam et al . , 2007 ) for the measurement of btv - induced oncolysis are currently under optimization and further development and adaptation . btv infection of many cultured human tumor cells has led to the production of many inflammatory cytokines such as tnf - alpha and il-6 which strongly activate the kinase activity of p38 mapk and erk1/2 . these cytokines also assist the subsequent activation of p38-dependent mapk and pkr pathways which can bring about the necrosis and apoptosis of cancer cells . this strongly suggests that btv can prime and direct the immune system of cancer patients against certain solid tumors such as breast and prostate cancer . repeat screening of over 500 human cytokines using biotin label - based cytokine antibody array is currently in progress and data are under comparative analysis . construction and rescue of recombinant infectious btv ( boyce and roy , 2007 ) expressing the enhanced green fluorescent protein ( egfp ) , using a protocol similar to one recently published by our lab for human parainfluenza virus type 3 ( roth et al . comparison of oncolytic reovirus and bluetongue virus . * reovirus references : strong et al . an activated ras / ralgef / p38 pathway is potentially involved with the permissiveness of host cells to reovirus infection ( norman et al . , 2004 ; shmulevitz et al . , 2005 ; alain et al . , 2007 ; kim et al . , 2007 ; marcato et al . , 2007 ) . it has been shown that the c - k - ras gene inside the lung carcinoma cell line a549 we used has mutation at codon 12 ( valenzuela and groffen , 1986 ) . since the ras / ralgef / p38 signal pathway inside the a549 cell has been activated , this might allow btv to degrade human tumor cells efficiently similar to reovirus ( stott et al . , 1985 ; shmulevitz et al . , 2005 , 2010 ; kim et al . , 2007 ; marcato et al . , 2007 we have further hypothesized that the genetic alterations / mutations of different human cancer cells , such as signals within a ras - activated pathway , play a key role in the susceptibility of different oncolytic viruses in addition to specific viral receptors which might be present in human cancer cells but not in normal human cells . identification of btv receptor(s ) on the surface of human cancer cells is also currently in progress . comparison of oncolytic potentials between reovirus and btv is summarized in table 2 . with regard to the potential cytotoxic effect mechanisms of btvs , the gp5 protein of btv identified by our lab ( li and yang , 1992 ; yang and li , 1992 , 1993 ; yang et al . , 1992a ) has recently been shown to induce or trigger apoptosis in mammalian cells ( mortola et al . , 2004 ) . furthermore , the non - structural btv protein , ns-3 , has been shown to be a viroporin , the polymerization of which forms pores on the surface of btv - infected cells , leading to severe cellular leakage , and potential cell death ( han and harty , 2004 ) . we hypothesize that these two btv proteins and other(s ) are involved with the cytotoxic effect of human cancer cells after btv infection . individual btv gene silencing with rnai to determine the contribution of each of the btv gene products to onclysis of human cancer cells are also in progress . the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s . however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal . toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) . tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens . thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells . to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai . human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) . preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny . cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines . experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated . several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) . the protein toll ( means weird in german ) was discovered in the fruit fly drosophila melanogaster in 1980s . however , if mutations in toll occurred in the fruit fly , the infection by aspergillus fumigates ( a fungus ) is lethal . toll - related molecules involved with human innate immunity and coined as toll - like receptors ( tlrs ) were then discovered in mid-1990s by ruslan medzhitov and charles janeway ( medzhitov and janeway , 2002 ; norman et al . , 2004 ) . tlrs are molecular pattern recognition receptors and can recognize molecules that are broadly shared by many pathogens . thus , tlrs pathways play a key role in both innate immunity and normal immune physiology that can sense different pathogens and cancer cells . to determine whether mutated tlrs are involved with the recognition of btvs , we have initiated the silencing of each 1 of the 10 known human tlrs tlr- 110 using rnai . human cancer cell lines , a498 and hek 239 , are transfected with dna plasmids containing sirna sequences against human tlrs ( invivogen , ca , usa ) . preliminary data indicated that endosome - located tlr-3 which specifically recognizes ds - rna is potentially involved since cells transfected with this tlr-3 rnai plasmid produced substantially less btv 17 and 10 progeny . cells transfected with tlr-2 , 4 , 7 , 8 , and 9 rnai plasmids produced slightly less viral progeny in these two human cancer cell lines . experiments are now underway to determine what types of mutation are in the tlr-3 in these four human cancer cell lines and this might provide a potential explanation of why normal human cells are not infected by btv because the regular tlr-3 has not been modified nor mutated . several signal pathways related to tlrs are currently under screening using pathway screen arrays ( qiagen / sabiosciences ) . human cancers can be divided into two major types , 90 and 10% of which are solid ( carcinoma and sarcoma ) or liquid ( leukemia ) tumors , respectively . recent data and epidemiological analysis of many tumors reveal that most fda - approved cancer therapeutic drugs can only provide small incremental improvement and survival to cancer patients . companies that make many unrealistic claims for human cancer cures in scientific tabloid media do so strictly for financial reasons since these expensive cancer drugs can generate great revenues via physicians who make drug recommendation and to whom drug companies provide substantial and financial however , the 90% of deaths of cancer patients are primarily caused by those cancer stem cells that have metastasized to other regions to form metastatic tumors . humans are sero - negative toward btvs since btvs do not infect normal human cells . infection and selective degradation of several human cancer cell lines , but not normal cells , in vitro by five us btv serotypes have been investigated by our lab for the last few years when btv oncolytic potential was discovered . we have also recently found that direct single injection of oncolytic btv into the human tumor xenografts in mice , have led to cytotoxic destruction and preferential lysis of cancer cells within the xenograft but not in normal healthy human or mouse cells ( preliminary data ) . thus , oncolytic btvs are safer and more effective than most current cancer treatments . however , we believe that oncolytic btv is not a stand - alone therapeutic and that simultaneous combination of btv with either radiation or chemotherapy can obliterate the tumors more rapidly and extensively . cancer patients should have fewer side effects since small dosage radiation and anti - cancer drugs are used for this combined treatment . related to the oncolytic btvs , the following questions have been frequently asked : is widely curing cancer by oncolytic viruses possible or just hype ? big is the gap between in vitro / in vivo studies and human application ? is oncolytic btv a vital alternative for cancer therapy ? what are the pathways btv used to carry out this task ? overall , we believe that the oncolytic ability of btv represents a significant potential to selectively degrade human cancer cells alone and would even be greater when combined with simultaneous anti - cancer drug and radiation treatment . our group and collaborators are currently engaged in several types of investigations with these goals : ( 1 ) study the mechanistic basis of lung , kidney , and liver cancer destruction by btvs , ( 2 ) examine and determine biological profiles of three types of solid tumors after btv infection , ( 3 ) correlate the responses of these human cancer cells to changes at the cellular dna , rna and protein level , and ( 4 ) construction and rescue of an infectious recombinant btv 17 expressing the enhanced egfp using the method that we have developed for the human parainfluenza virus type 3 ( roth et al . , 2010 ) with btv ds - rna fragments ( kowalik et al . , 1990b ; boyce and roy , 2007 ) , the success of which can further reveal the oncolytic pathways used by btvs . taking the next step in the investigation of btv oncolysis is very challenging since no one can individually excel in all the technical , scientific , and clinical avenues involved in this endeavor . our ongoing in vitro work with different human cancer cell lines will assist us in elucidating the potential mechanisms of the btv oncolytic activities . no cpe was detected nor viral progeny obtained when several primary human cell lines were infected by btv . when xenografts implanted in mice were injected directly with btv , no pathological damages were detected in any tissue or organs 721 days post - infection ( preliminary data ) . thus , the potential risks of btv oncotherapy is substantially lower than risks associated with other oncolytic viruses . however , this author sincerely hopes that expert advice and suggestions will be forthcoming from different champions after they have read this brief review . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

humans are sero - negative toward bluetongue viruses ( btvs ) since btvs do not infect normal human cells . infection and selective degradation of several human cancer cell lines but not normal ones by five us btv serotypes have been investigated . we determined the susceptibilities of many normal and human cancer cells to btv infections and made comparative kinetic analyses of their cytopathic effects , survival rates , ultra - structural changes , cellular apoptosis and necrosis , cell cycle arrest , cytokine profiles , viral genome , mrnas , and progeny titers . the wild - type us btvs , without any genetic modifications , could preferentially infect and degrade several types of human cancer cells but not normal cells . their selective and preferential btv - degradation of human cancer cells is viral dose dependent , leading to effective viral replication , and induced apoptosis . xenograft tumors in mice were substantially reduced by a single intratumoral btv injection in initial in vivo experiments . thus , wild - type btvs , without genetic modifications , have oncolytic potentials . they represent an attractive , next generation of oncolytic viral approach for potential human cancer therapy combined with current anti - cancer agents and irradiation .

leprosy is a well - known chronic infectious disease that is caused by mycobacterium leprae ( m. leprae ) . the incidence of leprosy has declined worldwide after the introduction of multidrug therapy ( mdt ) by the world health organization ( who ) in 1982;1 however , it is still a public health problem in many countries , which have a high rate of endemic infection.2 this was an indicator to modify or change who mdt , aiming at a more effective , safe , compliant , and shorter - duration regimen that is free from the fear of the emergence of resistant lepra bacilli.3 advances in , and deep understanding of , immunology , pathogenesis , and genetics of leprosy could improve the ability to fight against this potentially devastating infectious disease and may lead to the development of better protocols for treatment and prevention of the disease.4 the status of dermal vasculature was considered an important underlying pathogenic factor in leprosy , and the study of dermal vascular changes in leprosy was a matter of interest in previous reports.5 it was observed that there is narrowing , tortuosity , dilatation , or occlusion of the skin s blood vessels of lepromatous leprosy patients , especially those located in the extremities.6 it was proposed that there are two microvascular architectural patterns observed in cutaneous lesions of leprosy : a dense and tortuous mesh of microvessels among the granulomatous infiltrate in lepromatous leprosy and a microvessel network restricted to the periphery of the granulomas in tuberculoid leprosy.7 moreover , the proliferation and migration of endothelial cells result in the formation of new blood vessels from pre - existing vessels , a process that is known as angiogenesis , which has also been studied in leprosy . it was proposed that there is a significant correlation between angiogenesis and bacterial load in cutaneous lesions of lepromatous patients.8 in this study we assessed angiogenesis in lepromatous leprosy patients before and after treatment with mdt alone and in combination with minocycline , a drug known to have an antiangiogenic effect . moreover , we correlated the changes in angiogenesis with the changes in bacterial density ( bd ) in the same cutaneous lesions . a multicenter study was carried out on 40 lepromatous leprosy patients recruited from the outpatient clinics of the dermatology departments in al - hussein , tanta , and al- zahraa university hospitals , egypt . all the patients included were newly diagnosed , and none of them had received any treatment for leprosy prior to the commencement of the study . the diagnosis of leprosy was based on the clinical presentation of bilateral , symmetrical , nonscaly erythematous nodular skin lesions , which was confirmed by the detection of m. leprae in direct nasal or slit skin smear . exclusion criteria included pregnancy , lactation , known hypersensitivity to any of the therapeutic drugs , and serious illness or bad general condition . written consent was signed by each patient after full explanation of the nature of the study , expected benefits of treatment , and possible side effects . group a ( n = 20 ) received who mdt in the form of monthly observed doses of rifampicin ( 600 mg ) and clofazimine ( 300 mg ) and an unobserved daily dose of dapsone ( 100 mg ) and clofazimine ( 50 mg ) . group b ( n = 20 ) received who mdt therapy in addition to a monthly observed dose of minocycline ( 100 mg ) . a 4 mm punch biopsy was taken from each patient before treatment and 6 months after . the first biopsy was usually taken from the largest nodular lesion , and the second biopsy was taken from nearly the same site . from each specimen , one for routine hematoxylin and eosin staining for diagnosis and evaluation of the granulomatous reaction , one for modified ziehl - neelsen stain ( wade - fite method ) to record the bd ( assessed by bacterial index [ bi ] ) , and two for immunohistochemical staining with anti - cd31 and anti - cd34 monoclonal antibodies to assess microvascular density ( mvd ) . immunohistochemical staining was performed using an avidin - biotin peroxidase complex method on formalin - fixed , paraffin - embedded tissue sections,9 using a 1/50 dilution of monoclonal cd31 and cd34 antibodies ( dako , glostrup , denmark ) . briefly , tissue sections were mounted on 3-aminopropyl - triethoxysilane - coated slides and dried overnight at room temperature . subsequently , they were dewaxed in xylene and rehydrated in graded ethanol . after being rinsed with phosphate buffered saline , they were immersed in 0.01 mol / l citric acid titrated to ph 6.0 and heated twice for 10 minutes in a microwave oven . diaminobenzidine was used as a chromagen , and the slides were counterstained with mayer s hematoxylin . microvessel density was assessed by immunostaining for cd31 and cd34 according to weidner.10 areas with higher vascularity ( so - called hot - spots ) were located at low magnification ( 40 ) and then were counted at 400 magnification . each positive endothelial cell or group of cells in contact with a spot was counted as an individual vessel . the mean vessel count from three fields was used as cd34 microvessel density or cd31 microvessel density . the assessment of both bd and mvd was performed only in the dermal granuloma without assessment of the stromal changes . bd in each wade - fite stained specimen was assessed by bi following ridley s logarithmic scale ( from 1 to 6 ) . the number of solid staining ( viable ) lepra bacilli was visually calculated in ten different high power fields ( hpf ) , and the mean standard deviation ( sd ) was recorded . for quantitative data , the mean and sd were calculated . follow - up of the patients was done monthly for observation of the monthly doses of treatment and recording of any side effects , complications , or lepra reaction . we reported two patients in group a who developed erythema nodosum leprosum during the first month of treatment ( 2 and 3 weeks , respectively ) . the treatment was continued in both patients with the addition of oral corticosteroids ( 40 mg daily for 15 days then gradually withdrawn ) . all the patients included were newly diagnosed , and none of them had received any treatment for leprosy prior to the commencement of the study . the diagnosis of leprosy was based on the clinical presentation of bilateral , symmetrical , nonscaly erythematous nodular skin lesions , which was confirmed by the detection of m. leprae in direct nasal or slit skin smear . exclusion criteria included pregnancy , lactation , known hypersensitivity to any of the therapeutic drugs , and serious illness or bad general condition . written consent was signed by each patient after full explanation of the nature of the study , expected benefits of treatment , and possible side effects . group a ( n = 20 ) received who mdt in the form of monthly observed doses of rifampicin ( 600 mg ) and clofazimine ( 300 mg ) and an unobserved daily dose of dapsone ( 100 mg ) and clofazimine ( 50 mg ) . group b ( n = 20 ) received who mdt therapy in addition to a monthly observed dose of minocycline ( 100 mg ) . a 4 mm punch biopsy was taken from each patient before treatment and 6 months after . the first biopsy was usually taken from the largest nodular lesion , and the second biopsy was taken from nearly the same site . from each specimen , one for routine hematoxylin and eosin staining for diagnosis and evaluation of the granulomatous reaction , one for modified ziehl - neelsen stain ( wade - fite method ) to record the bd ( assessed by bacterial index [ bi ] ) , and two for immunohistochemical staining with anti - cd31 and anti - cd34 monoclonal antibodies to assess microvascular density ( mvd ) . immunohistochemical staining was performed using an avidin - biotin peroxidase complex method on formalin - fixed , paraffin - embedded tissue sections,9 using a 1/50 dilution of monoclonal cd31 and cd34 antibodies ( dako , glostrup , denmark ) . briefly , tissue sections were mounted on 3-aminopropyl - triethoxysilane - coated slides and dried overnight at room temperature . subsequently , they were dewaxed in xylene and rehydrated in graded ethanol . after being rinsed with phosphate buffered saline , they were immersed in 0.01 mol / l citric acid titrated to ph 6.0 and heated twice for 10 minutes in a microwave oven . diaminobenzidine was used as a chromagen , and the slides were counterstained with mayer s hematoxylin . microvessel density was assessed by immunostaining for cd31 and cd34 according to weidner.10 areas with higher vascularity ( so - called hot - spots ) were located at low magnification ( 40 ) and then were counted at 400 magnification . each positive endothelial cell or group of cells in contact with a spot was counted as an individual vessel . the mean vessel count from three fields was used as cd34 microvessel density or cd31 microvessel density . the assessment of both bd and mvd was performed only in the dermal granuloma without assessment of the stromal changes . bd in each wade - fite stained specimen was assessed by bi following ridley s logarithmic scale ( from 1 to 6 ) . the number of solid staining ( viable ) lepra bacilli was visually calculated in ten different high power fields ( hpf ) , and the mean standard deviation ( sd ) was recorded . follow - up of the patients was done monthly for observation of the monthly doses of treatment and recording of any side effects , complications , or lepra reaction . we reported two patients in group a who developed erythema nodosum leprosum during the first month of treatment ( 2 and 3 weeks , respectively ) . the treatment was continued in both patients with the addition of oral corticosteroids ( 40 mg daily for 15 days then gradually withdrawn ) . both patients showed significant relief of symptoms within 3 weeks . in group b , no significant side effects or complications were encountered during the treatment period . their ages ranged from 25 years to 53 years with a mean of 32 4.5 years . their ages ranged from 28 years to 49 years with a mean of 34 2.5 years . the duration of the disease ranged from 3 weeks to 11 months with a mean of 14 3 weeks . in 29 patients ( 72.5% ) skin lesions were located on the trunk and extremities , whereas in eleven patients ( 27.5% ) the lesions were distributed mainly on the extremities . facial involvement was reported in 13 patients ( 32.5% ) and nerve thickening in 27 patients ( 67.5% ) . m. leprae were detected in 16 patients ( 40% ) by nasal smear and in 24 patients ( 60% ) by slit skin smears . in group a , the mvd ( number of vessels [ v]/hpf ) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment , whereas after treatment the mvd decreased to 16.5 2.7 ( ranged from 13 v / hpf to 19 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 1 ) . in group b , the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment , which decreased to 7.6 1.9 after treatment ( ranged from 5 v / hpf to 10 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 2 ) . in group a , the mean mvd significantly decreased from 42.2 3.1 before treatment ( figure 1a ) to 18.8 2.4 after treatment ( figure 1b ) ( table 1 ) . in group b , the mean mvd significantly decreased from 43.7 2.3 before treatment ( figure 2a ) to 11.5 1.6 after treatment ( figure 2b ) ( table 2 ) . there was a statistically significant higher reduction ( p < 0.001 ) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a ( table 3 ) . in group a , bi ranged from 4 ( 10100 bacilli / hpf ) to 6 ( > 1000 bacilli / hpf ) with a mean of 5.1 0.4 before treatment , which was significantly decreased to 2.3 0.4 after treatment ( table 1 ) . in group b , the mean bi significantly decreased from 4.9 0.3 before treatment ( figure 3a ) to 1.4 0.2 after treatment ( figure 3b ) ( table 2 ) . there was a statistically significant higher reduction ( p < 0.001 ) in bd in group b compared with in group a ( table 3 ) . their ages ranged from 25 years to 53 years with a mean of 32 4.5 years . their ages ranged from 28 years to 49 years with a mean of 34 2.5 years . the duration of the disease ranged from 3 weeks to 11 months with a mean of 14 3 weeks . in 29 patients ( 72.5% ) skin lesions were located on the trunk and extremities , whereas in eleven patients ( 27.5% ) the lesions were distributed mainly on the extremities . facial involvement was reported in 13 patients ( 32.5% ) and nerve thickening in 27 patients ( 67.5% ) . m. leprae were detected in 16 patients ( 40% ) by nasal smear and in 24 patients ( 60% ) by slit skin smears . in group a , the mvd ( number of vessels [ v]/hpf ) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment , whereas after treatment the mvd decreased to 16.5 2.7 ( ranged from 13 v / hpf to 19 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 1 ) . in group b , the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment , which decreased to 7.6 1.9 after treatment ( ranged from 5 v / hpf to 10 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 2 ) . in group a , the mean mvd significantly decreased from 42.2 3.1 before treatment ( figure 1a ) to 18.8 2.4 after treatment ( figure 1b ) ( table 1 ) . in group b , the mean mvd significantly decreased from 43.7 2.3 before treatment ( figure 2a ) to 11.5 1.6 after treatment ( figure 2b ) ( table 2 ) . there was a statistically significant higher reduction ( p < 0.001 ) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a ( table 3 ) . in group a , the mvd ( number of vessels [ v]/hpf ) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment , whereas after treatment the mvd decreased to 16.5 2.7 ( ranged from 13 v / hpf to 19 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 1 ) . in group b , the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment , which decreased to 7.6 1.9 after treatment ( ranged from 5 v / hpf to 10 v / hpf ) . this reduction was statistically highly significant ( p < 0.001 ) ( table 2 ) . in group a , the mean mvd significantly decreased from 42.2 3.1 before treatment ( figure 1a ) to 18.8 2.4 after treatment ( figure 1b ) ( table 1 ) . in group b , the mean mvd significantly decreased from 43.7 2.3 before treatment ( figure 2a ) to 11.5 1.6 after treatment ( figure 2b ) ( table 2 ) . there was a statistically significant higher reduction ( p < 0.001 ) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a ( table 3 ) . in group a , bi ranged from 4 ( 10100 bacilli / hpf ) to 6 ( > 1000 bacilli / hpf ) with a mean of 5.1 0.4 before treatment , which was significantly decreased to 2.3 0.4 after treatment ( table 1 ) . in group b , the mean bi significantly decreased from 4.9 0.3 before treatment ( figure 3a ) to 1.4 0.2 after treatment ( figure 3b ) ( table 2 ) . there was a statistically significant higher reduction ( p < 0.001 ) in bd in group b compared with in group a ( table 3 ) . the major role of angiogenesis was reported in relation to neoplastic conditions and malignancies that need an ongoing blood supply to grow and expand.11 abnormal angiogenesis was also implicated in other conditions such as rheumatoid arthritis , inflammation , and degenerative eye conditions , in addition to many biological processes such as development , reproduction , and wound repair.12 in dermatological disorders , angiogenesis was implicated mainly in psoriasis,13 and to a lesser extent in skin aging and photoaging.14 drugs that inhibit angiogenesis were used primarily in the treatment of malignancy . the role of these antiangiogenic or antivascular therapies in the treatment of cancer became important after the establishment of the relationship between angiogenesis and tumor growth.15 inhibition of angiogenesis growth factors and transfer of antiangiogenesis genes were proposed as basic mechanisms of antiangiogenic drugs.16 the target receptors of antiangiogenic drugs include ptdins- 4,5-p2 that regulate vessel stability,17 vascular endothelial growth factor receptor key proteins,18 and cap43 calcium - inducible genes.19 there are certain drugs which have an antiangiogenic effect and are used in the treatment of some skin diseases , such as chloroquine , which was suggested to have a beneficial effect in the treatment of discoid lupus erythematosus due to its antiangiogenic properties.20 thalidomide also has an antiangiogenic effect and is currently used in the treatment of different dermatological conditions including lepra reaction,21 sclerodermatous cutaneous reaction of graft versus host disease,22 and jessner s lymphocytic infiltration of the skin.23 recently , rifampicin , which is a component of mdt for leprosy , was suggested to have promising antiangiogenic effects that may enable its use as an effective antitumor agent.24 minocycline was also reported as one of the drugs that inhibit angiogenesis,25 and it is known to have an important role in the treatment of leprosy as a main component of rifampicin , ofloxacin , and minocycline ( rom ) therapy.26 however , the mechanism of action of both drugs in the treatment of leprosy remains unclear . is it due to their antimicrobial effect only or due also to their antiangiogenic effect ? to our knowledge , no previous studies have evaluated the efficacy of these antiangiogenic drugs on angiogenesis in cutaneous lesions of lepromatous leprosy patients , but a few studies monitor angiogenesis in the spectrum of leprosy . bhandarkar et al8 studied angiogenesis in 32 leprosy patients using cd31 as a marker for microvessel density . the mvd of lepromatous leprosy lesions was ( 44.0 9.8 v / hpf ) . we found a lower mvd value with a cd31 marker in lepromatous leprosy ( 39.1 3.1 in group a and 38.3 2.5 in group b ) , and with a cd34 marker we found a higher value ( 42.2 3.1 in group a and 43.7 2.3 in group b ) , which was close to that encountered by bhandarkar et al . this may suggest the benefit of using more than one marker ( cd31 , cd34 ) in the assessment of angiogenesis . kim et al27 also found a significant increase of mvd in lepromatous leprosy lesions compared with in normal skin , with an overall increase in the mean number of vessels from tuberculoid leprosy through borderline ( tuberculoid and lepromatous ) to lepromatous leprosy lesions . the mvd values in lepromatous leprosy were higher ( 104.40 27.71 ) compared with our results . this can be explained by the use of a different vascular marker ( factor viii - related antigen ) or a different counting system . these studies suggested that angiogenesis could be evaluated by using different vascular markers . in the current study we preferred to use two markers ( cd31 and cd34 ) in the assessment of angiogenesis for proper evaluation of mvd and to minimize false positive results . both cd31 and cd34 are known as effective markers in identifying blood vessels and were proposed also to be good prognostic markers of neoangiogenesis.28,29 the significant reduction of mvd and bd in group a , which was treated with mdt , may prove the possible anti - angiogenic effect of rifampicin in addition to its antimicrobial effect . rifampicin was suggested to exert its antiangiogenic effect through inhibition of the expression of angiogenesis - associated genes with downregulation of proangiogenic genes and inhibition of microvascular endothelial cell proliferation.24 in a manner comparable with the action of endostatin , the endogenous angiogenesis inhibitor is known to downregulate a variety of growth and angiogenesis - related genes in a wide range of endothelial lineage cells.30 in group b , minocycline 100 mg , which is known to have antiangiogenic properties , was added to mdt as a monthly dose , aiming to potentiate the antiangiogenic efficacy of the classic mdt regimen . although minocycline has different mechanisms of action from its antimicrobial properties , such as inhibition of collagenase activity,31 and its anti - inflammatory effect through reduction of cytokines and pro - inflammatory protein expression,32 it also possesses inhibitory properties of angiogenesis.25 this antiangiogenic effect of minocycline may contribute to the direct inhibition of matrix metalloproteinase activity and also to the inhibition of vascular endothelial growth factor - induced smooth muscle cell migration.33 in the present work the reduction of mvd was significantly higher in group b than in group a using both markers . these results may suggest the presence of a synergistic effect of both minocycline and rifampicin as antiangiogenic drugs in addition to their antimicrobial effect . moreover , the absence of any side effects or lepra reaction in group b compared with in group a may add a new benefit for the combination of mdt with minocycline . another important finding in this work is the significantly higher reduction in bi in group b compared with in group a. this may suggest that the addition of minocycline to the mdt can cause a more rapid elimination of lepra bacilli from cutaneous lesions of lepromatous leprosy patients . this rapid elimination of lepra bacilli from cutaneous lesions and also from mucous membranes is helpful in decreasing the transmission of the disease and maintaining the standard target of elimination of leprosy . to our knowledge , this is the first work that assesses angiogenesis in leprosy patients after treatment with antiangiogenic drugs . the synergistic antiangiogenic effect of both minocycline and rifampicin was found to be promising and effective in the treatment of leprosy . moreover , this combination showed an advantage of rapid elimination of m. leprae from the cutaneous lesions of lepromatous leprosy patients and decreased the possibility of the occurrence of lepra reaction . we hope that this regimen will be considered in wide - scale studies , especially in endemic areas of leprosy .

backgroundangiogenesis was suggested to have a significant role in the pathogenesis of leprosy . however , the benefit of inhibiting angiogenesis in lepromatous leprosy patients has not previously been studied . the purpose of this study was to evaluate angiogenesis in leprosy patients before and after treatment with multidrug therapy ( mdt ) with and without minocycline.methodsa total of 40 patients with lepromatous leprosy were enrolled in this study . they were categorized into two equal groups ( a and b ) , each formed of 20 patients . group a received world health organization mdt , and group b received mdt combined with minocycline , which has a known antiangiogenic effect . microvascular density ( mvd ) in dermal granuloma was evaluated in both groups by immunostaining with cd31 and cd34 markers before and after 6 months of treatment.resultswith cd31 immunostaining , the mean mvd in group a significantly decreased from 39.1 3.1 vessels ( v)/high power field ( hpf ) to 16.5 2.7 v / hpf , and in group b it significantly decreased from 38.3 2.5 v / hpf to 7.6 1.9 v / hpf . cd34 immunostaining also showed a significant decrease of mvd from 42.2 3.1 v / hpf to 18.8 2.4 v / hpf in group a , and in group b it significantly decreased from 43.7 2.3 v / hpf to 11.5 1.6 v / hpf . the reduction of mvd was significantly higher in group b compared with in group a ( p < 0.0001 ) . moreover , there was a significant reduction in bacterial density ( assessed by bacterial index ) in the cutaneous lesions of in group b ( decreased from 4.9 0.3 to 1.4 0.2 ) compared with in group a ( decreased from 5.1 0.4 to 2.3 0.4).conclusionthe synergistic effect of mdt and minocycline seems to be promising in the treatment of lepromatous leprosy . it significantly reduces angiogenesis and rapidly eliminates lepra bacilli from the skin that enables a rapid control and elimination of the disease .

the original technique of staining the anterior capsule with trypan blue ( tb ) in eyes with no red reflex involves the injection of the dye with a cannula after filling the anterior chamber with an air bubble.1 the stability of the anterior chamber can be compromised by air escaping when the cannula is introduced and the dye is injected . moreover , the superficial tension of air bubble and capillary action between tb and the metal cannula can force the dye to build up within the aqueous meniscus surrounding the air bubble , making selective staining of the capsule difficult . several techniques using viscoelastic substances as an alternative to air have been presented to increase the stability of the anterior chamber.25 however , staining under viscoelastic material can be time - consuming and expensive , as it requires the mechanical spreading of tb onto the anterior capsule and the replacement of the viscous solution when an excessive diffusion of dye affects its transparency . we present an under - air staining technique of the anterior capsule using one drop of tb injected with a 30 g needle through the peripheral cornea . our procedure prevents the exit of air during the injection of the dye , resulting in increased stability of the anterior chamber . moreover , it allows selective staining of the capsule using the needle and avoids an excessive buildup of tb in the anterior chamber . connect a 30 g needle to a 2.25 ml luer lock syringe containing trypan blue 0.06% ( visionblue ; dutch ophthalmic research center international , zuidland , netherlands ) . make a standard side - port incision with a 15 blade . use a 25 g cannula connected to a 5 ml syringe to inject a big air bubble in the anterior chamber . introduce the 30 g needle bevel - up in the anterior chamber through the peripheral cornea between the side - port incision and the site chosen for the corneal tunnel . push the needle forward in the air bubble right between the anterior capsule and posterior corneal surface until the tip reaches the center of the pupil . unlike what occurs using a cannula , the air can not escape from the anterior chamber . inject gently , allowing the tb volume to grow within the needle bevel ( figure 1 ) . rotate the syringe 90 , letting the drop fall on the anterior capsule ( figure 2 ) . the needle tip allows precise control on the injected dye , similarly to a dropper . if the staining is not sufficient , it is possible to inject one more drop of tb on the unstained area . once the needle is removed , the perforation will close spontaneously in a few seconds , and if necessary an absorbent stick can be used to stop tiny leakages of air and/ or dye . inject viscoelastic substance in the anterior chamber through the side - port incision , allowing the air and the leftover dye to exit the eye . tb is the most used dye to perform capsulorhexis in eyes with no red reflex.6 the main staining techniques involve tb injection under air,1 under viscoelastic material,25 or directly under aqueous humor . an air bubble within the anterior chamber reduces the contact between the dye and the corneal endothelium and allows the delivery of undiluted tb onto the anterior capsule . however , the air bubble makes the anterior chamber unstable and prone to collapse when the cannula is introduced to inject the dye . in such an event , the anterior chamber has to be reinflated , thus increasing the duration of surgery . moreover , sudden reductions in volume of the anterior chamber can cause narrowing of the pupil and damage to the corneal endothelium . to prevent the air bubble from escaping the anterior chamber , it has been proposed that a small amount of high - density viscoelastic material be placed near the side - port incision.7 in our technique , we inject the dye in the anterior chamber , introducing a 30 g needle through the peripheral cornea . in this way , the air can not escape from the side - port incision , as often occurs when a cannula is used . once the needle is pulled out , the corneal perforation spontaneously closes in a few seconds . when the dye is injected with a blunt cannula through the side - port incision , the superficial tension of the air bubble spreads the dye into the thin liquid layer between the air and the anterior capsule.8 in this phase , a meniscus of tb forms between the metal cannula and the anterior capsule due to the capillary action and adhesive forces . to obtain the right staining , the anterior capsule must be swept thoroughly with the cannula , allowing the meniscus of tb to contact the largest possible area . if too much dye is injected , it can migrate back between the cannula and the capsule , escaping from the paracentesis , or it could build up in the aqueous meniscus surrounding the air bubble . to avoid this , toprak et al8 modified a cannula by bending it to increase the surface contact between the dye and the capsule . in our procedure , we use a very sharp - tipped needle , which reduces the adhesive forces with tb and allows the drop of dye to fall easily over the anterior capsule . many authors have described alternative staining techniques based on viscoelastic substances instead of air.25 these techniques have the advantage of making the anterior chamber more stable and of better dosing the amount of injected dye . however , when the anterior chamber is filled with viscoelastic substance , it is difficult to deliver the dye onto the capsule . the surgeon has to mechanically spread the dye on the largest possible surface of the capsule , and at times the dyed viscoelastic material or pockets of dye can affect the visibility of the capsule beneath . to avoid these problems , special cannulae have been proposed.2,4 rarely , an exchange of the viscoelastic material is needed to restore the transparency before the capsulorhexis . we injected tb with a 30 g needle in seven consecutive patients with white cataract and normal anterior chamber depth . in this small group of patients , we have not observed intra- or postoperative complications . after surgery , all patients showed marked improvement in their visual acuity . in six patients , we performed a regular capsulorhexis after injecting only one drop of dye . in a single patient , one more drop of tb had to be injected to build up the staining of the capsule . an additional corneal access and a sharp needle in the anterior chamber are the major disadvantages of our technique . in eyes with shallow anterior chamber or with excessive posterior pressure , the use of the 30 g needle technique could be dangerous and should be avoided . in conclusion , in a small series of seven patients , the staining of the anterior capsule using tb under air with a 30 g needle allowed for enhanced stability of the anterior chamber and the use of the smallest quantity of dye .

the original technique of staining the anterior capsule of the lens with trypan blue involves the injection of an air bubble in the anterior chamber . a drawback of this technique is the possible instability of the anterior chamber caused by the sudden exit of air when the dye is injected with the cannula through the side - port incision . other staining techniques that use viscoelastic substances to increase the stability of the anterior chamber and to dose the injected dye have been described . the authors present an under - air staining technique of the anterior capsule using one drop of trypan blue injected with a 30 g needle through the peripheral cornea . this procedure prevents the air bubble from escaping the anterior chamber and allows fast and selective staining of the capsule .

it is estimated that in the year 2005 , 63,210 new cases of bc will be diagnosed in the united states alone . approximately one fifth of these patients will undergo radical cystectomy ( rc ) and urinary diversion ( ud ) . rc has become an established standard of care for patients with muscle invasive bc in the united states . despite our better understanding of pelvic anatomy and improved surgical techniques , therefore , a need persists for technological advances that would minimize intraoperative blood loss and decrease perioperative complications in patients undergoing rc . the most notable of recent advances in surgical techniques is the increased application of minimally invasive laparoscopic surgery in the management of urological disorders . the established advantages of laparoscopic surgery include decreased pain , shorter hospital stay , and decreased intraoperative blood loss compared with these things in conventional open surgery . there is still room for improvement , however , as the current laparoscopic technology is limited due to the lack of 3-dimensional visualization and poor ergonomics . recently , the da vinci surgical robotic system ( dsrs ) has been added to the armamentarium of minimally invasive surgery . the dsrs adds the much needed 3-dimensional vision , 6 degrees of freedom of movement , and improved ergonomics to laparoscopic techniques . the dsrs is being increasingly used to perform complex urological procedures including radical prostatectomy , radical cystectomy , and urinary diversions . menon et al first reported the use of the dsrs to perform rc both in men and women with decreased blood loss and operative time less than 4 hours . however , most of the published studies are limited to a small cohort of patients without comparison with the conventional open method ( om ) . to establish the efficacy of robotic radical cystectomy , we compared the early perioperative outcomes of patients undergoing rc by either om or the robotic method ( rm ) . this study includes a cohort of 37 consecutive patients undergoing rc by om or rm over the same time period by a single surgeon , thereby minimizing the influence of selection bias and multiple surgeons on perioperative outcome . all relevant clinical information on patients undergoing robotic urological procedures at our institution is maintained in a prospectively established microsoft access database . an institutional review board ( irb ) exempt status was obtained because all patient identifiers are deleted after pertinent clinical information is obtained . this study consists of 37 consecutive patients undergoing open and robotic rc since the approval of dsrs for human use in the year 2000 . the procedures were carried out at 2 teaching institutions , a university hospital and veterans administration medical center , by a single surgeon . because the dsrs was available only at the university hospital , the option to undergo rc by rm was offered to patients at the university site with the following exclusion criteria : morbid obesity ( generally body mass index>35 ) , prior pelvic radiation , or significant medical comorbidities including pulmonary obstructive airway disease . variables analyzed included age , sex , body mass index ( bmi ) , operative time , postoperative analgesic use , estimated blood loss ( ebl ) , blood transfusion rate , hospital stay , final pathology results , and early perioperative complications before discharge from the hospital . the patients , while under general anesthesia , were placed in the lithotomy and steep head down position with a nasogastric tube and urethral catheter in place . the procedure was carried out intraperitoneally following insufflation of the abdomen with co2 up to 15 mm hg pressure obtained using a veress needle . five ports were used including a 12-mm left paraumbilical camera port , two 8-mm robot working ports placed approximately 8 cm from the camera port on either side forming a 15 to 30 angle inferiorly and 2 additional 5-mm to 12-mm working ports ( one step port , u.s . operative steps in male patients , in order , included dissection of the vas deferens on both sides leading posterior to seminal vesicles , dissection of the prerectal space posterior to the prostate , bilateral ureteral mobilization from close to the lower pole of the ipsilateral kidney to the ureterovesical junction ( uvj ) before clipping , division close to the uvj and tagging using 12-inch long 2 0 absorbable sutures , incision of endopelvic fascia on both sides , securing the lateral pedicles to the bladder using endovascular staplers , division of the medial umbilical ligaments to enter the retropubic space , division of deep dorsal venous complex , completely freeing up the specimen before trapping in an endocatch ii bag ( u.s . surgical , norwalk , ct ) and removal of the intact specimen through an approximately 5-cm midline periumbilical or suprapubic incision . the rent in the anterior vaginal wall was closed primarily with dsrs using 2 0 vicryl sutures supported by greater omental mobilization done extracorporeally at the end of the procedure . a bilateral limited pelvic lymph node dissection was carried out intracorporeally , which included obturator and external iliac groups of lymph node . the wilcoxon rank sum test was used to compare distributions of continuous variables between the om and the rm . the patients , while under general anesthesia , were placed in the lithotomy and steep head down position with a nasogastric tube and urethral catheter in place . the procedure was carried out intraperitoneally following insufflation of the abdomen with co2 up to 15 mm hg pressure obtained using a veress needle . five ports were used including a 12-mm left paraumbilical camera port , two 8-mm robot working ports placed approximately 8 cm from the camera port on either side forming a 15 to 30 angle inferiorly and 2 additional 5-mm to 12-mm working ports ( one step port , u.s . operative steps in male patients , in order , included dissection of the vas deferens on both sides leading posterior to seminal vesicles , dissection of the prerectal space posterior to the prostate , bilateral ureteral mobilization from close to the lower pole of the ipsilateral kidney to the ureterovesical junction ( uvj ) before clipping , division close to the uvj and tagging using 12-inch long 2 0 absorbable sutures , incision of endopelvic fascia on both sides , securing the lateral pedicles to the bladder using endovascular staplers , division of the medial umbilical ligaments to enter the retropubic space , division of deep dorsal venous complex , completely freeing up the specimen before trapping in an endocatch ii bag ( u.s . surgical , norwalk , ct ) and removal of the intact specimen through an approximately 5-cm midline periumbilical or suprapubic incision . the rent in the anterior vaginal wall was closed primarily with dsrs using 2 0 vicryl sutures supported by greater omental mobilization done extracorporeally at the end of the procedure . a bilateral limited pelvic lymph node dissection was carried out intracorporeally , which included obturator and external iliac groups of lymph node . the wilcoxon rank sum test was used to compare distributions of continuous variables between the om and the rm . during our study period , 37 patients underwent rc , 24 ( 64.9% ) by om and 13 ( 29.7% ) by rm . , 6 patients underwent ileal conduit , 5 underwent ileal neobladders , and 2 underwent indiana pouch urinary diversions . in the open group , 16 patients underwent ileal conduits , 7 underwent ileal neobladders , and 1 underwent an indiana pouch urinary diversion . the bmi , age , sex , blood transfusion rate , and drop in hemoglobin were comparable between the 2 groups ( table 1 ) . median ebl and length of hospital stay for rm were significantly lower compared with that for om ( p=0.0002 and p=0.044 , respectively ) . operating time for rc and urinary diversion ( ud ) was significantly longer in the rm group ( p=0.0002 ) . there were 3 ( 12.5% ) positive margins in the om group and none in the rm group ( p=0.54 ) . four ( 16.7% ) perioperative complications occurred in the om group ; and 2 ( 15.4% ) occurred in the rm group , p=1.0 ( table 2 ) . one perioperative death caused by central venous line sepsis occurred in the om group . the incidence of organ - confined ( t2n0mx ) and nonorgan - confined disease ( t3 ) was 9 ( 37.5% ) and 15 ( 62.5% ) in the om group compared with 7 ( 53.8% ) and 6 ( 46.2% ) in the rm group ( p=0.49 ) . comparison of characteristics of patients undergoing radical cystectomy by open or robotic methods early perioperative morbidities following radical cystectomy by open or robotic methods because rc is usually performed in older patients with malignancy and associated nutritional deficiencies , rc is often associated with high postoperative complication rates . in spite of several modifications to the open surgical techniques , chang et al reported median blood loss of 600 ml in a series of over 300 patients undergoing rc . the increased use of minimally invasive surgical techniques to perform major urological operations , such as radical prostatectomy and nephrectomy , over the last decade has resulted in significantly decreased intraoperative blood loss compared with their respective open methods . several published reports establish the feasibility of safely performing robotic radical cystectomy ( table 3 ) . comparison of reports of early perioperative outcomes following robotic radical cystectomy plnd = pelvic lymph node dissection ; eplnd = extended pelvic lymph node dissection ; ic = ileal conduit ; onb = orthotopic neobladder ; rc(p ) = radical cystectomy and cystoprostatectomy ; ud = urinary diversion . our current study demonstrates a similar and significant decrease in ebl in the rm group compared with that in the om group ( p=0.0002 ) with a consequent decrease in blood transfusion rates . in this study , 18/24 ( 75% ) patients in the om group received blood transfusions compared with 7/13 ( 53.8% ) in the rm group despite the fact that only 4 patients in the rm group had blood loss over 500 ml . blood transfusion rates in this study are clearly much higher than rates of about 30% in other published studies . at our institution , blood transfusions are carried out without the benefit of well - established critical care pathways . in addition , because of the lack of prior experience in accurately estimating blood loss during robotic rc , patients received blood transfusions more readily , which accounts for the high blood transfusion rates in this series of patients . for purposes of calculation , because several patients had received intraoperative or immediate postoperative blood transfusions , the lack of significance of postoperative hemoglobin change may not accurately reflect intraoperative blood loss . the operative time for performing rc and urinary diversion was significantly longer in the rm group then in the om group . although several of the initial cases of rc by rm lasted for more then 4 hours , rc including pelvic lymph node dissection in the last 3 patients was completed consistently in less than 4 hours . the improving operative time corroborates the published conclusions in the literature that robotic surgery can be performed efficiently with operative time comparable to that of om with increasing experience . in this study , we do not report the operative time for rc and urinary diversions separately because of lack of such data for the om cases . in spite of the minimally invasive nature of rm , the difference in hospital stay in the rm group was only marginally better although statistically significant , median of 8 days ( range , 4 to 23 ) compared with 10 days ( range , 6 to 35 ) in the om group ( p=0.044 ) . following rc by rm , the specimen was removed through a small midline abdominal incision , and ud was performed extracorporeally in all patients . the patients hospital stay was directly related to the time required for return of the bowel function . therefore , irrespective of the method of rc the return of bowel function was comparable between the 2 groups resulting in a minimal difference in hospital stays . although we have previously reported on totally intracorporeal robot - assisted laparoscopic ileal conduit urinary diversions , we resisted our temptation to perform urinary diversions totally intracorporeally to limit operative time . the sample size is relatively small and the follow - up data are currently not mature enough to evaluate oncological outcomes . all 3 cases of positive margins occurred with om , 1 in a patient with locally advanced prostatic adenocarcinoma and the other 2 in patients with locally advanced pt3b and pt4a transitional cell carcinoma of the bladder . although not statistically significant , almost two thirds of patients undergoing rc by om had nonorgan - confined disease compared with about half in the rm group ( p=0.49 ) , which could possibly account for the increased positive margin rate in those undergoing om . alternatively , the dsrs provides excellent visualization by 10x magnification that facilitates meticulous dissection around the tumor , which could have contributed to decreased positive margin rates . although , a large randomized cohort of patients comparing rc by om or rm is necessary to resolve the issue , it nevertheless raises an interesting hypothesis that rc by rm may decrease positive surgical margins . overall perioperative complication rates were comparable between the 2 groups ; 4(16.7% ) and 2(15.4% ) in om and rm groups , respectively ( p=1.0 ) . one perioperative death in the om group resulted from central venous line sepsis . several studies evaluating outcomes of patients undergoing radical surgery for prostate and bladder cancer by open methods have demonstrated that the performing surgeon is an independent predictor of outcome . by comparing the perioperative outcomes using a contemporary cohort of patients undergoing rc by conventional om and rm performed by a single surgeon using our prospectively established lrusp institutional database , we minimized the influence of multiple surgeons performing the procedure at different institutions at different time periods on perioperative outcome . therefore , our data suggest that the same surgeon familiar with the surgical techniques can perform rc by either rm or om with similar efficacy . in this study , we have not compared postoperative analgesic use between the 2 methods because several patients in the om group received epidural analgesia compared with none in the rm group . although we focused on early perioperative complications , longer follow - up is necessary to evaluate long - term complications . although the demographics of both groups were comparable , morbidly obese patients were not offered rm as a treatment option , which may have biased the results of this study . furthermore , the operative time discussed in this study includes the time taken for performing urinary diversions , which limits our ability to comment specifically on the operative time for performing rc alone . nevertheless , our study establishes the feasibility of performing rc by rm with efficacy comparable to that of conventional om . a major impetus to explore the feasibility and efficacy of robotic rc is persistently high complication rates , such as increased intraoperative blood loss in patients undergoing rc by conventional om , even at centers where a high volume of procedures are performed . despite being a technically demanding operation , early reports of rc by rm , including the current study have demonstrated excellent perioperative outcomes including significantly decreased blood loss . moreover , robotic pelvic surgery including radical prostatectomy is being increasingly performed , and it may only be a matter of time before rm is more widely used to perform rc . therefore , pioneering work such as this study can contribute towards establishing the safety , efficacy , and technical standards for performing rc by rm . radical cystectomy ( rc ) with urinary diversion ( ud ) is relatively commonly performed and arguably one of the most complex of urological operations . rm is technically demanding , but clearly this procedure can be done with early perioperative results comparable to results of conventional open methods . currently , the operative time is longer with rm ; however , it is associated with decreased ebl and hospital stay . longer follow - up and a larger cohort of patients are required to establish oncological outcomes .

objective : we analyzed early perioperative outcomes following radical cystectomy by the robotic method compared with the conventional open method.methods:all relevant clinical information was entered in a microsoft access database and queried . p<0.05 were considered statistically significant.results:the study cohort comprised 37 consecutive patients undergoing radical cystectomy ; 24 ( 64.9% ) cases were performed by the conventional open method and 13 ( 29.7% ) by the robotic method . body mass index , age , sex , blood transfusion rate , and median decrease in hemoglobin were comparable between the 2 groups . the robotic method resulted in significantly lower median estimated blood loss , shorter hospital stay , and longer operating time compared with the open group ( p<0.05 ) . four ( 16.7% ) perioperative complications occurred in the open group compared with 2 ( 15.4% ) in the robotic group ( p=1.0 ) . the incidence of organ - confined ( t2n0mx ) disease was 9 ( 37.5% ) and 7 ( 53.8% ) in the open and robotic groups , respectively ( p=0.49).conclusions : radical cystectomy by the robotic method produces early perioperative results comparable to those of the open method . although intraoperative estimated blood loss and hospital stay were significantly lower in the robotic group , operative time was longer which likely reflects our early operative experience with radical cystectomy by the robotic method .

problems with shoe wear have long been recognized as an endemic issue among the geriatric population with a prevalence rate of nearly 80% . women in particular are more susceptible to these problems than men . individuals having foot pathology are often severely physically impaired , making it increasingly difficult to perform activities of daily living . consequentially , this leads to physical inactivity , which is cited as one of the first signs of deterioration and the overall decrease in quality of life . moreover , some studies have linked physical inactivity to suicide , depression , and increased risk of cardiovascular - related problems . individuals with diabetes , chronic disease , nondiabetic neuropathy , and inflammatory conditions are at a severe disadvantage . further complications of foot pathology , which include cellulitis , ulcerations , and difficulty in maintaining balance , have increased the risk of serious injuries and fractures from falls . studies have shown that adults older than 65 years fall at least once per year on average , some of which are attributed to generalized thinning of skin and fat pad atrophy . the increased risk of falls may highlight the need to promote preventative measures to combat this issue . unfortunately , little current data or research focuses on the role of footwear in the prevention of inactivity in the elderly patients . common foot pathologies like corns , hallux valgus ( bunions ) , and hammertoes have been known to increase plantar pressure , cause discomfort , pain , and swelling . multiple etiologies have been noted to cause foot problems , and studies have indicated ill - fitting shoes as one of the major underlying cause . a prevailing hypothesis is that wearing the appropriate footwear will improve factors such as plantar pressure , thereby mitigating pathology while facilitating balance . this review aims to form a general basis of understanding footwear - associated foot pathologies pertaining to the elderly patients . elderly individuals with preexisting clinical conditions such as diabetes , neuropathies , and musculoskeletal disorders are at a higher risk of developing foot problems when compared to normal , healthy individuals . furthermore , factors such as exercise and living conditions contribute to the development of foot problems . finally , the uneven pressure provoked by ill - fitting footwear has been documented as a key factor that can cause , accelerate , or exacerbate foot - related conditions . some common foot pathologies include hallux valgus ( bunions ) , corns ( callus ) , and hammertoes . callus formations , or corns , result from continuous pressure from tight footwear , leading to hyperkeratosis of the skin . if left unattended , callus formations can lead to ulcerations , which are particularly troublesome for diabetic patients . the hallux valgus deformity , or bunion , is also frequently linked to ill - fitting ( ie , tight , narrow ) footwear . it is caused by the lateral deviation of the great toe , which causes a valgus deformity in the first metatarsophalangeal ( mtp ) joint . lesser toe deformities , such as hammertoes , are also associated with ill - fitting footwear . these deformities of the second , third , fourth , and fifth toe are caused by contracture of the phalangeal joints . clinical presentations include the deformity accompanied by pain over the dorsal surface of the foot , which tends to worsen with ill - fitting footwear . one of the unifying elements to these 3 common forms of foot pathology is their link with ill - fitting footwear and their tendency to go unnoticed in the elderly population . nonoperative approaches have been employed as the first line of treatment for the most common foot pathologies . measures are taken to adjust or alleviate pressure from the affected area . as a result , foot inserts ( ie , dr scholls ) are also used to facilitate balance by improving arch support , which can decrease the width of the foot during weight bearing maneuvers . furthermore , met pads and splits operative management is considered only when conservative measures fail to relieve progressed , advanced - stage pathology . usually , symptomatic relief can be achieved with more accommodating shoe wear after the soft tissue envelope around the foot is stabilized . this process of relieving the pressure areas and encouraging natural healing of ulcerations or soft tissue problems to heal before using modified shoe wear and orthotics often involves close follow - up in the office or wound care center . special consideration is given to diabetic patients , who have a higher risk of developing ulcerations due to peripheral neuropathy . therefore , routine physical examination is encouraged to detect and address these issues early before they lead to progressive deterioration . studies have shown that women are more susceptible than men to problems associated with inappropriate footwear in terms of the choice of footwear in the elderly patients , lord et al concluded that individuals had better balance when wearing shoes with higher collars than with lower ones . wide shoes are effective against bunions , and extra - depth shoes are more appropriate if the individual has hammertoes or mid - foot arthrosis . low - heeled footwear is known to reduce the risk of falls , likely due to the associated lowering of the center of gravity . while assessing different footwear , athletic and canvas shoes ( sneakers ) are associated with the lowest risk of a fall when compared to other types of shoes . moreover , some suggested that sole hardness have a little effect on overall balance in the absence of a specific condition , such as hallux rigidus , which may benefit from application of hard sole . as an extra preventative measure , better fitting footwear with slip - resistant soles is recommended both inside and outside the household to reduce the risk of falls . taken together , these studies seem to support the use of footwear with low heels , slip - resistant soles and wider frames to ensure comfort , better balance , and reduced risk of injury in the elderly population . in the event of multiple deformities , an individual may benefit from the combination of both extra - depth , wide , and low - heeled footwear to increase foot stability and facilitate balance . the use of appropriate shoe wear in the elderly patients is related to the socioeconomic factors . millions of elderly individuals live at or below the poverty level . in fact , women older than 75 years are 3 times more likely to become poor . without established financial programs such as social security , nearly half of all elderly individuals would be considered poor today . changes in living conditions ( ie , community homes , nursing homes , etc ) can often worsen the socioeconomic status of elderly patients . deteriorating health conditions followed by reduced physical activity can lead to a shift in clinical care , one that emphasizes the management of illness . in the face of such socioeconomic constraints , furthermore , retail stores often do not stock the customized shoes ( ie , extra wide ) needed for elderly customers , making availability an issue for elderly patients . the neglect of appropriate footwear in the elderly has been linked to early amputation , and in rare cases , early demise . to help alleviate the financial burden , elderly patients enrolled in medicare part b may benefit from the medical coverage of certain medical supplies and preventative services . eligible patients typically have diabetes or other severe foot diseases . under the medicare part b provision , such patients are entitled to a pair of custom - molded shoes , 3 pairs of inserts , or a pair of extra - depth shoes per calendar year . the patient will be held responsible for only 20% of the cost while medicare covers the rest . . some of these hazards such as wet pavements and bad weather conditions are capable of causing serious injury . these pathologies , in return , have contributed to the growing risk of even more serious injuries such as falls and fractures . ultimately , the noticeable decrease in physical activity in the elderly patients has been linked to depression and an overall decline health and quality of life . clinically , preventative measures such as appropriate footwear should be continually emphasized to stop or delay the progression of some foot problems . while recommending appropriate footwear is a cost - effective approach when compared to other options , it is often hindered by socioeconomic obstacles . nevertheless , studies have shown that it can be a vital step to ensure prolonged musculoskeletal and overall health among the elderly patients .

foot pathologies are common in nearly 80% of all elderly patients , and studies have indicated inappropriate footwear as one of the major underlying cause . it has been postulated that ill - fitting shoe wear affects plantar pressure , thus exacerbating weak balance . complications arising from foot pathologies , which include difficulty in maintaining balance , have increased the risk of falls that can result in fractures and other serious injuries . the link between footwear and the onset or progression of certain foot pathologies has emphasized the need to explore and promote preventative measures to combat the issue . wider and higher toe boxed shoes , along with sneakers , are examples of footwear documented to evenly distribute plantar pressure , increase comfort , and facilitate appropriate balance and gait . ultimately , the use of appropriate footwear can help to better stabilize the foot , thus reducing the risk of sustaining debilitating physical injuries known to drastically decrease the quality of life among the geriatric population .

to develop a compact system for basic experiments , we chose a sealed x - ray tube with a molybdenum target ( oxford instruments inc . , ca ) . the voltage we could apply ranged from 10 to 50 kvp ( voltage - peak ) , and the maximum current was 1 ma . we clearly observed strong characteristic radiations of 17.479 kev and 19.608 kev , which corresponded to the k and k , respectively , of molybdenum . the bremsstrahlung broadband spectrum was also generated by bombarding of the target material with electrons . the x - rays generated from the target were reflected on the inner wall of each capillary and they passed through the parabolic polycapillary optics . as the shape of the each capillary was a parabola , the x - rays radiated from a focus of the parabola passed through each capillary and they were parallel to the optical axis . in other words , the polycapillary optics transferred the divergent x - rays to a parallel beam along the optical axis . the outer diameter and length of the parabolic polycapillary optics were 10 mm and 133 mm , respectively . for mo k radiation , the critical angle of each capillary was 1.7 mrad and the reflectivity of a single reflection on the capillary was greater than 0.8 . the incidence x - rays with the critical angle were reflected approximately 17 times on the inside wall of each capillary , and in this case , the reflectivity was approximately 0.02 . however , when the incidence x - rays with less than the critical angle were included , the final transmission efficiency of the generated x - rays could be as high as 0.3 . taking into consideration the transmission efficiency , the applied tube voltage was 40 kvp which was higher than the applied tube voltage for conventional mammography . in this study , the operating condition of the x - ray tube for imaging test objects was fixed at a tube voltage of 40 kvp and a current of 0.5 ma , and the exposure time to let the image develop was three seconds . the parabolic polycapillary cut the high energy of more than 20 kev by its x - ray reflection properties , but the low energy x - ray of less than 20 kev was passed by the parabolic polycapillary . thus , a tube voltage more than 40 kvp could be applied and the number of photons generated on the x - ray tube could be increased . in a tube voltage of 40 kvp with a current of 0.5 ma , the number of photons generated by the molybdenum target x - ray tube in front of the polycapillary optics was ~ 610 photons / sec . it meant that the number of photons on the detector was enough to make a digital image . in our experiment , , nj ) with a 24 m 24 m pixel size was used for imaging . the nyquist frequency of the captured image using the ccd detector was 21 lp / mm for which the spatial resolution was limited by the pixel size of the ccd detector . in order to obtain a higher spatial resolution of 21 lp / mm in our system configuration , the pixel size of the ccd detector would have to be less than 24 m 24 m . the ccd chip was cooled down to 223 k to reduce the dark noise of the ccd detector . figure 1 shows the experimental setup used to evaluate the performance of the small - field digital mammography . parallel x - ray beams by the parabolic polycapillary optics entered a filter and then passed through a test sample . all images used in these experiments were corrected by post - image processing , and the contrast ( imax - imin ) / imin , was calculated by maximum and minimum pixel values . the objects on an image are distinguished by their contrast difference and achievable spatial resolution of the system . the mtf of an imaging system shows a performance of the spatial resolution connected with the contrast ( 12 ) . we directly examined the mtf to verify the spatial resolution of the small - field digital mammographic imaging system by taking images of the line - pairs instead of indirectly obtaining the images from an edge spread function . the exposure time was three seconds when we took the linear pattern images of a phantom of a gold - line . x - ray exposure gives rise to energy accumulation in an object , and the energy can destroy biological tissue and its chemical state . when x - ray photons n with a wavelength are irradiated on the area d of a sample , then the dose of the specimen with the thickness and density can be written as : which is the radiation dose for a single x - ray wavelength or energy of hc/ , and where is the linear absorption coefficient of the sample . all the x - rays that arrive on the sample contribute to the radiation dose of the sample . therefore , the radiation dose d for a general x - ray tube source can be expressed as : we measured the radiation exposure to enter the test sample using an ionization chamber ( fluke corp . , oh ) with an exposure time of three seconds and measured the spectrum of incident x - rays to the test sample together . the absolute number of x - ray photons with energy hc/ was known by the radiation exposure and the x - ray spectrum , and the radiation dose of the test sample was obtained from equations and and the material information of the test sample . this method is similar to the dose measurement in which x - ray tube voltage and half - value layer ( or equivalent other parameters ) should be known . we obtained an image more than 10 mm in diameter by scanning a test standard phantom of breast and combining each scanned image . breast tissue which was fixed by paraffin and proven on biopsy to be a ductal carcinoma in situ was imaged with the small - field digital mammographic imaging system . a conventional projection - type digital mammography of the breast tissue sample was taken to compare with the findings of the small - field digital mammographic imaging system . the breast tissue study was carried out in accordance with the regulations of the institutional review board of university . there were various choices of filters for our mammographic imaging system , e.g. , molybdenum ( mo ) and rhodium ( rh ) . the spatial resolution of 12 lp / mm for the molybdenum and rhodium filters with a 25 m thickness was achieved at a mtf of 10% as shown in figure 2 . the spatial resolution of 12 lp / mm corresponded to 41.7 m in spatial resolution . from the standpoint of the ccd detector , this spatial resolution was matched to almost two pixels . when we used molybdenum filters with 25 m and 50 m thickness , the mtf values for each filter were n't very different . however , the contrasts for the molybdenum filter with a 25 m and 50 m thick was 1.7 and 1.5 , respectively . the contrast was measured with a step - like acrylic object . in the case of a 25-m molybdenum filter , the contrast for was 12% higher than that for a 50-m molybdenum filter . the image contrast was proportional to the square of the photon number detected on the ccd camera . thus , the thick filter led to a lower contrast . when a 100-m - thick molybdenum filter was used in the mammographic imaging system , the contrast rapidly decreased to 0.48 and the spatial resolution was 11 lp / mm . figure 4 shows the radiation dose using rhodium filters of 25-m thickness for an acrylic phantom with a 44 mm thickness ( mammographic accreditation phantom , cirs , va ) , equivalent to a 42 mm compressed breast with 50% glandular tissue and 50% adipose tissue , as a function of the tube current and fixed voltage for molybdenum . the radiation dose , as a function of the voltage at a fixed current , increased quadratically . the radiation dose shown in the table 1 is when the parabolic polycapillary optics was not combined with the x - ray tube in our system configuration . when no parabolic polycapillary optics was used , the system became a conventional projection - type digital mammography . the radiation dose of the system without the parabolic polycapillary optics was three times higher than with the parabolic polycapillary optics . the field that was exposed to the radiation dose for the small - field digital mammographic imaging system was 3% of the field for the projection - type system . the small - field digital mammographic imaging system compared to the projection - type digital mammography had advantages in terms of the radiation dose , dose field and absorbed x - ray energy as shown in table 1 . radiation dose was calculated from the measured x - ray energy absorbed and the measured dose field which led to mass information . in the mammography system without the parabolic polycapillary optics , the field illuminated x - ray could be controlled by lead block or collimator . in this case , the radiation field could be small . however , the radiation dose was still three times higher because the radiation dose was not proportional to the field size but to the accumulated x - ray energy and mass of the object . in our small - field mammographic imaging system , the radiation doses for the molybdenum and the rhodium filters were 0.18 mgy and 0.13 mgy , respectively . theoretical estimation of the radiation dose ( 13 , 14 ) for the phantom was 0.32 mgy at a radiation exposure of 0.15 r , a half - value layer of 0.41 , and a tube voltage of 35 kvp for a mo / rh tube - filter combination . the value of 0.32 mgy was very close to the 0.38 mgy in table 1 . in addition , the american college of radiology advocates that the maximum value of the mean glandular dose must not exceed 3 mgy ( 15 ) . we compared spectra for x - ray beams passing through parabolic polycapillary optics combined with the x - ray tube and direct x - ray beam as shown in figure 5a . the high energy x - rays larger than 20 kev were almost removed by propagating the parabolic polycapillary optics although low energy x - rays were also reduced simultaneously . the cut - off of the high energy could also be verified by the calculation of the x - ray reflectivity on the inner surface of the parabolic polycapillary optics ( 16 ) . figure 5b shows the x - ray reflectivity as a function of x - ray energy at the fixed incidence angle of 1.7 mrad . after two reflections , the high energy x - rays of more than 20 kev were almost removed . the x - ray generated from the x - ray tube could be reflected up to 17 times on the inner surface of the parabolic polycapillary optics . low energy x - rays have a larger critical angle than that of high energy x - rays , but the reflectivity of the low energy x - rays decrease at a larger incidence angle than its critical angle . thus , the intensity of low energy x - rays passing through the parabolic polycapillary optics with various incident angles was also reduced as shown in figure 5a . additionally , the molybdenum and rhodium filters remarkably reduced the low energy of the x - ray beam which passed through the parabolic polycapillary optics removing the high energy larger than 20 kev of the generated x - rays because the critical angles for the high energy x - rays were smaller than that for molybdenum radiation . because of the parabolic polycapillary optics and the filter , the radiation doses were remarkably reduced , and the fact that the rhodium filter allowed a lower radiation dose could be verified by measuring the energy spectra shown in figure 6 . within the overall energy region , the number of photons for the rhodium filter was lower than that for the molybdenum filter . we were able to obtain an enlarged image more than 10 mm in diameter by scanning a test object and combining each scanned image . each scanned image was overlapped by approximately 30% of the field in order to guarantee that no scan field was missed on the test sample . x - ray exposure to make an image was three seconds and the object was moved for next imaging during one second . figure 7a presents the scanned and recombined phantom image of 0.32-mm - al2o3 specks which simulate the microcalcifications of breast cancer . the 44-mm - thick acrylic phantom contained fibers , specks and masses that were composed of various materials . figure 7b shows an image of the same specks taken on projection - type digital mammography . the system produced a magnified image of 1.07 x , and the spatial resolution of the system was 11 lp / mm which was tested by a line - pair phantom . the spatial resolution was limited by the spot size of 100 m rather than the pixel size of the detector because of the cone beam . comparing both sets of images in figure 7a , b , the ratio of background noise , which was defined as small - field mammographic imaging system / projection - type digital mammography , was 0.8 . it meant that the image contrast of the small - field mammographic imaging system might be 1.2 times higher than that of the projection - type , digital mammography . figure 8a clearly shows microcalcifications within breast tissue which was fixed by paraffin and proven by biopsy to be ductal carcinoma in situ . a small figure located on the left upper position in figure 8b shows a full image of the breast tissue sample taken with a projection - type digital mammography , and the outer black circle in figure 8b was the same field taken with the small - field scanning mammographic imaging system . calcifications located within two small circles in figure 8a were clearly observed . in figure 8b , it was difficult to distinguish calcifications located at the same positions because of lower image contrast . however , the same objects in figure 8b seemed to one unseparated object . because of the high spatial resolution of 12 lp / mm for the small - field mammographic imaging system , two microcalcifications could be distinguished . however , the projection - type digital mammography could not distinguish two objects because of the separation distance was at the resolution limit of 11 lp / mm . there were various choices of filters for our mammographic imaging system , e.g. , molybdenum ( mo ) and rhodium ( rh ) . the spatial resolution of 12 lp / mm for the molybdenum and rhodium filters with a 25 m thickness was achieved at a mtf of 10% as shown in figure 2 . the spatial resolution of 12 lp / mm corresponded to 41.7 m in spatial resolution . from the standpoint of the ccd detector , this spatial resolution was matched to almost two pixels . when we used molybdenum filters with 25 m and 50 m thickness , the mtf values for each filter were n't very different . however , the contrasts for the molybdenum filter with a 25 m and 50 m thick was 1.7 and 1.5 , respectively . the contrast was measured with a step - like acrylic object . in the case of a 25-m molybdenum filter , the contrast for was 12% higher than that for a 50-m molybdenum filter . the image contrast was proportional to the square of the photon number detected on the ccd camera . thus , the thick filter led to a lower contrast . when a 100-m - thick molybdenum filter was used in the mammographic imaging system , the contrast rapidly decreased to 0.48 and the spatial resolution was 11 lp / mm . figure 4 shows the radiation dose using rhodium filters of 25-m thickness for an acrylic phantom with a 44 mm thickness ( mammographic accreditation phantom , cirs , va ) , equivalent to a 42 mm compressed breast with 50% glandular tissue and 50% adipose tissue , as a function of the tube current and fixed voltage for molybdenum . the radiation dose , as a function of the voltage at a fixed current , increased quadratically . the radiation dose shown in the table 1 is when the parabolic polycapillary optics was not combined with the x - ray tube in our system configuration . when no parabolic polycapillary optics was used , the system became a conventional projection - type digital mammography . the radiation dose of the system without the parabolic polycapillary optics was three times higher than with the parabolic polycapillary optics . the field that was exposed to the radiation dose for the small - field digital mammographic imaging system was 3% of the field for the projection - type system . the small - field digital mammographic imaging system compared to the projection - type digital mammography had advantages in terms of the radiation dose , dose field and absorbed x - ray energy as shown in table 1 . radiation dose was calculated from the measured x - ray energy absorbed and the measured dose field which led to mass information . in the mammography system without the parabolic polycapillary optics , the field illuminated x - ray could be controlled by lead block or collimator . in this case , the radiation field could be small . however , the radiation dose was still three times higher because the radiation dose was not proportional to the field size but to the accumulated x - ray energy and mass of the object . in our small - field mammographic imaging system , the radiation doses for the molybdenum and theoretical estimation of the radiation dose ( 13 , 14 ) for the phantom was 0.32 mgy at a radiation exposure of 0.15 r , a half - value layer of 0.41 , and a tube voltage of 35 kvp for a mo / rh tube - filter combination . the value of 0.32 mgy was very close to the 0.38 mgy in table 1 . in addition , the american college of radiology advocates that the maximum value of the mean glandular dose must not exceed 3 mgy ( 15 ) . we compared spectra for x - ray beams passing through parabolic polycapillary optics combined with the x - ray tube and direct x - ray beam as shown in figure 5a . the high energy x - rays larger than 20 kev were almost removed by propagating the parabolic polycapillary optics although low energy x - rays were also reduced simultaneously . the cut - off of the high energy could also be verified by the calculation of the x - ray reflectivity on the inner surface of the parabolic polycapillary optics ( 16 ) . figure 5b shows the x - ray reflectivity as a function of x - ray energy at the fixed incidence angle of 1.7 mrad . after two reflections , the high energy x - rays of more than 20 kev were almost removed . the x - ray generated from the x - ray tube could be reflected up to 17 times on the inner surface of the parabolic polycapillary optics . low energy x - rays have a larger critical angle than that of high energy x - rays , but the reflectivity of the low energy x - rays decrease at a larger incidence angle than its critical angle . thus , the intensity of low energy x - rays passing through the parabolic polycapillary optics with various incident angles was also reduced as shown in figure 5a . additionally , the molybdenum and rhodium filters remarkably reduced the low energy of the x - ray beam which passed through the parabolic polycapillary optics removing the high energy larger than 20 kev of the generated x - rays because the critical angles for the high energy x - rays were smaller than that for molybdenum radiation . because of the parabolic polycapillary optics and the filter , the radiation doses were remarkably reduced , and the fact that the rhodium filter allowed a lower radiation dose could be verified by measuring the energy spectra shown in figure 6 . within the overall energy region , the number of photons for the rhodium filter was lower than that for the molybdenum filter . we were able to obtain an enlarged image more than 10 mm in diameter by scanning a test object and combining each scanned image . each scanned image was overlapped by approximately 30% of the field in order to guarantee that no scan field was missed on the test sample . x - ray exposure to make an image was three seconds and the object was moved for next imaging during one second . figure 7a presents the scanned and recombined phantom image of 0.32-mm - al2o3 specks which simulate the microcalcifications of breast cancer . the 44-mm - thick acrylic phantom contained fibers , specks and masses that were composed of various materials . figure 7b shows an image of the same specks taken on projection - type digital mammography . the system produced a magnified image of 1.07 x , and the spatial resolution of the system was 11 lp / mm which was tested by a line - pair phantom . the spatial resolution was limited by the spot size of 100 m rather than the pixel size of the detector because of the cone beam . comparing both sets of images in figure 7a , b , the ratio of background noise , which was defined as small - field mammographic imaging system / projection - type digital mammography , was 0.8 . it meant that the image contrast of the small - field mammographic imaging system might be 1.2 times higher than that of the projection - type , digital mammography . figure 8a clearly shows microcalcifications within breast tissue which was fixed by paraffin and proven by biopsy to be ductal carcinoma in situ . a small figure located on the left upper position in figure 8b shows a full image of the breast tissue sample taken with a projection - type digital mammography , and the outer black circle in figure 8b was the same field taken with the small - field scanning mammographic imaging system . calcifications located within two small circles in figure 8a were clearly observed . in figure 8b , it was difficult to distinguish calcifications located at the same positions because of lower image contrast . because of the high spatial resolution of 12 lp / mm for the small - field mammographic imaging system , two microcalcifications could be distinguished . however , the projection - type digital mammography could not distinguish two objects because of the separation distance was at the resolution limit of 11 lp / mm . previous studies ( 7 - 9 ) have demonstrated the potential for x - ray capillary optics , non - parabolic polycapillary optics , aligned between the object and computed radiography phosphor imaging plate , to be incorporated into digital mammographic image . consequently , the post - object capillary optics had the potential to increase image contrast by eliminating scattered x - rays . a scanning process is also required to obtain an image because the size of the capillary optics is small . in the system configuration , the capillary optics played a role as a small ' grid ' , and there was no improvement on the spatial resolution which was approximately 5 lp / mm because the divergent x - ray source of 100 m spot size illuminated the object and the pixel size of detector was large . in addition , the system configuration could not avoid a high radiation dose because of long exposure time , i.e. , six seconds per one scanned image during scanning . in the scanning process , the capillary optics and the detector were combined with each other and moved together . we modified the system configuration to improve the spatial resolution and to reduce radiation dose instead of sacrificing the contrast slightly ( fig . the spot size of the x - ray tube used in this system was not an important factor in effecting the spatial resolution due to the use of a parallel beam . for the parallel beam , there was no image blurring by finite spot size of the x - ray tube , and the pixel size of the detector was a critical factor to determine the spatial resolution . the illumination area of the parallel beam was small because the diameter of the parabolic polycapillary optics was small . however , there was enough space to increase the field of view by scanning the object . when the x - ray tube of 100-m - spot size without the parabolic polycapillary optics and the same ccd detector are used to make a projection - type x - ray image , we may obtain a similar spatial resolution of 12 lp / mm at a magnification of 1.48 . in the imaging system using the divergent x - ray beam , the edge gradient blurring for the spot size and the magnification may correspond with the two pixels of the ccd detector which is almost equal to the spatial resolution . if the magnification is larger than 1.48 , the spatial resolution will be worse , and if the magnification is smaller than 1.48 , the spatial resolution will not improve because one line - pair needs two pixels to separate the pattern at least . in this sense , only similar spatial resolution of 12 lp / mm will be available at a magnification of less than 1.48 for the divergent x - ray beam . in addition , a high radiation dose compared to the low radiation from small - field digital mammographic imaging system can not be avoided for the divergent beam . in general , a thick filter considerably reduces low - energy x - rays including also characteristic radiations and requires a longer exposure time to obtain the same number of photons on the ccd detector compared to a thin filter . with using a thick filter , the detector noise could be increased and the image quality might be reduced if the x - ray source was not strong enough . spatial resolutions of 12 lp / mm for molybdenum and rhodium filters with a 25-m thickness were obtained using the small - field digital mammographic imaging system . the resolution in the digital mammographic imaging system strongly depended on the pixel size of the detector . the resolution should be improved in future with advances in the manufacturing technology for ccd or cmos detectors thereby creating a smaller pixel size . the small - field digital mammographic imaging system was able to diminish the radiation dose compared to that of the conventional projection - type mammography , for which an entire object receives the radiation dose . in addition , the parabolic polycapillary optics itself plays a role of a low - pass filter to eliminate x - rays which have higher energy than molybdenum radiation . image blur and radiation dose by x - ray beams of high energy which are not removed by conventional molybdenum and rhodium filters the x - ray beams of high energy larger than 20 kev removed contributes to improve image quality and to reduce the radiation dose . in other applications , e.g. , conventional radiography , high energy x - ray , for instance , 75 kev , is required . to propagate the high energy x - ray , each diameter of glass capillary in the polycapillary optics should be very small , i.e. , sub - micrometer size , because the critical angle for the high energy x - ray is extremely small . in current manufacturing technology thus , general x - ray imaging using parallel beam through the parabolic polycapillary optics can be restricted . therefore , to obtain a parallel beam of high photon flux , the x - ray tube should have a high capacity . in conclusion , due to its high spatial resolution and low radiation dose , a small - field digital mammographic imaging system has the potential to be used for examining local volumes of breast tissue . a relatively large field image this system can be a useful diagnostic tool for examining a small field of breast cancer with a low radiation dose to the patient after conducting conventional mammography studies . to improve the small - field digital mammographic imaging system using parabolic polycapillary optics , large

objectiveto evaluate the efficacy for spatial resolution and radiation dose of a small - field digital mammographic imaging system using parabolic polycapillary optics.materials and methodswe developed a small - field digital mammographic imaging system composed of a ccd ( charge coupled device ) detector and an x - ray source coupled with parabolic polycapillary optics . the spatial resolution and radiation dose according to various filters were evaluated for a small - field digital mammographic imaging system . the images of a test standard phantom and breast cancer tissue sample were obtained.resultsthe small - field digital mammographic imaging system had spatial resolutions of 12 lp / mm with molybdenum and rhodium filters with a 25-m thickness . with a thicker molybdenum filter ( 100 m thick ) , the system had a higher spatial resolution of 11 lp / mm and contrast of 0.48 . the radiation dose for a rhodium filter with a 25-m thickness was 0.13 mgy within a 10-mm - diameter local field . a larger field image greater than 10 mm in diameter could be obtained by scanning an object . on the small - field mammographic imaging system , microcalcifications of breast cancer tissue were clearly observed.conclusiona small - field digital mammographic imaging system with parabolic polycapillary optics may be a useful diagnostic tool for providing high - resolution imaging with a low radiation dose for examination of local volumes of breast tissue .

if medical radiation sciences is to evolve as an evidencebased profession , we must increase research productivity and publication in our field . the importance of research collaboration has been recognised and strongly supported by research organisations and policy makers alike1 and there has been a marked rise in international collaboration in numerous industries over the last three decades.2 international collaboration is recognised for enhancing the ability to approach complex problems from a variety of perspectives , increasing development of a wider range of research skills and techniques , and improving publication and acceptance rates.3 these benefits could be leveraged to increase research capacity in medical radiation science and support the further development of our professional knowledge base . the aim of this paper is to describe the current status of international collaboration in medical radiation science and compare this to other allied health occupations . individuals may be identified as research collaborators if they : ( 1 ) work on the same project , ( 2 ) aid in the research proposal , and/or ( 3 ) make decisions regarding the plans for the research project.4 kimiloglu et al.3 studied the most crucial requirements for collaborative research . they determined that for collaboration to be effective , each researcher should be allocated clearly defined tasks , prior to conducting research ( i.e. all members agreeing how responsibilities will be shared ) and collaborators should willingly share their knowledge , experience and resources with all group members . as interest has grown over the years , collaboration with one or more institution(s ) in other countries excluding all other national institutions. with the recent intensification of collaboration , specific groups and countries have been more engaged in the trend than others . additionally , some fields are more international than others.6 the rate of international collaboration has grown significantly over the last 35 years . there are a variety of reasons countries and fields become involved with international collaborations , ranging from sharing expensive research equipment to using unique countryspecific data . researchers in some countries may seek to improve a lagging research capacity in a field by cooperating with leading researchers in other countries . international collaboration may also allow researchers to partner with others with their own specialised focus who are not available in geographic proximity . externally , increased financial support for international collaboration from nations and sponsors may have played a role in the increase , and the growth of internetbased communication systems has certainly facilitated effective international collaboration . historical ties and geographic proximity seem to play a decreasing role in the promotion of international collaboration , but may arise more from geographic funding such as that provided by the european commission , than from a preference for colocation . interactions may also be facilitated or discouraged by language , culture and political climate.5 while we know that international scientific collaboration is on the rise , little is known about international collaboration within the medical radiation sciences community or how it compares to other health professions . this study utilised a content analysis approach where coauthorship of a journal article was used as a proxy for research collaboration , and the papers were assigned to countries based on the corporate address given in the byline of the publication . this method of analysis is well established and accepted across studies of research activity.8 the content analysis focused on the major professional journals published in the countries represented within our research team australia , the united kingdom ( uk ) and the united states of america ( usa ) . for medical radiation sciences , the three journals chosen for analysis were journal of medical radiation sciences the joint journal of australia and new zealand ( aus / nz ) ( previously the radiographer ) , radiography from the uk and radiologic technology from the usa . a convenience sample method was employed and articles published in the three major medical radiation science journals over a 3year period from 2012 to 2014 were inspected manually for author and author affiliation . allied health professions australia defines allied health professionals as a subset of healthcare professionals that do not include those of medicine , nursing or dentistry.9 physiotherapy , speech pathology and occupational therapy were chosen as comparisons for medical radiation science for international collaboration as all these occupations are included as allied health professionals . nursing was also selected for comparison as this is a longer established health profession with a longer history of academic research . the journals chosen for comparison to the medical radiation science journals are detailed in table 1 . the content analysis on the frequency of international coauthorship was performed on all of these journals . number of articles published ( pub ) , number of articles with international collaboration ( ic ) and the percentage of articles with international collaboration ( rate ) . only articles where all authors did not share a country affiliation were counted as representing international collaboration in the study . letters to the editor , corrigenda or errata , newsletters and conference papers were excluded to limit the study to only original published articles . in total , table 1 presents the breakdown of publications by professional field , publication country of origin , and publication year . to determine the rate of international collaboration , the number of articles having authors from two or more countries was divided by the total number of articles reviewed . while rates of publication vary by country and field , by examining the number of internationally collaborative articles as a percentage of the total articles published within that journal , we correct for variances introduced by fields and countries with differing publication rates and health occupation populations . the data was analysed using minitab 17 statistical software . rates of international collaboration across the health professions have increased over the past 3 years . when all the professions were combined , the rates of international collaboration steadily rose over the 3year period from 7.2% to 10.3% ( see fig . this figure demonstrates the overall change in international collaboration seen across the allied health occupations over the 3year period studied . likewise , rates of international collaboration in medical radiation science journals from aus / nz , the uk and the usa have steadily increased over the 3year period sampled from 2012 to 2014 ( see fig . 2 ) . overall , the journal published in the uk had the highest average rate of international collaboration ( 11.0% ) , the usa the lowest ( 5.1% ) with aus / nz demonstrating an average of 8.5% . this figure illustrates the changes seen in international collaboration rate within the medical radiation sciences over the 3year period studied . the pooled standard deviations were used to calculate the intervals . using oneway analysis of variance ( anova ) analysis , this analysis employed the null hypothesis that all means are equal and the alternative hypothesis that at least one mean is different . the mean international collaboration rate for nursing was significantly lower than that for speech pathology , occupational therapy and physiotherapy . medical radiation science demonstrated lower average rates of international collaboration ( 7.9% ) than the other allied health occupations sampled , but there was not a statistically significant difference in the means between medical radiation science and any of the other fields . speech pathology had the highest average rate with 15.1% , followed by physiotherapy ( 12.5% ) and occupational therapy ( 12.4% ) . the average rate of international collaboration in nursing of 2.7% was far below that of the allied health occupations sampled ( see fig . this figure compares rates of international collaboration for the fields of nursing , occupational therapy ( ot ) , physiotherapy ( pt ) , medical radiation sciences ( mrs ) , and speech language pathology ( slp ) . the same trends as those seen in the medical radiation science journals by country of publication continued when all the professions were combined . while the differences in the means were not significantly different , those published in the uk had the highest average rate of international collaboration of 11.8% , followed by australia ( 9.8% ) , then the usa ( 8.0% ) ( see fig . 4 ) . boxplot of international collaboration rate for all allied health occupations by country of publication . this figure illustrates the differences and overlap between international collaboration rates by country of publication australia ( aus ) , the united kingdom ( uk ) and the united states of america ( usa ) . the increase in the rate of international collaboration in medical radiation science is to be expected as it mirrors the trends in numerous other branches of science over recent years.2 , 10 , 11 the increase is closely aligned with the increase of globalisation and improved it communication facilities so to see a decrease in international collaboration would defy the global trend . however , the medical radiation science community is lagging in developing international collaborations in comparison to other allied health fields . while the differences are not currently significant , we should act to remedy the situation before the gap widens . analysis of strategies employed by other health professions can be instructive for improving international collaboration in medical radiation sciences . initiatives developed within the european medical radiation sciences community may account for the increased rate of international collaboration growth in the uk in comparison to australia and the usa . the usa is the third largest country by area,12 the third most populated in the world13 and has over 50 jurisdictions.14 a country with the diversity of the usa would allow ample opportunity to collaborate on research projects without the need to seek coresearchers from overseas , which may contribute to the lower rates of international collaboration found in journals from that country . historically , the usa is a very nationalistic country15 , which may be another possible explanation for their lower rates of international collaboration . there are several possible explanations why the uk has a higher average rate of international collaboration . time zone differences are a significant barrier to international collaboration16 and the uk 's geographical location with its proximity to numerous other countries with comparable time zones lessens this burden . the european union has also developed programmes to promote international collaboration like the european institute of innovation and technology , the european higher education area and the erasmus mundus programme.17 the greatest increase in the rate of international collaboration in medical radiation science journals sampled was in the uk journal radiography from 2013 to 2014 increasing from 5.6% to 16.2% , the highest rate in the medical radiation science journals sampled , close to the rates of other allied health occupations . this increase coincided with the first offering of the annual optimisation of image quality and xradiation dose in medical imaging ( optimax ) residential summer school held at the university of salford in manchester , england.18 due to the increasing emphasis on collaboration and raising the profile of research in the medical radiation science profession,19 , 20 the optimax summer school programme was initiated . the programme is open to bsc , msc and phd students from the disciplines of radiography , nuclear medicine , biomedical science and physics , and is funded by the erasmus mundus programme . the programme strives to develop teambased radiography research on an international level by enabling students to experience international collaboration firsthand and develop multinational / cultural partnerships early in their careers.18 , 21 the universities and professional associations can play a large part in raising the profile of research in the medical radiation sciences throughout the world by promoting international collaboration within the profession . universities can follow the lead taken by those who formed the optimax summer school programme , while medical radiation science professional associations can encourage collaboration with similar associations as has been done in physiotherapy22 and occupational therapy23 , which may contribute to those professions ' higher rate of international collaboration . this study focused on internationally collaborative articles , published in the most widely accepted professional journals in only three countries studied , as these are the most likely to impact practice in the allied health fields of those countries . this could have resulted in missing some significant collaborations with medical radiation science professionals published in less specific journals . however , it is the evidence informing professional practice that we most hoped to capture . this study focused on internationally collaborative articles , published in the most widely accepted professional journals in only three countries studied , as these are the most likely to impact practice in the allied health fields of those countries . this could have resulted in missing some significant collaborations with medical radiation science professionals published in less specific journals . however , it is the evidence informing professional practice that we most hoped to capture . overall , medical radiation science is lagging in international collaboration in comparison to other allied health fields in their respective journals , although the mean international collaboration rate for the medical radiation sciences journal in the uk is quite competitive with physiotherapy and occupational therapy . the medical radiation science community needs to examine the strategies for encouraging international collaboration if goals of increasing research capacity in the profession and developing a more robust professional knowledge base are to be realised .

abstractintroductioninternational collaboration is recognised for enhancing the ability to approach complex problems from a variety of perspectives , increasing development of a wider range of research skills and techniques and improving publication and acceptance rates . the aim of this paper is to describe the current status of international collaboration in medical radiation science and compare this to other allied health occupations.methodsthis study utilised a content analysis approach where coauthorship of a journal article was used as a proxy for research collaboration and the papers were assigned to countries based on the corporate address given in the byline of the publication . a convenience sample method was employed and articles published in the professional medical radiation science journals in the countries represented within our research team australia , the united kingdom ( uk ) and the united states of america ( usa ) were sampled . physiotherapy , speech pathology , occupational therapy and nursing were chosen for comparison.resultsrates of international collaboration in medical radiation science journals from australia , the uk and the usa have steadily increased over the 3year period sampled . medical radiation science demonstrated lower average rates of international collaboration than the other allied health occupations sampled . the average rate of international collaboration in nursing was far below that of the allied health occupations sampled . overall , the uk had the highest average rate of international collaboration , followed by australia and the usa , the lowest.conclusionoverall , medical radiation science is lagging in international collaboration in comparison to other allied health fields .

since minimally invasive surgery ( mis ) is performed using an endoscope and several thin instruments through small incisions made in patients , it provides patients with some advantages like shorter recovery time , less postoperative pain , earlier resumption of normal activity , and cost savings . however , it is more ergonomically challenging to performing surgeons due to its inherent complexity such as limited work volume and degree of freedom . specifically , such complexity induces abnormal movements of arm and shoulder while holding certain body postures , for example , head and torso , for prolonged time [ 13 ] . importantly , this complexity often exposes performing surgeons to increased ergonomic risks including muscle fatigue that can result in critical errors during surgical procedures [ 46 ] . the vulnerability to ergonomic risk is well confirmed in a literature stating that performing laparoscopic surgery is significantly more stressful for the surgeon than open surgery . in addition , it has been reported that mis carries more complications than open surgery [ 8 , 9 ] . therefore , it is important to quantitatively measure ergonomic risks on performing surgeons , particularly muscle fatigue during mis procedures . note that timely intervention with information about muscle fatigue can ensure improved quality of mis procedures . some existing study for fatigue analysis has focused on quantitative measures and their applications for isometric or isotonic contractions during relatively short bouts of high - force activities . among these studies , frequency banding analysis predicted muscle fatigue when lower frequency bands increase and higher frequency bands decrease . standard discomfort analysis was also used to detect the level of muscle fatigue on subjects , which may lack objective analysis . in addition , heart rate and tissue oxygen saturation were used as indicators of fatigue development . importantly , power spectral density ( psd ) of transformed emg data through fast fourier transform approach was widely used to detect muscle fatigue [ 1416 ] . in this approach , frequency and amplitude changes are considered as a result of a reduction in conduction velocity in the muscle fibers and larger motor unit synchronization . as mis operations can be considered as prolonged light muscle activation , psd based measure , which is mainly for static activities , may not be efficiently used to predict muscle fatigue during mis operations . specifically , when psd based measures are used for light muscle activations , conflicting results and complicated relationship between subjective and objective fatigue have been reported [ 17 , 18 ] . such conflicts result from stationary data requirement for psd based measures to be successfully used . in fact , emg data collected from light muscle activations are nonstationary due to changing distance between muscle and emg sensor , as well as changing muscle length . from the literature , it is noted that fatigue mechanism during these activities is not well understood in comparison to high - force ( or intense ) muscle activities . although there are no significant reductions in muscle force level during mis operations , there are some physiological changes in muscles that can lead to muscle fatigue affecting the capacity of muscles and the performance of subjects [ 20 , 21 ] . uhrich et al . assessed the muscle fatigue during a relatively short time during simulated laparoscopic surgery . in their study , the effects of fatigue , monitor placement , and surgical experience have been compared . after obtaining the emg activity and muscular discomfort scores before and after a fatigue session , it was found that the emg data and discomfort scores demonstrated a fatigue response in several muscle groups . they found minimal differences between the two monitor positions and less muscle activity and discomfort in the attending surgeons . in another study , slack et al . the length of the operations varied within 110 hr , but only one minute period before and after operations were analyzed using frequency analysis . in addition , the muscle fatigue increases proportional to time . on the other hand , recurrence quantification analysis ( rqa ) has been tested on biceps brachii and shown to be more sensitive to muscle fatigue than fft variable spectral center frequency ( fc ) . in another study , filligoi and felici showed that determinism% , which is one of the rqa variables , is more effective than median frequency to detect emg signal changes in biceps brachii muscle . for the first time , this novel data analysis method is used in our study to quantify any possible muscle fatigue in real laparoscopic surgery operations which is known as prolonged and low - force muscle activity compared to some intense tasks which involve isometric muscle contraction throughout the whole task . the main goal of this study is to answer the following questions : can objective manifestations of muscle fatigue be detected from emg data during a laparoscopic surgery as prolonged light muscle activation?what is time - to - fatigue for the muscles experiencing fatigue during laparoscopic surgery?which muscle has the highest possibility level of fatigue in laparoscopic surgery among the tested muscle groups ? can objective manifestations of muscle fatigue be detected from emg data during a laparoscopic surgery as prolonged light muscle activation ? what is time - to - fatigue for the muscles experiencing fatigue during laparoscopic surgery ? which muscle has the highest possibility level of fatigue in laparoscopic surgery among the tested muscle groups ? as the first step , emg data for fatigue analysis was collected . specifically , surface emg electrodes were attached to upper arm muscles of participants to collect muscle activations while performing various mis procedures . next , emg data was converted into higher dimensional data using time shift and represented by recurrence plots that facilitate recurrence quantification analysis ( rqa ) , particularly computation of determinism values . then , moving average technique was applied for trajectories of determinism values to detect any changes as the indicator of muscle fatigue . under an irb - approved protocol , five right - hand - dominant expert laparoscopic surgeons , who have performed more than 100 laparoscopic surgeries , performed fifteen mis procedures in a local hospital . , a total of eight surface emg ( semg ) electrodes were attached to the following four bilateral muscles including bicep , triceps , deltoid , and trapezius . note that since lower arms are scrubbed from the fingertips to the elbow , electrodes were not placed on these sterilized muscle compartments . tricep and bicep were selected due to their high activity level during arm flexion and extension . since the shoulder and neck are the common area of muscle fatigue , trapezius and deltoid were also included in our study . all semg data were collected using an 8-channel bioradio 150 physiologic data acquisition system ( great lakes neurotechnologies , incorporated , cleveland , oh ) . after wiping skin surfaces overlying target muscle groups with rubbing alcohol and allowing the alcohol to dry , two 1 1 mvap - ii electrodes ( mvap medical supplies , incorporated , newbury park , ca ) were placed over the muscle bellies of each muscle group and connected to the positive and negative input poles for each channel . an electrode was also attached to the right elbow and connected to the ground input on the bioradio 150 to complete the input circuit . the biocapture data acquisition software package ( great lakes neurotechnologies , incorporated , cleveland , oh ) more specifically , semg data was sampled at frequency of 256 hz from each channel . digital signal processing filters were then applied to exclude the noise that are at low ( < 10 hz ) and high frequency ( > 127 hz ) signals . rqa is a time series analysis method and detects the deterministic structure of the dynamical systems . let x(i ) be the ith point on the orbit describing a dynamical system in d - dimensional space , for i = 1 , n square , where a dot is placed at ( i , j ) whenever x(j ) is sufficiently close to x(i ) . in order to obtain a recurrence plot from a time series { ui } , the following procedures are required . first , we choose an embedding dimension d and construct the d - dimensional orbit of x(i ) by the method of time delays : if u and i are scalar , x(i ) = ( ui , ui+1 , , ui+d1 ) . next , we choose r(i ) such that the ball of radius r(i ) centered at x(i ) in r contains a reasonable number of other points x(j ) of the orbit . finally , we plot a dot at each point ( i , j ) for which x(j ) is in the ball of radius r(i ) centered at x(i ) . we call this picture a recurrence plot ( rp ) and an example of rps is shown in figure 2 . note that i and j are , in fact , times ; therefore , a recurrence plot describes natural time correlation information . recurrence plots tend to be fairly symmetric with respect to the diagonal i = j because if x(i ) is close to x(j ) , then x(j ) is close to x(i ) . there is , however , no complete symmetry because we do not require r(i ) = r(j ) . when n is more than 2 dimensions in a phase space , projection is performed . starting from the time series s(t ) = { s1 , , sn } , the attractor of the underlying dynamics is reconstructed in a phase space by applying the time - delay vector method by takens . the reconstructed trajectory x can be expressed as a matrix where each row is a phase space vector : ( 1)x = x1,x2, xmt , where xi = [ s1 , si+t, si+(de 1)t ] , m = n ( de 1)t , de is the embedding dimension , and t is the delay time . the recurrence plot is a tool that can be used to investigate higher dimensional dynamics through a two - dimensional binary plot of its recurrences . any recurrence of state i with state j is pictured on a boolean matrix expressed by(2)ri , jde,=xixj , where xi , j r are the embedded vectors , i , j n , ( ) is the heaviside step function , and is an arbitrary threshold . in the graphical representation , each nonzero entry of ri , j is marked by a black dot in the position i , j. since any state is recurrent with itself , the recurrent plot ( rp ) matrix fulfills ri , j = 1 which hence contains the diagonal line of identity ( loi ) . the percentage of determinism ( % det ) derived from diagonal lines and related to predictability of the system is an important parameter in rqa which quantifies the ratio of the recurrence points . the following formula is typically used to calculate % det from a recurrence plot : ( 3)%det=l = lminnlpll=1nlpl100%,where p(l ) is histogram or frequency distribution of diagonal line lengths , n is the length of a data series , and lmin is predefined minimal length of a diagonal line . in this study , the above % det for emg data was used as a muscle fatigue indicator . cross recurrence plot ( crp ) toolbox available in matlab was used for rqa analysis . in order to ensure optimized results , rqa parameters including embedding dimension ( de ) , time delay ( t ) , and threshold ( ) should be selected carefully . in the crp toolbox , one can set the parameters of optimal embedding dimension and time delay to the obtained values from false nearest neighbors ( fnn ) and average mutual information ( ami ) function which may lead to the optimal recurrence plots as shown in figure 3 . using these functions , embedding dimension and time - delay parameters to facilitate optimal threshold value , it has been suggested to consider the threshold value only a few percent of the maximum phase space diameter . therefore , the threshold value was chosen according to the 10% of the minimum value of maximum phase space diameter of the data . in this study , the whole duration of mis operations summarized in table 1 was analyzed by applying rqa for each minute of the operation with a window length of 15,360 data points . then , % det value was derived from rqa analysis and the results were plotted against operation time . finally , a moving average analysis with the interval ( or window size ) of ten was applied to the % det values to determine time - to - fatigue . figure 4 shows % det values of all eight muscles for surgeon 1 performing case 3 for 132 minutes as an example . it was observed from figures 4(g ) and 4(h ) that moving average of % det of bilateral trapezius muscles increased after 4550 minutes of operation . in addition , moving average of % det of bilateral deltoid increased after 5560 minutes of operation as shown in figures 4(e ) and 4(f ) . between deltoid and trapezius , bilateral trapezius became more deterministic with % det close to 75% at the end of operation while bilateral deltoid was less deterministic with % det value close to 50% . in this study , the increase in the moving average of % det values is considered as the development of muscle fatigue . thus , it is conjectured from these observations that trapezius became more fatigued than deltoid for surgeon 1 performing case 3 . however , there were no changes in moving averages of % det of bilateral bicep and triceps throughout the entire operation as shown in figure 4 . as the next step , % det was tested for all the mis operations and results are summarized in table 2 . det results did not detect any changes in any of the muscles for surgeon 2 in case number 1 which had the least completion time of 55 minutes . also , surgeon 4 in case number 2 as well as surgeon 5 in case number 3 did not experience any fatigue in their deltoid muscle . specifically , no fatigue signs were detected on bilateral bicep and triceps in any of the subjects . figure 5 shows the mean of moving average values for det% of all the operations and subjects . table 2 also shows that fatigue signs are developed at least 45 minutes after operations begin and trapezius gets fatigue sign earlier than deltoid . the moving average of % det for bilateral trapezius deltoid increased after 4550 and 5560 minutes of operation , respectively . this might be caused due to the following nature of mis procedures : ( i ) during mis , surgeons should maintain their head positions at certain orientation to keep watching the monitor and this may impose significant stress on trapezius muscle , and ( ii ) as the surgeon operates thin and long instrument through a small incision , they often use excessive shoulder movements to overcome the limited degree of freedom and this may impose fatigue on deltoid muscle . in this study , higher % det value implies that the emg data are getting more deterministic and periodic . this deterministic and periodic pattern is the result of synchronization of motor units in fatiguing stage . in order to supply necessary force to continue certain tasks , if almost all motor units have already been involved , motor unit synchronization takes place to continue task operation and prevent the failure . the fact that more motor units are recruited and synchronized during the course of muscle fatigue has been shown in literature . for conventional occupational tasks , increase in synchronization of motor units has been successfully detected using % det values through a computer simulation model . on the other hand , shoulder disorder is considered as one of most important musculoskeletal disorders , especially in prolonged repetitive activations with high precision [ 3335 ] . an increase in interstitial potassium concentrations in trapezius muscle can be the cause of decreased conduction velocity . also , the intra- and extracellular sodium and potassium concentration changes can be the main reason for changes in emg data which are related to muscle fatigue . although the recovery of these metabolic changes is quick , in prolonged light muscle activation , muscles need longer recovery times in order to regain the primary force capacity [ 37 , 38 ] . because of orderly recruitment of motor units , low threshold fibers are vulnerable in prolonged muscle activation , which necessitates the importance of recovery time to avoid myalgic disorders in trapezius [ 39 , 40 ] . it can be summarized from the literature review that fatigue analysis is highly difficult for prolonged light muscle activation tasks such as mis procedures . this study shows that trapezius was the first muscle to show fatigue sign in prolonged light muscle activation during mis procedures . it is interesting to note that similar results were reported in a light assembly task in industrial operations . while the existing study also suggested recovery / break time for workers at 90 minutes after the task begins , the results in this study suggest recovery / break time after 4550 minutes after mis operations begin . more likely , this difference results from intensity and precision level of two tasks : industrial and surgical . this might be because of less muscle activation and less muscle contraction in laparoscopic surgery for these two muscles . as muscles are vulnerable to fatigue during mis operations which have prolonged and intense nature , detecting the muscle fatigue and estimation of time - to - fatigue are of great importance . to meet this need , this study proposed and tested a novel measure that can be efficiently used for detection of muscle fatigue and time - to - fatigue from emg data . in the future study , correlation between objective and subjective results would be important . here , objective results mean fatigue analysis using the proposed measure of this study and subjective results correspond to survey analysis to subjects asking whether they feel fatigue symptoms during mis procedures . also , it would be interesting to compare performance analysis with fatigue analysis in the future , since the final goal for muscle fatigue analysis is to detect any possible effect on surgeon 's performance . this will emphasize the importance of quantitative muscle fatigue analysis , although it might be challenging to apply performance analysis to real surgical operations . however , performance analysis can be easily applied to dry lab experiments such as fundamentals of laparoscopic surgery ( fls ) tasks , since there are validated methods to analyze the performance of subjects . therefore , the combination of rqa , as a quantitative muscle fatigue analysis method , and standard performance analysis of fls tasks will open up new doors for future studies related to muscle fatigue and performance analysis of real mis operations which would be very beneficial for surgical community . also , future studies should include larger number of subjects and more surgical operations in order to confirm findings of this study . in this study , recurrence quantification analysis ( rqa ) was applied to emg data recorded from eight muscle groups of five surgeons while doing fifteen mis operations . the results showed that this novel measure could detect the sign of muscle fatigue on bilateral deltoid and trapezius at 4555 minutes after operations began , and no sign of fatigue was found on other muscles . it was found from this study that trapezius and deltoid were the most vulnerable muscles among all eight muscle groups tested . here , it is worthwhile to note the nature of mis procedures such that surgeons manipulate long and thin instruments for the frequent changes of their orientation and position mainly using bicep and triceps and maintain their arm posture using deltoid while gazing through monitor using trapezius muscle for prolonged time . considering this nature , deltoid and trapezius may show clear sign of fatigue while other muscles do not show any sign of fatigue as tested in this study . based on the results , it could be suggested that surgeons need to take break time at 4550 minutes after mis operations begin in order to minimize muscle fatigue and other possible muscle disorders . to have a better understanding about the effect of recovery time on surgeons doing such a complicated laparoscopic operation , future research might test different recovery times to find an optimum amount of time for muscle relaxation before muscles fatigue . in addition , ergonomic improvement of surgical equipment to reduce the intensity level might be efficient in reducing muscle fatigue or at least increasing time - to - fatigue .

due to its inherent complexity such as limited work volume and degree of freedom , minimally invasive surgery ( mis ) is ergonomically challenging to surgeons compared to traditional open surgery . specifically , mis can expose performing surgeons to excessive ergonomic risks including muscle fatigue that may lead to critical errors in surgical procedures . therefore , detecting the vulnerable muscles and time - to - fatigue during mis is of great importance in order to prevent these errors . the main goal of this study is to propose and test a novel measure that can be efficiently used to detect muscle fatigue . in this study , surface electromyography was used to record muscle activations of five subjects while they performed fifteen various laparoscopic operations . the muscle activation data was then reconstructed using recurrence quantification analysis ( rqa ) to detect possible signs of muscle fatigue on eight muscle groups ( bicep , triceps , deltoid , and trapezius ) . the results showed that rqa detects the fatigue sign on bilateral trapezius at 47.5 minutes ( average ) and bilateral deltoid at 57.5 minutes after the start of operations . no sign of fatigue was detected for bicep and triceps muscles of any subject . according to the results , the proposed novel measure can be efficiently used to detect muscle fatigue and eventually improve the quality of mis procedures with reducing errors that may result from overlooked muscle fatigue .

magnetization transfer ( mt ) imaging is a quantitative approach for detecting subtle or occult abnormalities in brain tissue . in previous studies , the magnetization transfer ratio ( mtr ) , an index of mt imaging , was sensitive to brain changes in patients with mild cognitive impairment , an alzheimer 's disease prodrome [ 1 , 2 ] , to new lesions in patients with multiple sclerosis , and to changes associated with progression in chronic neurological disorders . the higher magnetic field strength afforded by 3 t allows mt image resolution to be augmented compared with conventional mt acquisition at 1.5 t [ 57 ] . we developed a high resolution mt technique to detect subtle changes in anatomically small , functionally eloquent brain structures . the increased field strength affords whole - brain coverage with considerably thinner slices , potentially reducing partial volume artifacts . however , even among healthy subjects , numerous factors may introduce variability in measures derived from magnetic resonance ( mr ) data , such as static field b0 signal dropout and rf nonuniformity . measurement variation may be introduced by scan repetitions , repositioning at different time points , and image post - processing . moreover , 3 t may be susceptible to variation associated with increased field strength . such variability may pose limitations when conducting clinical comparisons to differentiate normal and diseased brains or in developing statistically predictive algorithms . to validate high resolution mt for detecting early disease or for monitoring progression in chronic neurological disease , it is necessary to collect information on normative values and to evaluate the reliability and reproducibility of the measurements when measured across time in healthy controls . this investigation evaluated observer - agreement of high - resolution mt measurements determined from repeated brain scans of 9 healthy volunteers . we evaluated the reliability and reproducibility of the high resolution mt measurements in 12 brain regions of interest ( rois ) , applied statistical measures to the data and used complex multivariate mixed - effects models to test the statistical significance of several effects due to region , subject , observer , time , and manual repetition . the study was approved by the irb at the north shore university health system , and conducted following the ethical principles outlined in the declaration of helsinki . eleven healthy adult volunteers were randomly selected from a database maintained at the center for advanced imaging , radiology department , northshore university health system provided written informed consent and evaluated for eligibility criteria . to protect the subjects ' confidentiality , all data were de - identified and handled according to the guidelines specified by the health insurance portability and accountability act ( hipaa ) in the usa . brain images were acquired using a 3 t general electric ( ge ) hdx system ( waukesha , wi , usa ) . each volunteer was scanned twice in a randomly - selected time interval between 1 to 4 weeks . methods for reducing random errors in image acquisition included the use of a body - coil for excitation to control b1 non - uniformities and an 8-channel quadrature receive - only coil . mt pulses with ( ms ) and without saturation ( m0 ) were applied at an offset frequency from water resonance . to accelerate the scan for whole - brain coverage , while maintaining thin slices , the image protocol was optimized based on 3 t using 3d spgr . the gaussian sinc mt pulse was applied in 8 ms at a 1200 hz offset . the stability of the scanner and set - up procedure were addressed with a fixed set of parameters per subject . mt pulse was based on a three - dimensional spoiled gradient recalled ( 3d spgr ) acquisition . the image protocol included the following parameters : tr 34 to 35 ms , te 4 to 8 ms , imaging fa 5 , bandwidth 15.6 khz , 0.75 nex , phase fov 0.75 , voxel dimensions 0.9 0.9 0.9 ~ 1.3 mm . the whole brain was covered in 90 to 140 slices with acquisition time ranging from 7 minutes 40 seconds to 10 minutes 20 seconds using a partial k - space acquisition . mtr maps were generated off - line on a general electric aw workstation ( general electric , milwaukee , wi , usa ) using the standard equation : ( 1)mtr = m0msm0100% , where ms and m0 were the signal intensities in a given voxel obtained , with and without the mt saturation pulse , respectively . the 12 rois were : genu , splenium , left and right hemispheres of the hippocampus , caudate , putamen , thalamus , and cerebral white matter . each roi was sized approximately 30 to 43 mm and manually and independently placed by observers 1 and 2 ( authors s.s . and y.w . after an initial consensus decision was drawn regarding the sizes and locations of the 12 rois , the observers performed manual segmentations of the roi independently on each set of images . mtr values were extracted using the manually - defined rois with the combinations of observer , time point , and repetition ( table 1 ) . the mean and sds of the roi values were calculated . meta - data were stored in a sas 9.1 ( sas , cary , nc , usa ) dataset , with individual volunteer identification numbers withheld and replaced by a sequence of 1 to 9 for each subject . statistical analyses were performed using sas 9.1 ( sas institute , cary , nc , usa ; http://www.sas.com ) . proc univariate , proc means , proc corr , and proc mixed . let y = yijklm having the indices described in table 1 be a random variable representing the mean roi value . for the mth roi , we first computed the sample mean and standard deviation of all mean roi values : ( 2)mean^(ym)=ym=1nml=12k=12j=12i=19yijklm , sd^(ym)={var ^(ym)}1/2={1nm1l=12k=12j=12i=19(yijklmym)2}1/2 , where nm = i j k l = 9 2 = 72 measurements and the operator the 95-percentile normality range was approximately within the following interval , with the following lower and upper bounds : ( 3)(mean^(ym)2sd^(ym ) , mean^(ym)+2sd^(ym ) ) . the term normality range as used in europe , could be arbitrarily - defined according to the number of standard deviations away from the mean . thus , it should not be viewed as the range of the entire dataset , but rather an interval useful for estimating the population value by one or several standard deviations away from the mean . here the critical value of 2 was chosen as recommended by bland and altman . additionally , we justified using a student 's t - distribution with nm 1 = 71 degrees of freedom . for any tail probability of /2 ( e.g. , 0.025 for a 95-percent normality range ) , we used the quantile of the corresponding to particular t - distribution , such that ( 4)tnm11(12)=t711(0.975)=1.994 , this value happened to be close to the recommended multiplier of 2 . we first explored and measured the concordance between the various measurements fully nonparametrically via spearman 's rank correlation coefficient . suppose that we correlated the roi values by observers j = 1 and j = 2 , then denoted the marginal ranks , rijklm = ranki(yijklm ) and rijklm = ranki(yijklm ) , respectively , for all j j with j = 1 and j = 2 . the sample version of pearson 's product - moment correlation coefficient between the ranks of the data was equivalent to spearman 's rank correlation coefficient : ( 5)cor^(rijklm , rijklm)=(nm/2)l=12k=12i=19(ri1klmri2klm)l=12k=12i=19ri1klml=12k=12i=19ri2klm{(nm/2)l=12k=12i=19ri1klm2}1/2{(nm/2)l=12k=12i=19ri2klm2}1/2,=l=12k=12i=19(ri1klmri2klm)(nm/2)ri1klmri2klm(nm/21)sd(ri1klm)sd(ri2klm ) . where denotes ( l = 1k = 1i = 1ri1klm ) and denotes ( l = 1k = 1i = 1ri2klm ) . assuming that there was no presence of any ties since the roi values were of continuous random variables , the spearman 's rank correlation coefficient between observers j and j was ( 6)corr(rijklm , rijklm)=16l=12k=12i=19diklm2(nm/2)(nm2/41 ) , where the difference of an arbitrary pair of marginal ranks for observer j and j was denoted by diklm = rijklm rijklm , for all j j. consequently , all of the raw mean roi values were converted to their marginal ranks and the differences between the ranks of each observation on the two variables spearman 's rank correlation coefficient was also computed for the roi values between any two different time points k = 1 and k = 2 . bar diagrams were made to display the spearman 's rank correlation coefficients between observers or time points for each roi . we used the normalized measure of dispersion of a distribution to evaluate the reproducibility of the measurement . the measure was the coefficient of variation ( cv ) , defined as the ratio of the sd to the mean . ( 7)cv^(ym)=sd^(ym)mean^(ym ) , where both the numerator ( i.e. , sample sd ) and the denominators ( i.e. , sample mean ) in the above expression for cv are provided in ( 2 ) . skewed data , such as those generated by an exponential distribution for which the underlying population mean and standard deviation would be equal , and thus the cv became 1 . < 1 would generally represent low variability , and cv > 1 would represent high variability . as in ( 4 ) and ( 6 ) , further stratified computations of cv for different observers , time point , or repetitions were achieved using formulae similar to ( 7 ) . as overall variability was likely a result of the effects illustrated in table 1 . we employed a multivariate mixed - effects regression analysis to direct model the roi values . a variance - component approach has advantages over many stratified analyses , especially studying studies with a limited sample size . here , because of the novel imaging modality using mt and 3 t acquisitions with labor - intensive manual segmentation procedures , large number of subjects would not have been feasible . to conduct an analysis of variance ( anova ) based on the various effects , a distributional assumption of normality was necessary and convenient . we would demonstrate ( see section 3.4 ) that the normality assumption was generally satisfactory . thus , we could then consider adopting a linear random - effects model with all pair - wise interactions , in addition to a third - order interaction term : ( 8)yijklm=m+si+oj+tk+rl + sioj+sitk+sirj+ojtk + ojrl+tkrj+ojtkrl+ijklm , i=1, ,9 , j=1,2 , k=1,2 , l=1,2 . the effects represented the following : m as intercept , si as subjects , oi as observers , ti as time points , ri as repetitions , and ijklm as the error team . a random - effects model assumed that each of the effects would have independent normal distributions with mean and variance . if normality had failed and because the data were mean roi values that were positively - valued , we would recommend a box - cox transformation , h(yijklm , ) , of the outcome variable with an optimal power coefficient [ 1719 ] . note that the log - normal becomes a special case when the power coefficient = 0 . this normality transformation is given by : ( 9)yijklm=h(yijklm,)={yijklm1,0log ( yijklm),=0i=1, ,9 , j=1,2 , k=1,2 , l=1,2 . a profile log - likelihood , llik of given the observations yijklm , would be maximized to estimate an optimal box - cox transformation via a nonlinear minimization routine , where the log - likelihood was ( 10)llik(yijklm)=nmlog { sd(yijklm)}+(1){i=1nmlog ( yijklm)}+c , where c was a constant free of the power coefficient to be optimized . due to the limited number of subjects , however , even with an optimal normality transformation , over - fitting and non - convergence might be issues . alternatively , we could regard all of the observers , time points , and repetitions as fixed and specify a mixed - effects model . the significances of the sources of variability were tested via a restricted maximum likelihood ( reml ) approach . for our multivariate analysis , the significance threshold for two - tailed p - values was set if p .05 . stratified by the time points within each roi , a two - way anova was performed by regarding all of the observers , time points , and repetitions as fixed . we specified a mixed - effects model for simplicity . due to the complexity of the variance components for example , all subjects were segmented by the same observers who were from an entire population of observers . in other words , the subject effect was always assumed to be random , while the remaining effect ( e.g. , here the observer ) was assumed to be fixed . we simplified our notations by only keeping the indices for the subject and observer effects of interest . we decomposed the data as follows : ( 11)yij=+si+oj+sioj+ij , i=1, ,9 , j=1,2 , where the subject effect si was assumed to be random in an upper - case letter , which had a normal distribution with mean 0 and variance s , for all i = 1 , i = 9 ) ; the observer effect oj was considered to be a fixed effect in a lower - case letter , with the constraint j = 1oj = 0 , with the corresponding parameter to the variance being o = ( 1/(j 1))j = 1oj , for all j = 1 , , j ( here j = 2 ) ; the interaction term between the subject and the observer si oj was the degree to which the jth observer departed from his or her usual rating tendencies for the ith subject , which had a normal distribution with a mean of 0 and variance so ; the errors terms ij were assumed to have an independent and identical distribution ( iid ) normal distribution with a mean of 0 and variance e . for the same ith subject , the effects are further assumed to be subjected to the constraint j = 1(so)ij = 0 over all of the observers . shrout and fleiss gave the true definition of icc using the variance ratio of the subject variance over the total variance , with its estimated version using the quantities via anova ( table 3 ) : ( 12)icc=s2so2/(j1)s2+so2+e2,icc^(3,1)=bsmsemsbsms+(j1)ems . similar to the analysis described above , we adopted a hybrid approach by considering two effects at once , with the subject effect always assumed to be random and the time point assumed to be fixed . the associate model was given by ( 13)yij=+si+tk+sitk+ik , i=1, ,9 ; k=1,2 . as in ( 12 ) , the estimated intraobserver agreement and its estimate were provided by : ( 14)icc=s2st2/(k1)s2+st2+e2,icc^(3,1)=bsmsemsbsms+(k1)ems , where the interaction term the interaction term between the subject and the time si tk had a normal distribution with a mean of 0 and variance st . we performed a sensitivity analysis by computing 6 different icc values shrout and fleiss previously proposed assumptions for iccs ( table 4 ) . a sas macro , written by professor robert hamer , university of north carolina school of medicine , chapel hill , nc , usa ( http://www.bios.unc.edu/~hamer ) , was run to perform the various icc computations . eleven healthy adults provided written informed consent to be evaluated and 9 underwent brain scans . mean age of participants who received scans was 37.9 14.2 years ; 7 participants were men and 2 were women . the mean roi values varied across different region ( table 5 ) . the left and right hemispheres tended to yield similar results when the average over these healthy subjects was considered . spearman 's rank correlation coefficients showed that a majority of correlations within each observer was above 0.5 , suggesting a moderate to high concordance ( figure 3 ) . time point 2 tended to yield higher concordance between the observers , which suggested a possible learning effect over time ( figure 4 ) . due to limited sample sizes in this pilot study , in figures 3 and 4 , we demonstrated the effect of observers by averaging over repetitions by each observer . similarly , we demonstrated the effect of time points by averaging over repetitions at each time point . overall , cvs ranged from 1.2% in the genu for observer 2 to 7.0% in the right hippocampus for observer 1 ( table 6 ) . since all of the cvs were within 7% , that is , the tests of the normal distribution assumption marginally using the shapiro - wilk test indicated that only occasionally ( e.g. , for left caudate , left and right putamen , and right hippocampus ) , this assumption was not met ( see table 7 ) . therefore , it was reasonable to specify linear mixed - effects modeling and two - way anova reported in sections 3.5 and 3.6 . at time point 1 , iccs were greater than 0.7 in regions of genu , left and right putamen , whereas iccs were from 0.5 to 0.7 in regions of splenium , left and right hippocampus , left caudate , and right cerebral white matter ( table 8) . these results indicated moderate to strong interobserver reliability . in comparison , at time point 2 , iccs were greater than 0.7 in regions of genu , splenium , left and right caudate , putamen and cerebral white matter , and left hippocampus and thalamus , while iccs were from 0.5 to 0.7 in right hippocampus and thalamus . however , for some rois such as the left cerebral white matter , right caudate , right thalamus , iccs increased from 0.2 ( at time point 1 ) to 0.9 ( at time point 2 ) , making it difficult to determine whether this represents a learning effect . at each time point , intraobserver agreement was at least 0.5 for a majority of the regions ( table 9 ) . six different methods for generating iccs exhibited similar patterns for high vs. low reliability results in different rois ( table 10 ) . these mathematical and statistical methods may easily be generalized to practical studies with larger sample sizes or to studies of patients with active disease . we acquired repeat brain measurements based on a high resolution mt imaging protocol at 3 t in 9 healthy adults . our results indicate moderate to high reproducibility , supporting the validity of this method for further studies . overall , higher intraobserver reliability was observed at the second time point than that at the initial time point , suggesting a possible learning curve effect for both observers . interobserver reliability was generally lower than intraobserver variability , suggesting a strong observer effect in this comparison , which may be a factor in future investigations using mt imaging . our analyses examined different aspects in a typical observer - agreement study , using measures for concordance , reproducibility , reliability , variance - component analysis , and multivariate analysis . in other studies , all or some of such methods may be considered . however , with a simpler study of either several observers , or one observer with several repetitions at different sessions or time points , then these scenarios may only require several of our methods . only a small sample of healthy volunteers was evaluated in this initial pilot study . therefore , the generalization of the 95-percentile normality range may be limited with respect to the wider spectrum of brain mechanisms represented in the broader population . for instance , demonstrating summary measures using all possible observer and time point combinations may not lead to meaningful interpretations in all cases . nevertheless , since the technology is new , this research may provide useful pilot information for future investigations . moreover , the statistical methods employed and illustrated here may easily be generalized to studies with larger sample sizes and diseased subjects . another limitation was that this study aimed to evaluate only the reproducibility and reliability , rather than the accuracy in a more comprehensive validation study . in the absence of a true gold standard , such as one based on digital phantoms where realistic variability may still not be simulated , or on histopathology , improved reliability further research would benefit from a useful algorithm to perhaps statistically and optimally estimate the underlying spatial ground truth [ 22 , 23 ] . finally , future research may be directed to evaluating the diagnostic utility of high resolution mt for early detection of alzheimer 's disease , multiple sclerosis or other neurological disorders and for monitoring progression across the clinical course .

magnetization transfer imaging ( mt ) may have considerable promise for early detection and monitoring of subtle brain changes before they are apparent on conventional magnetic resonance images . at 3 tesla ( t ) , mt affords higher resolution and increased tissue contrast associated with macromolecules . the reliability and reproducibility of a new high - resolution mt strategy were assessed in brain images acquired from 9 healthy subjects . repeated measures were taken for 12 brain regions of interest ( rois ) : genu , splenium , and the left and right hemispheres of the hippocampus , caudate , putamen , thalamus , and cerebral white matter . spearman 's correlation coefficient , coefficient of variation , and intraclass correlation coefficient ( icc ) were computed . multivariate mixed - effects regression models were used to fit the mean roi values and to test the significance of the effects due to region , subject , observer , time , and manual repetition . a sensitivity analysis of various model specifications and the corresponding iccs was conducted . our statistical methods may be generalized to many similar evaluative studies of the reliability and reproducibility of various imaging modalities .

nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively . they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) . all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study . the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) . they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) . the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) . the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat . the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility . two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) . the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) . cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue . the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt , the riders performed a self - paced warm - up ( approximately two laps on the tt course ) . the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt , the riders had access to hr , speed , distance , and power output data . wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively . environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively . relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air ) , the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively . power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt . hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) . rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) . in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter . body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) . continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova . anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) . effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) . data are presented as mean sd along with the mean differences ( 95% confidence interval ) . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) . speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) . speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) . average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) . this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) . 1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction . the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig . 1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig . power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig . power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig . power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively . 2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) . the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) . consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) . hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig . 2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig . 2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 , the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance . the current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists . the cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc . however , while maintaining similar hr , they subsequently experienced a marked decrease in power output and speed in the heat . these decrements were progressively restored with heat acclimatization , despite core temperature in all tt in the heat increasing significantly more than that in cool conditions . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) . in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants . these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) . however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined . our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 ) relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement , our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) . one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) , this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig . 1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig . our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity . indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate . although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) . it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig . moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) . rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) . given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) . this similar work rate adopted seems to correspond to a critical power ( 16 ) . given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) . however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . initial power output was similar between trials and decreased thereafter in relation to acclimatization state . indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) . consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow . the current study is the first to investigate cycling performance during outdoor tt in a hot environment . however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study , the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) . the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% . consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study , air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) . thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different . the novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output , irrespective of the environmental conditions or previous experience . consequently , non heat - acclimatized cyclists are incapable of sustaining this absolute effort . however , the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk . furthermore , our data seem to confirm that sustainable power output is related to a given relative intensity ( 24 ) , which is partly reflected in the maintenance of hr within a certain range . finally , because of the reduction in air density associated with cycling in hot ambient conditions , speed was not different between tth-3 and ttc .

abstractpurposethis study aimed to determine the effects of heat acclimatization on performance and pacing during outdoor cycling time trials ( tt , 43.4 km ) in the heat.methodsnine cyclists performed three tt in hot ambient conditions ( tth , approximately 37c ) on the first ( tth-1 ) , sixth ( tth-2 ) , and 14th ( tth-3 ) days of training in the heat . data were compared with the average of two tt in cool condition ( approximately 8c ) performed before and after heat acclimatization ( ttc).resultstth-1 ( 77 6 min ) was slower ( p = 0.001 ) than tth-2 ( 69 5 min ) , and both were slower ( p < 0.01 ) than ttc and tth-3 ( 66 3 and 66 4 min , respectively ) , without differences between ttc and tth-3 ( p > 0.05 ) . the cyclists initiated the first 20% of all tt at a similar power output , irrespective of climate and acclimatization status ; however , during tth-1 , they subsequently had a marked decrease in power output , which was partly attenuated after 6 d of acclimatization and was further reduced after 14 d. hr was higher during the first 20% of tth-1 than that in the other tt ( p < 0.05 ) , but there were no differences between conditions from 30% onward . final rectal temperature was similar in all tth ( 40.2c 0.4c , p = 1.000 ) and higher than that in ttc ( 38.5c 0.6c , p < 0.001).conclusionsafter 2 wk of acclimatization , trained cyclists are capable of completing a prolonged tt in a similar time in the heat compared with cool conditions , whereas in the unacclimatized state , they experienced a marked decrease in power output during the tth .

nonalcoholic steatohepatitis ( nash ) is a progressive form of non - alcoholic fatty liver disease ( nafld ) . the pathogenesis of nash is multifactorial , indicating a significant association with inflammation . in its severe form , nash can lead to cirrhosis , with permanent liver damage ( 1 , 2 ) . patients with nash show significantly higher serum levels of tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) , compared with patients with simple steatosis ( 3 ) . tumor necrosis factor - alpha is produced by several types of inflammatory cells and tissues , such as monocytes , macrophages , neutrophils , endothelial cells , neuronal tissue , t cells , hepatocytes , and kupffer cells ( 4 ) . the tnf- is significantly involved in the development of nash and nafld in animal models and humans ( 5 - 8 ) . interleukin-6 plays a pivotal role in liver pathology and synthesis of acute phase proteins , such as c - reactive protein and serum amyloid a ( 9 ) . on one hand , il-6 can improve liver regeneration in il-6 knockout mice with diet - induced nash and , on the other hand , it can promote hepatocyte apoptosis in patients with nash ( 10 - 12 ) . interleukin 8 ( il-8 ) is produced by peripheral blood mononuclear cells , macrophages , and infiltrated lymphocytes . ( 13 ) reported that il-8 , alanine aminotransferase ( alt ) , age , and adiponectin were independently associated with nash . pentoxifylline ( ptx ) is a methylxanthine derivative and an antitnf- agent , which is reportedly administered for the treatment of nash . it can decrease the production of several pro - inflammatory cytokines , including tnf- and shows anti - inflammatory properties , through the inhibition of nuclear factor - kappa b ( nf-b ) ( 14 - 16 ) . the results of several studies , involving the use of ptx in patients with nash , have shown the effect of ptx in nash . published studies have several limitations , due to variation in characterization of patients , such as those with biopsy - proven nash or nash based on sonography and alt and aspartate transaminase ( ast ) levels . differences in study designs , such as prospective cohort studies , with control group , or randomized and controlled trials and , also , difference in sample size are other reasons for different findings of studies on the effects of ptx , in patients with nash ( 17 - 24 ) . the primary aim of this study was to evaluate the effectiveness of ptx for 6 months , in a randomized , double - blind , placebo - controlled clinical trial , in patients with nash , in the city of kerman , in south - eastern iran . the other aims were to assess the effect of ptx on the serum levels of alt , ast , tnf- , il-8 , and il-6 in patients with nash . thirty patients with nash were recruited at the kerman university of medical sciences , besat clinic , kerman , south - eastern iran . written informed consent was obtained from each participant in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki . patients were considered for the study if they had a diagnosis of nash , based on sonography , and a 1.5-fold mean change from baseline of ast and alt levels . patients were excluded if they had a past history of excessive alcohol consumption or any cigarette smoking or narcotics use . patients with hypertension ( 140/90 mmhg ) and those who tested positive for hepatitis c virus or hepatitis b virus infection were also excluded . additionally , patients were excluded if they had high serum levels of alpha-1-antitrypsin , ceruloplasmin , anti - smooth muscle antibody , and antinuclear antibody . patients were also excluded if they were taking anti - hypertensive , anti - hyperlipidemic , anti - hyperglycemic medications , or amiodarone or oral contraceptive pills . patients who were suffering from acute or chronic liver disease were excluded before and during the intervention . the study was designed as a randomized , double - blind , placebo - controlled trial . the study protocol was approved by the ethics committee of kerman university of medical sciences , kerman , iran . the study was designed for 30 patients ; 15 patients ( frequency : male = 80% , female = 20% , age = 35.7 6.6 years ) in the intervention group , who received a ptx dose of 400 mg thrice a day , for 6 months , and 15 patients ( male = 80% , female = 20% , age = 39.4 9.4 years ) in the placebo group , who received a placebo for 6 months . all patients attended the gastroenterology clinic , at afzalipour hospital , and were given advice concerning low calorie diet and the importance of daily exercise by a resident in gastroenterology . there were no differences with regard to shape , taste , and color between the placebo and ptx . the placebo and ptx were labeled by an unblinded pharmacist , as a and b , respectively , before random allocation to the patient . patients , the medical examiner , laboratory and pharmacy staff did not know the nature of the drugs . on completion of data collection , statistical analysis was performed by a statistician who was unaware of the nature of the compounds represented by a and b. fifteen untreated healthy subjects were enrolled to determine the serum levels of cytokines , to present a cut - off point when comparing with that of the patients . demographic data for the 15 controls were as follows : males = 36% , females = 64% the healthy controls , selected from kerman blood transfusion centre , kerman , iran , had no history of systemic or organ specific autoimmune diseases , allergy , infectious diseases , cancer , and metabolic disorders . laboratory studies were performed to measure levels of ast and alt , in patients with nash , at the baseline , 3 months , and 6 months . the serum levels of il-6 , il-8 , and tnf- were also measured in patients at baseline and 6 months . weight and height of the patients were measured , to determine body mass index ( bmi ) at baseline and during the clinical trial at 3 and 6 months . the height of placebo and intervention groups at baseline , 3 months and 6 months was 171.2 9.8 and 172.8 7.2 , respectively . the weight of the placebo group at baseline , 3 months and 6 months was 82.2 13 , 80.2 13 and 77.7 13.1 kg , respectively , and the weight of the intervention group at baseline , 3 months and 6 months was 80.8 12.5 , 78.2 13.1 and 73.9 12.7 kg , respectively . sampling lasted 6 months . during this period , five patients in the intervention group stopped treatment , four of which because of adverse effects and one for personal reasons . nine patients in the placebo group stopped treatment , five of which because of problems similar to adverse effects of ptx and four for personal reasons . liver aminotransferases , including alt and ast , were measured for patients with nash by enzymatic photometry . serum levels of il-6 , il-8 , and tnf- were determined for patients with nash and healthy controls by an enzyme - linked immunosorbent assay ( elisa ) technique , according to the manufacturer s instructions ( bosterbio , pleasanton , ca , usa ) . according to the previous study ( 18 ) , by considering alpha error of 5% and the power of 90% and also employing the formula for two - sample comparison of means , the sample size was calculated to be nine , for each group . statistical analyses , such as descriptive statistics , repeated measurement test , one - way anova , and independent t - test were performed by spss software version 17.0 ( spss inc . , thirty patients with nash were recruited at the kerman university of medical sciences , besat clinic , kerman , south - eastern iran . written informed consent was obtained from each participant in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki . patients were considered for the study if they had a diagnosis of nash , based on sonography , and a 1.5-fold mean change from baseline of ast and alt levels . patients were excluded if they had a past history of excessive alcohol consumption or any cigarette smoking or narcotics use . patients with hypertension ( 140/90 mmhg ) and those who tested positive for hepatitis c virus or hepatitis b virus infection were also excluded . additionally , patients were excluded if they had high serum levels of alpha-1-antitrypsin , ceruloplasmin , anti - smooth muscle antibody , and antinuclear antibody . patients were also excluded if they were taking anti - hypertensive , anti - hyperlipidemic , anti - hyperglycemic medications , or amiodarone or oral contraceptive pills . patients who were suffering from acute or chronic liver disease were excluded before and during the intervention . the study was designed as a randomized , double - blind , placebo - controlled trial . the study protocol was approved by the ethics committee of kerman university of medical sciences , kerman , iran . the study was designed for 30 patients ; 15 patients ( frequency : male = 80% , female = 20% , age = 35.7 6.6 years ) in the intervention group , who received a ptx dose of 400 mg thrice a day , for 6 months , and 15 patients ( male = 80% , female = 20% , age = 39.4 9.4 years ) in the placebo group , who received a placebo for 6 months . all patients attended the gastroenterology clinic , at afzalipour hospital , and were given advice concerning low calorie diet and the importance of daily exercise by a resident in gastroenterology . there were no differences with regard to shape , taste , and color between the placebo and ptx . the placebo and ptx were labeled by an unblinded pharmacist , as a and b , respectively , before random allocation to the patient . patients , the medical examiner , laboratory and pharmacy staff did not know the nature of the drugs . on completion of data collection , statistical analysis was performed by a statistician who was unaware of the nature of the compounds represented by a and b. fifteen untreated healthy subjects were enrolled to determine the serum levels of cytokines , to present a cut - off point when comparing with that of the patients . demographic data for the 15 controls were as follows : males = 36% , females = 64% . the healthy controls , selected from kerman blood transfusion centre , kerman , iran , had no history of systemic or organ specific autoimmune diseases , allergy , infectious diseases , cancer , and metabolic disorders . laboratory studies were performed to measure levels of ast and alt , in patients with nash , at the baseline , 3 months , and 6 months . the serum levels of il-6 , il-8 , and tnf- were also measured in patients at baseline and 6 months . weight and height of the patients were measured , to determine body mass index ( bmi ) at baseline and during the clinical trial at 3 and 6 months . the height of placebo and intervention groups at baseline , 3 months and 6 months was 171.2 9.8 and 172.8 7.2 , respectively . the weight of the placebo group at baseline , 3 months and 6 months was 82.2 13 , 80.2 13 and 77.7 13.1 kg , respectively , and the weight of the intervention group at baseline , 3 months and 6 months was 80.8 12.5 , 78.2 13.1 and 73.9 12.7 kg , respectively . sampling lasted 6 months . during this period , five patients in the intervention group stopped treatment , four of which because of adverse effects and one for personal reasons . nine patients in the placebo group stopped treatment , five of which because of problems similar to adverse effects of ptx and four for personal reasons . liver aminotransferases , including alt and ast , were measured for patients with nash by enzymatic photometry . serum levels of il-6 , il-8 , and tnf- were determined for patients with nash and healthy controls by an enzyme - linked immunosorbent assay ( elisa ) technique , according to the manufacturer s instructions ( bosterbio , pleasanton , ca , usa ) . according to the previous study ( 18 ) , by considering alpha error of 5% and the power of 90% and also employing the formula for two - sample comparison of means , the sample size was calculated to be nine , for each group . statistical analyses , such as descriptive statistics , repeated measurement test , one - way anova , and independent t - test were performed by spss software version 17.0 ( spss inc . , the mean ages of the patients in the intervention and placebo groups were 35.7 and 39.4 years , respectively . the difference between the two groups , concerning age , was insignificant ( p = 0.220 ) . the means of bmi in the intervention and placebo groups were 26.9 and 26.6 , respectively , and the difference was statistically insignificant ( p = 0.912 ) . data regarding laboratory parameters and ultrasonography of the liver , for patients with nash are presented in table 1 . the levels of ast and alt , measured at the baseline , 3 and 6 months , are presented in table 2 . results in table 2 were presented after repeated measure anova with greenhouse - geisser correction . post - hoc tests were performed to obtain mean studied parameters between time points and results were statistically significant after bonferroni correction . abbreviations : alt , alanine transaminase ; ast , aspartate transaminase ; bmi , body mass index . significant difference was detected from comparison between the baseline and 3 months and also between the baseline and 6 months . there was no significant difference with regard to the serum level of alt , between the placebo and intervention groups , at baseline ( p = 0.217 ) , at 3 months ( p = 0.446 ) or at 6 months ( p = 0.236 ) . similarly , no significant difference was observed in the serum level of ast between the groups , at baseline ( p = 0.701 ) , at 3 months ( p = 0.857 ) or at 6 months ( p = 0.125 ) . the bmis obtained at baseline , 3 and 6 months , are presented in table 2 . there was no significant difference in term of bmi between the groups , at baseline ( p = 0.912 ) , at 3 months ( p = 0.346 ) , and at 6 months ( p = 0.391 ) . the serum levels of il-6 , il-8 , and tnf- , as mean sd , in healthy controls , were 6.3 3.9 , 18.0 7.5 , and 16.8 11.3 pg / ml , respectively . levels of these cytokines were significantly higher in patients with nash , compared with healthy controls . serum levels of these cytokines were unchanged between baseline and 6 months in the intervention and placebo groups , with the exception of il-8 , where a statistically lower level in the intervention group at baseline had increased at 6 months ( table 3 ) . abbreviations : il-6 , interleukin 6 ; il-8 , interleukin 8 ; tnf , tumor necrosis factor . most clinical trials on the effect of ptx in patients with nash have sufficed to compare the cytokine levels in the intervention and placebo groups , without including the healthy controls . in the present study , patients with nash had significantly higher serum levels of il-6 , il-8 , and tnf- , as compared to healthy individuals , which confirmed the important role of inflammation in the pathogenesis and progression of nash ( 11 , 12 ) . in the present study , ptx decreased the serum levels of liver transferases in patients with nash , without any significant differences between the intervention and placebo groups . the results of a study by van wagner et al . which measured and compared the serum levels of alt , ast , tnf- , and il-6 , in patients with nash during 12 months , shows that their intervention versus placebo clinical trial is in accordance with our findings ( 25 ) . reported serum levels of liver transferases , as well as serum levels of tnf- and il-6 in patients with nash , which were decreased in intervention and placebo groups in a 3-month , randomized , double - blind , placebo - controlled trial ( 17 ) . they recommended daily exercise and a low - calorie diet to both the groups and reported no significant difference between the groups , with regard to the serum levels of tnf- , il-6 , ast , and alt at the end of the clinical trial . their results are also in agreement with our findings . however , our findings are in disagreement with the results of another randomized , double - blind , placebo - controlled trial , which was performed by buranawui et al . on 32 patients with nash ( 26 ) . they reported a significant decrease of alt and ast in the intervention group , as compared to that in the placebo group . they also stated no significant differences in tnf- and bmi between the groups , at the end of a 6-month clinical trial , which is in agreement with our findings . our results are in disagreement with the findings of zein et al . , who showed that liver fibrosis and the histological features of nash could be improved by ptx administration for one year ( 27 ) . georgescu ef and georgescu m carried out a non - randomized and uncontrolled prospective study on an outpatient database to determine the efficiency of several agents , including ptx , for the treatment of nash ( 19 ) . the bmi , serum levels of alt , glutamyl transpeptidase , alkaline phosphatase , total cholestrol , triglycerides and liver biopsies were determined at baseline and at the end of the treatment . they concluded that ptx showed efficacy in treating non - hypertensive / non - dyslipidemic patients with nash . however , this study did not control for the confounding effect of dietary and lifestyle interventions . performed a systematic review on the treatment with ptx in patients with nash until june 2010 ( 28 ) . they extracted their results from six studies , including two randomized , double - blind , placebo - controlled trials and four prospective cohort studies . they showed that the serum levels of ast and alt were significantly decreased in ptx - treated patients . however , there were no significant differences between the serum levels of il-6 and tnf- , in the intervention group , as compared to that in the placebo group . they have suggested performing more well - designed , randomized , controlled clinical trials , with a larger sample size , as well as more prospective observational studies , to investigate the effect of ptx in reducing levels of cytokines . they reviewed all relevant controlled trials of the effect of ptx in patients with nafld / nash from 1997 to july 2013 . two prospective cohort studies with controls and three randomized , double - blind , placebo - controlled trials were included in their meta - analysis . their findings indicated that ptx administration results in improved liver function and histological changes , as well as weight loss , in patients with nafld / nash . one study , which was included in their meta - analysis , had a limitation , as the researchers ignored to evaluate the influence of exercise and a low - calorie diet in ptx - treated patients with nash . the small sample size of several trials and low number of trials were other limitations of the meta - analysis . recently performed a clinical trial to evaluate the effect of ptx on liver aminotransferases , in patients with nafld ( 30 ) . although the levels of liver aminotransferases were reduced during the trial in intervention and control groups , their results did not show significant differences between the groups , which are in agreement with our results . the lack of biopsies from the liver of patients with nash , as a gold standard confirmation method , was a limitation in our study . satapathy et al . showed that five out of six patients with nash improved histologically and with improved liver aminotransferases , particularly alt , after 12 months of ptx ( 21 ) . therefore , they suggested that measurement of liver transferases can be an acceptable criterion for evaluating patients with nash . in general , our results suggest that ptx administration does not significantly help in the treatment of patients with nash . however , other studies show different results and it might be premature to draw conclusions about the efficacy of ptx in the treatment of nash . hence , we suggest that a further randomized , double - blind , placebo - controlled trial should be performed , with the enrolment of more patients with nash from different races , to confirm the effect of ptx on liver aminotransferases , histological findings , and inflammatory cytokines , in patients with nash . employment of modern techniques , such as microarray to evaluate and define more inflammatory cytokines genes , which are potentially involved in nash , may help scientists and clinicians to address the efficacy of ptx therapy on inflammation in the future .

background : non - alcoholic steatohepatitis ( nash ) is a progressive form of nonalcoholic fatty liver disease . several studies suggest that pentoxifylline ( ptx ) can improve the disease outcome.objectives:we aimed to compare the effect of pentoxifylline with placebo on liver aminotransferases and cytokines , including interleukin 6 ( il-6 ) , tumor necrosis factor alpha ( tnf- ) , and interleukin 8 ( il-8 ) in patients with nash.patients and methods : thirty patients with nash were included in the study , based on ultrasonography and 1.5-fold mean change from baseline serum levels of liver aminotransferases . patients with nash were randomized to receive 1200 mg ptx ( the intervention group ) or placebo ( the placebo group ) for 6 months . the serum levels of liver aminotransferases and cytokines were compared between the intervention and placebo groups , at various time points.results:the serum levels of liver aminotransferases were significantly reduced at 3 months and at 6 months , compared with baseline , in both groups . the serum levels of il-6 were significantly decreased , in both groups , only at 6 months , compared with baseline . compared to the placebo group , the serum level of tnf- was significantly decreased in the intervention group , at 6 months . the serum level of il-8 was increased , in both groups , after 6 months , without reaching clinical significance . there was no significant difference in serum levels of liver aminotransferases and cytokines , between intervention and placebo groups.conclusions:decreases in the serum levels of liver aminotransferases and cytokines , in both groups , are related to low - calorie diets and exercise , rather than ptx .

compounds with metals as therapeutic agents for various diseases states have been investigated in the last few decades [ 13 ] . metals can react with different atoms of many amino acids residues in proteins providing therapeutic actions . because of their different mechanism of actions , the development of metal complexes for various drugs provides an alternative route of novel drug delivery system . binding of a drug to metalloelement can enhance or reduce its activity and in some cases the complex may have even such activity that the parent compound does not have . nonsteroidal anti - inflammatory drugs ( nsaids ) are some of the most prescribed drugs worldwide as antipyretic , analgesic , and anti - inflammatory agents . however , the major limitation to nsaid use is the gastric and intestinal mucosal damage . in the uk an estimated 12000 peptic ulcer complications and 1200 deaths per year are attributable to nsaids use . therefore , much has been studied so far to reduce the gastric toxicity of nsaids and in this regard , complex formation of nsaids with transition metals has long been recognized as an effective way of reducing gastric mucosal lesions caused by these drugs . thus , the present study is performed to synthesize transition metal complexes of naproxen ( figure 1 ) , to resolve their characterization , and to observe their relative stability by conducting forced degradation studies . forced degradation is an integral component of validating many analytical methods that indicate stability of the drug and detect different impurities coming from manufacturing processes [ 10 , 11 ] . they facilitate analytical methodology development and validation , better understanding of stability of drug molecules in different environments , and finding out the degradation pathways of drugs and byproducts [ 1214 ] . to the best of our knowledge , a combined study of synthesis , characterization , and forced degradation study of naproxen - metal complexes has never been done yet . but completed studies of the degradation of the drug substance and drug product are required at the new drug application ( nda ) stage . so in our current study we put our effort to synthesize and characterize different transition metal - naproxen complexes along with the determination of their relative stability under various stressed conditions . also , the rp - hplc method for analysis of naproxen outlined in usp has been verified for the drug - metal complexes . all the apparatus and reagents were in analytical grade of merck origin , used without purification , and were available in the laboratory of the department of clinical pharmacy and pharmacology , faculty of pharmacy , university of dhaka . 0.82 gm ( 0.1 m ) of naproxen ( ligand ) was dissolved with 0.1 m of sodium hydroxide solution in water to form the sodium salt of naproxen . the reaction mixture was put on a water bath to evaporate until a crystal film appeared ; upon cooling the white product separated out . equimolar metal salts dissolved in water were added to the above mixture so that the ratio n ( metal ) : n ( ligand ) of monovalent , divalent , and trivalent ions used was 1 : 1 , 1 : 2 and 1 : 3 , respectively , in each case and immediate precipitation occurred . then the solid complexes were isolated by filtration , washed until being free of chlorides with the corresponding solvent ( methanol or water ) , and finally dried at room temperature . hplc system ( shimadzu prominence ) , equipped with uv - visible detector , was used for the analysis of the samples . reversed phase c-18 column ( zorbax eclipse xbd - c18 , 150 4.6 mm , 5 m ) was used to analyze the standards and samples . the hplc assay method described in united states pharmacopoeia ( usp35 nf30 , volume 3 , page 3996 , 2012 ) was used to analyze the samples . it is important that more strenuous conditions than those used for accelerated studies ( 25c/60% rh or 40c/75% rh ) should be used while performing this study . in general , the following conditions were investigated : ( 1 ) acid and base hydrolysis , ( 2 ) hydrolysis at various ph , ( 3 ) thermal degradation , ( 4 ) photolysis , and ( 5 ) oxidation . it was focused on determining the conditions that degrade the drug by approximately 10% . however , beginning at extreme conditions ( 80c or even higher , 0.5 n naoh , 0.5 n hcl , 3% h2o2 ) and testing at shorter ( 2 hours , 5 hours , 8 hours , 24 hours , etc . ) the conditions listed in table 1 were followed in the current study . 1 mg / ml of solutions was prepared of each naproxen - metal chelates . then 1 ml of sample solutions and 4 ml of 1 m hcl were mixed and mixture was kept for 24 hours at room temperature . after 24 hours , the sample solutions were allowed to be neutralized by 1 m naoh to ph 7.0 and the volume was made up to 10 ml with diluting solution . 1 mg / ml of solutions was prepared of each naproxen - metal chelates . then 1 ml of sample solutions and 4 ml of 1 m naoh were mixed and mixture was kept for 24 hours at room temperature . after 24 hours , the sample solutions were allowed neutralized by 1 m hcl to ph 7.0 and the volume was made up to 10 ml with diluting solution . . 1 ml of samples and 9 ml of 10% h2o2 solution were mixed and the mixture was kept for 24 hours at room temperature . reduction of naproxen and its metal complexes was studied using 10% sodium bisulfite for 24 hours . 1 ml of samples and 9 ml of 10% sodium bisulfite solution were mixed and the mixture was kept for 24 hours at room temperature . 1 ml of samples and 9 ml of distilled water were mixed and the mixture was kept for 24 hours at room temperature . 5 mg of each naproxen - metal chelate was placed in an oven for 3 hours at 105c and then the heated samples were dissolved in 5 ml of diluting solution and allowed to attain the room temperature . the analytical method was verified according to united states of pharmacopeia ( usp ) 37 , general information , 1225 , validation of compendial procedures guideline with respect to some parameters used in method validation . according to 21 cfr 211.194(a)(2 ) of the current good manufacturing practice regulations , suitability of all compendia testing methods used shall be verified under actual conditions of use ( usp 37 , general information/1226 verification of compendial procedures ) . in current case , system suitability , solution stability , accuracy , precision , and robustness were performed for method verification . all the complexes synthesized were crystalline solids and soluble in common organic solvents but insoluble in ethanol and acetone . they were characterized by elemental analyses , ir spectra , thermal analysis , electronic photography ( sem ) , and magnetic properties ( nmr ) . the melting points or decomposition temperatures of the chelates are higher which suggests their thermal stability . naproxen decomposes at 153c where the complexes decompose in the range of 218250c ( figure 2(a ) ) followed by complete burning at above 700c . the representative equations for the formation of the complexes can be presented as(1)mn+clnhh2o+nnal = mlnmh2o+nnacl+hm2o ( where m = co , cu , zn , ag , fe ; n = 1 or 2 or 3 ; h = 0 , 2 , 4 , or 6 ; m = 0 or 2 or 3 ) . in this study , the carboxylic acid group of naproxen shows the (c = o ) stretching mode as a band at = 1729 cm . this was gone because of deprotonation and in the sodium salt there were two new bands at 15351546 and 14051414 range , the carboxylate antisymmetric and symmetric vibrations , respectively ( figures 2(b ) and 2(c ) ) . the coordination of the carboxylate ion to metal ion took place in three different ways . the difference between as(coo ) and s(coo ) in monodentate complexes was expected to be greater than 350 cm . when 200 < < 350 cm , anisobidentate was observed which means an intermediate state between monodentate and bidentate and when < 200 cm , the carboxylate groups were regarded as bidentate . these situations were observed in the relative position of the antisymmetric and symmetric stretching vibrations . the main ir bands in the spectra of the sodium salt and the complexes are listed in table 3 . there was a band observed in the region 31453455 which is certainly due to the absorption of crystal or coordination water . assignment of the carboxylate group of these metal complexes coordination depended on the position of both as and s bands and the values of [ 1721 ] . the values of all of these complexes lie in the range of 132159 cm which is close to that of sodium salt of naproxen indicating that the carboxylate group acts as a bridging ligand . in solid state , the synthesized complexes of carboxylate mostly form bridged dimers ( m2l2 or m2l4 ) and also polymeric networks . in the h - nmr spectrum of naproxen , the protons of methyl ( ch3 ) group have a sharp doublet at ~ 1.51.6 ; the methenyl ( ch ) proton has a triplet around 3.603.90 . in case of the methoxy ( ch3o ) protons , they exhibit a sharp singlet at 4.00 and the naphthyl protons appear at 7.107.80 as a multiplet . sequentially all of these protons shift upfield in complexes ; the methenyl proton displays the highest shift 0.250.30 , whereas the methoxy protons shift the least ~ 0.05 . this occurs because of the lesser electron withdrawing capacity of metal ions in the complexes relative to that of the carboxy proton in the ligand . the hydrogen atom of the cooh group is absent in the metal complexes of h - nmr spectra ( range of 1013 ppm ) . this data indicates coordination and the carboxyl group is not protonated and the complexation reaction takes place . scanning electron microscope ( sem ) images were taken in order to study the surface morphology of naproxen - metal complexes . the images showed particles with fiber - like morphology of the complexes compared to ligand ( naproxen ) which is homogenously distributed in the solid powder . the photograph clearly indicated that the complexes are hydrated and they formed dimer or even polymeric networks in micrometer range . the rp - hplc studies were performed in order to determine identity of the new synthetic products in comparison to the free ligand with respect to retention time . hplc methods were used to confirm the appearance of new products after the synthesis had been performed . the samples of ligand and complexes eluted close to each other with similar retention times ( figure 4 ) . in the forced degradation study it was found that naproxen - metal complexes were the most stable compounds against any type of forced degradation condition applied than parent naproxen . the highest degradation of naproxen was found by acid hydrolysis and it was only 7.92% . among the complexes , degradation levels are very close among all these complexes and it is due to the almost same coordination environment of the complexes . the most probable reason for their higher stability than naproxen is the possibility of forming dimer or even polymer structures that is shown in sem images . in dsc study , it was also revealed that the complexes have very high decomposition point than that of the parent naproxen . low quantity of % rsd of area changes demonstrated that the drugs were fairly stable in the diluting solution and in the mobile phase . results were shown in table 7 . accuracy or recovery study was performed and result found in acceptable range for all samples for different concentrations . the % rsd values found in precision study depicted in table 8 showed that the compendial method provides acceptable intra- and interday variations for samples . we changed ph 0.2 , flow rate 50% , wave length 3 nm , and solvent concentration 30% . search for drugs of higher efficacy and lower toxicity is a never ending effort . in our current research we were able to synthesize some naproxen - metal derivatives and to highlight their stability profile under stressed conditions with a view to facilitating the invention of novel nsaids with better therapeutic efficacy . but from the result of present study further useful information was achieved that the metal derivatives of naproxen were found more stable than naproxen itself . this finding suggests that the metal derivatives of naproxen can be more potent anti - inflammatory agent in human body with longer half - life as well as in the dosage form with longer shelf life when compared to the parent naproxen .

the aim of our current research was to synthesize some transition metal complexes of naproxen , determine their physical properties , and examine their relative stability under various conditions . characterizations of these complexes were done by 1h - nmr , differential scanning calorimetry ( dsc ) , ft - ir , hplc , and scanning electron microscope ( sem ) . complexes were subjected to acidic , basic , and aqueous hydrolysis as well as oxidation , reduction , and thermal degradation . also the reversed phase high - performance liquid chromatography ( rp - hplc ) method of naproxen outlined in usp was verified for the naproxen - metal complexes , with respect to accuracy , precision , solution stability , robustness , and system suitability . the melting points of the complexes were higher than that of the parent drug molecule suggesting their thermal stability . in forced degradation study , complexes were found more stable than the naproxen itself in all conditions : acidic , basic , oxidation , and reduction media . all the hplc verification parameters were found within the acceptable value . therefore , it can be concluded from the study that the metal complexes of naproxen can be more stable drug entity and offer better efficacy and longer shelf life than the parent naproxen .

primary mesenchymal tumors of the intestinal tract are a heterogeneous group of tumors with a wide clinical spectrum ranging from benign incidentally detected nodules to frank malignant tumors . traditionally the majority of these tumors were thought to be derived from smooth muscle cells . it has now been realized that the majority of these tumors are not smooth muscle or nerve sheath tumors , but represent a distinct clinico - pathological entity termed as gastrointestinal stromal tumor ( gist ) . it is currently believed that gist is a specific mesenchymal neoplasm and the term is used to refer to those mesenchymal neoplasms of the gastrointestinal tract ( git ) which express cd117 , a c - kit proto - oncogene protein , and show gain of function mutation of c - kit gene that encodes a growth factor receptor with tyrosine kinase activity . the introduction of a new targeted treatment for gist in the form of imatinib mesylate , a receptor tyrosine kinase inhibitor , has further validated this entity . gist can occur at all levels of git and may also arise in extra - gi locations , principally mesentery , omentum and retroperitoneum and occasionally in pancreas . approximately 50 - 60% of gists arise in the stomach , 20 - 30% in small bowel , 10% in large bowel and 5% in esophagus . about 30% of gist are malignant and liver is the most common site for metastasis.[710 ] the criteria for differentiation of benign from malignant gist remain controversial . many parameters have been proposed , tumor size and proliferative activity have been found to be the most important prognostic indicators . gist reported outside the git t as apparent primary tumors are designated as extra - gastrointestinal stromal tumor ( egist).[1214 ] the frequency of egist is only 5 - 7% . because of overlapping morphology , gists are cytologically difficult to distinguish from other gastrointestinal mesenchymal neoplasms including smooth muscle tumors and nerve sheath tumors.[1519 ] although many investigators have described various features used in the cytologic diagnosis of gist , few have reported their findings in patients with malignant gist . the present study is the largest series discussing the cytology of gists and egist from different anatomic sites including extremely rare sites like subcutaneous nodules , fluid cytology samples and metastasis from a primary pancreatic egist . in the present study we studied the cytomorphological features in 33 gist / egist from 27 patients . all patients of histologically and immunohistochemically confirmed gist and egist who underwent cytological examination over a period of ten years ( jan 2000-july 2010 ) were retrieved from the records of the department of pathology . 33 specimens from 27 patients including 26 gist ( 13 primary , 12 metastatic , one recurrent ) and seven egist ( 5 primary , 1 metastatic , 1 recurrent ) were identified . these included guided ( 28 ) and unguided ( 2 ) fine - needle aspirates , imprint smear ( 1 ) and fluid cytology ( 2 ) . fine needle aspiration cytology ( fnac ) was performed using 22 gauge needle attached to 10 ml disposable syringe under ultrasound guidance of intra - abdominal tumors . fna material was smeared on glass slides and slides fixed in 95% alcohol were stained with papanicolaou and hematoxylin and eosin stain while air dried smears were stained with may - grnwald - giemsa stain ( mgg ) . histological categorization of gist of different sites into various risk groups was done as suggested recently by miettinen et al . cytology was reviewed in all cases with emphasis on the following cytological features : overall cellularity , smear pattern ( cohesion vs. dispersed cells ) , palisading , crush artefact , prominent vascular pattern , spindle versus epithelioid cell morphology , nuclear grooves and inclusions , nuclear pleomorphism , presence of nucleoli , round or blunt - ended oval or wavy nuclei , multinucleation / bizarre cells / or giant cells , perinuclear vacuoles , cytoplasmic quality , mitoses and necrosis . the patients included 20 males and 7 females with male : female ratio of 2.67:1 . the mean age was 50.6 years ( range : 26 - 76 , median : 52 years ) . there were 18 primary tumours ( 5 gastric , 5 duodenum , 1 ileum , 1 ileocecal , 1 rectum , 4 intra - abdominal / retroperitoneal tumours , 1 mesentery ) , 9 hepatic metastases ( 5 gastric , 2 duodenum , 1 jejunum , 1 small bowel primary tumours ) , 1 ascitic fluid metastases ( gastric primary ) , 1 pleural fluid metastases ( jejunum primary ) , 1 subcutaneous nodule metastases ( gastric primary ) , 1 lump at base of penis ( rectum primary ) and 2 recurrences ( 1 ileum , 1 intra - abdominal / retroperitoneal ) . the smears were variably cellular . malignant and metastatic lesions were commonly highly cellular . in patients with predominantly spindle cells on cytology ( n = 18 ) smears showed cells often arranged in cohesive to loose three dimensional clusters and singly scattered or dispersed cells [ figure 1a and b ] . the tumor cells often formed fascicles with parallel , side - by - side arrangements of nuclei . in these fascicles tumor cells the stroma of the cohesive sheets present between the nuclei was loosely fibrillary and stained pink to magenta on mgg [ figure 1d ] . skenoid fibres were noted in a smear from liver metastasis of a patient with primary jejunal gist . spindle cells with high cellularity , closely packed to loose clusters and dyscohesive cells ( mgg , 100 ) ; ( a ) spindle cells displaying elongated to wavy nuclei with blunt to tapered ends ( mgg , 200 ) ; ( b ) fascicles with parallel , side - by - side arrangements of nuclei with scant cytoplasm . nuclear palisading is also observed ( mgg , 100 ) ; ( c ) abundant extracellular stromal material ( mgg , 200 ) ; ( d ) ( inset : spindle cells with bipolar cytoplasmic processes ( mgg , 400 ) focal to diffuse epithelioid cell morphology admixed with spindle cells were observed in twelve samples and 3 tumors had only epithelioid cell type [ figure 2a ] . plasmacytoid appearance was noted in eight samples [ figure 2c ] few demonstrated rossetting and acinar structure arrangement reminiscent of adenocarcinoma [ figure 2d ] . the nucleoli were indistinct in low grade tumors and prominent or multiple nucleoli were seen in high grade tumors irrelevant of cell type . the presence of marked cytologic atypia with presence of bizarre and giant cells was identified in 6 tumors . occasional tumors displayed bubbly appearance due to multiple cytoplasmic vacuoles . in 9 cases mitotic figures were observed . the pleural and ascitic fluid cytology smears exhibited loosely formed aggregates with epithelioid cell morphology . smear showing groups of epithelioid tumors cells with round nuclei ( mgg , 200 ) ; ( a ) multinucleation ( mgg , 400 ) ; ( b ) plasmacytoid tumor cells displaying eccentric round nuclei with smooth nuclear membrane and fine chromatin ( mgg , 400 ) ; ( c ) acinar arrangement of tumor cells mimicking epithelial tumor ( mgg , 400 ) ; ( d ) ( inset : mitotic figure in a mixed gist ( mgg , 400 ) histology sections of all primary gist and egist were reviewed . the majority ( n = 18 , 66.67% ) of tumors were classified as spindle cell type , while 3 ( 11.12% ) were classified as epithelioid type and 6 ( 22.23% ) as mixed cell type . the cellularity of the majority of tumors was subjectively assessed as moderate ( n = 17 ) . categorization of gist of different sites into various risk groups was performed as suggested recently by miettinen et al . there was only one low grade tumor , 4 tumors with moderate risk , and 22 tumors were categorized as high risk . immunohistochemically all tumors were positive for cd117 with strong intensity of positivity in the majority of tumors . the cytological studies on gist in literature are not comparable as many of the earlier published cytologic studies have combined gist into a group of neoplasms encompassing leiomyoma , schwannoma , leiomyosarcoma , and an epithelioid leiomyoblastoma.[1517 ] however recently , few authors have established fnac as a reliable method for diagnosing gist and egist before surgical procedure [ table 1].[1830 ] we observed several morphologic features suggestive of high grade gist and egist . in recurrent , metastatic and malignant gist however , it was difficult to find mitoses in the cytologic smears because most of the tumor cells occurred in closely packed cohesive thick tissue fragments . li et al . , found that mitoses in the resected malignant gists were seldom seen in fnac smear . we also found that malignant gists sometimes had no significant pleomorphism in the cytologic smears . nuclear inclusions were more commonly seen in malignant and metastatic tumors irrespective of cell type in our cases . dirty or necrotic background again was least reliable indicator of malignancy as it was seen in two samples only . we agree with earlier descriptions that fna findings alone can not reliably assess behavior of gist . prediction of behavior by cytological features is likely to result in either underestimation or overestimation of malignant potential.[1825 ] summary of various series of gist and egist cytology cases described in the literature the differential diagnosis between gists and gastrointestinal leiomyomas is difficult owing to their overlapping clinical and cytologic features . leiomyoma shows varying cellularity and are composed of bland spindle cells with abundant cytoplasm often having a fibrillary appearance . leiomyosarcomas shows three - dimensional , tightly cohesive , sharply marginated syncytia of spindle cells , often with nuclear crush artefact . benign and malignant nerve sheath tumors show fibrillar cytoplasm and wavy nuclei , similar to a subset of and characteristic features of nerve sheath differentiation , such as nuclear palisading , may be focal . epithelioid gists may cause significant diagnostic confusion with carcinomas , neuroendocrine tumors , and melanoma , particularly when metastatic and even hepatocellular carcinoma . the regular round nuclei , with finely granular chromatin seen in many of cases raised the possibility of neuroendocrine tumor . key features of melanoma are loose aggregates or isolated cells , cellular pleomorphism , enlarged nuclei with macronucleoli , binucleation , multinucleation and intracytoplasmic melanin . intranuclear inclusions , intracytoplasmic bile pigment and no bile duct epithelium are important distinguishing features . the separation of metastatic gist from other metastasis is important since these may respond to imatinib . to conclude , firstly cytology is a useful method for preoperative diagnosis and follow - up of gists and egist . gist show a broad morphologic spectrum on cytology and gist should be considered as a differential diagnosis of tumors with spindle or epithelioid morphology . in an appropriate clinical and radiologic setting the presence of closely packed spindle or oval cells forming fascicles with parallel side - by - side arrangements of nuclei suggests gist . the diagnosis of gist should also be considered in aspirates of the gastrointestinal tract , liver , mesentery , or abdominal wall mass lesions when epithelioid cells are the predominant cell type . the presence of cellular dyscohesion , nuclear pleomorphism , necrosis and mitosis suggest malignant behavior , however absence of these features on cytology is not diagnostic of low risk benign behavior .

background : the term gastrointestinal stromal tumors ( gist ) is used to refer to those mesenchymal neoplasms of the gastrointestinal tract ( git ) which express cd117 , a c - kit proto - oncogene protein.aims:to study the cytological features of gist and extra - gastrointestinal stromal tumors ( egist ) , to correlate them with histology and to determine cytological indicators of malignancy.materials and methods : cytological smears from patients diagnosed as gist / egist on histology were retrieved . from jan 2000 to july 2010 , 26 gist ( 13 primary , 12 metastatic , one recurrent ) and seven egist ( 5 primary , one metastatic , one recurrent ) cytologic samples from 27 patients were identified.results:the patients included 20 males and 7 females with a mean age of 50.6 years . tumor sites included stomach ( 5 ) , duodenum ( 5 ) , ileum ( 2 ) , ileocecal ( 1 ) , rectum ( 1 ) , liver ( 9 ) , retroperitoneum ( 5 ) , mesentery ( 1 ) , subcutaneous nodule ( 1 ) , supra - penile lump ( 1 ) , ascitic ( 1 ) and pleural fluids ( 1 ) . the smears were cellular with cohesive to loosely cohesive thinly spread irregularly outlined cell clusters held together by thin calibre vessels . the tumor cells were mild to moderately pleomorphic , spindle to epithelioid with variable chromatin pattern and variable cytoplasm . cellular dyscohesion , nuclear pleomorphism , intranuclear pseudoinclusions , prominent nucleoli , mitosis and necrosis were more prominent in malignant , metastatic and recurrent tumors.conclusions:gists show a wide spectrum of cytological features and the presence of mitosis , necrosis and nuclear pleomorphism can help in prediction of malignant behavior . further , cytology is a very useful screening modality in patients of gist and egist to detect early recurrence and metastasis at follow - up .

dissemination and implantation of gastrointestinal malignancies throughout the peritoneal cavity result in peritoneal carcinomatosis ( pc ) , which is the second most frequent cause of death in colorectal cancer after metastatic disease to the liver . in 25% of colorectal cancer cases , the peritoneal cavity is the only site of metastatic disease . despite recent advances in the development of systemic chemotherapeutic agents , pc attempts have been made in the past decades to increase long - term survival in patients with pc by combining cytoreductive surgery ( crs ) to remove visible disease and hyperthermic intraperitoneal ( ip ) chemotherapy ( hipec ) to eradicate microscopic viable residual disease , with elevated and persistent drug concentration in the peritoneal cavity . the peritoneal route of chemotherapy administration has the advantage of delivering the agent to the entire peritoneum after all adhesions have been lysed and before they have the opportunity to reform . the peritoneal - plasma division concept allows a tenfold higher concentration of the chemotherapy to be reached in the abdominal cavity in direct contact with residual cancerous cells while minimal systemic absorption and adverse effects are expected . the addition of hyperthermia results in a potentiation of cytotoxicity , improving its capability of penetration into tumoral masses and serving to rewarm the patient after a significant open procedure . the ideal ip chemotherapeutic agent should be one that has maximal efficacy , offering optimal regional therapeutic benefits , while minimizing systemic toxicity . mitomycin c ( mmc ) has been the most frequently used ip chemotherapy for gastrointestinal malignancies . the high molecular weight of mmc limits its systemic absorption and toxicity after ip administration . more recently , based on the efficacy of new systemic agents for patients with metastatic colorectal cancer , oxaliplatin and irinotecan have emerged as good options for peritoneal perfusion [ 6 , 7 ] . although there are no randomized trials comparing mmc with oxaliplatin as ip chemotherapy , a retrospective study with 539 patients with pc associated with colorectal cancer showed that both agents achieve similar median overall survival ( 32.7 vs. 31.4 months ; p = 0.925 ) . however , due to shortage of mmc in brazil , we have been using oxaliplatin as the preferred agent . despite all those potential benefits associated with crs with hipec , morbidity and mortality rates are substantial . large series of patients undergoing crs with hipec report mortality rates ranging from 1 to 8% [ 9 , 10 , 11 , 12 , 13 ] . in the only randomized study ( n = 105 ) comparing systemic chemotherapy ( fluorouracil - leucovorin ) with or without palliative surgery with aggressive crs with hipec , followed by the same systemic chemotherapy regime , the mortality rate in the crs with hipec group was 8% . in addition , surgical morbidity , such as postoperative ileus , anastomotic fistula , and wound infection , as well as medical morbidity , including pharmacological toxicity , cytopenia , bone marrow aplasia , renal toxicity , and hydroelectrolytic disorders , are quite frequent following crs with hipec [ 3 , 9 ] . the most common reported complications include enteric fistula , intraabdominal abscess , pneumonia , small - bowel obstruction , pancreatitis , and neutropenia which often prolong the hospital stay . to the best of our knowledge , , we describe the case of a previously healthy young man who underwent crs with hyperthermic ip oxaliplatin and developed one episode of tonic - clonic seizure on the second postoperative day . a 26 year - old man presented in mid-2011 with increasing abdominal pain . in october 2011 he was found to have a poorly differentiated adenocarcinoma of the transverse colon with multiple peritoneal implants . it was consistent with a pt4 pn2 ( 4/28 ) pm1 colonic adenocarcinoma , kras wild - type . from january to july 2012 , he received 12 cycles of folfiri ( folinic acid , 5-fluorouracil , and irinotecan ) plus bevacizumab at another institution and achieved complete clinical response . at that time a pet - ct scan revealed lesions with increased fdg uptake in the peritoneum as well as in the colonic anastomosis . from june 2014 to june 2015 , he received 24 cycles of folfiri and cetuximab with initial response followed by stable disease . in september 2015 , he was first seen at our institution and , during a multidisciplinary meeting , it was decided to pursue crs with hipec ( oxaliplatin 300 mg / m ) , which was started on october 5 , 2015 . on the second postoperative day , the patient developed one episode of generalized tonic - clonic seizure which lasted for approximately 2 min . he was started on phenytoin to prevent new episodes of seizures during the postoperative period . on october 15 , due to no recurrence of seizures , the anticonvulsant was discontinued . he stayed at the icu for only 72 h and had the first bowel movement 6 days after surgery . on the 7th postoperative day , he developed fever with no identified origin and was started on piperacillin - tazobactam and vancomycin . at this time , the patient was also complaining of left scrotal pain and swelling . an ultrasound of the testis was performed on october 17 and showed normal testicles and increased volume of the left epididymis with heterogeneous echogenicity . in addition , analysis of the epididymal waveform revealed a low - resistance pattern suggestive of acute epididymitis . despite all cultures having remained negative , it was decided to switch antibiotics to amikacin for 10 days and to doxycycline for 14 days and treat the symptoms as infectious epididymitis . the patient was discharged home on october 22 , 2015 , to complete antibiotics at the outpatient unit . . ten percent of adults experience a seizure some time during their life . each year , 300,000 people experience a first epileptic seizure ; most of them are younger than 18 years old . seizures result from a shift in the normal balance of excitation and inhibition within the central nervous system as well as from abnormal brain function . studies indicate that 2530% of first seizures are acute symptomatic or provoked by a brain insult or a metabolic / toxic disturbance of brain function . provoking factors include fever , head injury , excessive alcohol intake , withdrawal from alcohol or drugs , hypoglycemia , electrolyte disturbance , brain infection , ischemic stroke , intracranial hemorrhage , and proconvulsive drugs ( such as clozapine , maprotiline , tramadol , theophylline , and baclofen ) . there are several alterations in the physiology of patients during crs and the administration of hipec . the body temperature increases significantly due to hipec , they experience transient hyperglycemia secondary both to stress from surgery and hyperthermia , as well as the presence of dextrose in the perfusate . however , none of those abnormalities has ever been reported to cause seizures , as seen in our case . there are no published articles regarding neurological complications such as seizures in patients who were submitted to hipec with oxaliplatin . there is , however , one case report of a 37-year - old female patient with a previous history of seizure disorder who underwent crs and hipec with mmc without intraoperative complications and developed signs and symptoms of cerebral edema 4 h after the operation was completed . she eventually died of cerebral edema , with no established etiology . despite a normal eeg , it was hypothesized that crs with hipec could have caused unrecognized partial complex seizures leading to acute cerebral edema . even though our patient had no history of seizure disorder , it is a possibility that the seizure threshold may have been lowered secondary to the stress of surgery and hyperthermia . in fact , seizures are a rare side effect of intravenous oxaliplatin , which are usually associated with posterior reversible leukoencephalopathy syndrome ( pres ) . pres is characterized by neurologic symptoms such as headache , altered mental status , visual disturbances , and seizures with typical lesions on neuroimaging shown as bilateral , subcortical , symmetric , and vasogenic edema . however , our patient had no symptoms or radiologic findings of pres . there is a published case of a previously healthy man with mixed adenoneuroendocrine carcinoma who had seizures after the third and fourth cycles of intravenous folinic acid , 5-fluorouracil , and oxaliplatin ( folfox ) with no signs of pres . after the chemotherapy regimen was switched to folfiri , he never had another event . in this case , the authors attributed the seizures to the intravenous oxaliplatin . our patient had no electrolyte abnormalities , no signs of infection , brain structural alteration , or other metabolic disorder that could possibly explain the single episode of seizure . therefore , it may be possible that the seizure was caused either by the physiological modifications that occurred during hipec or by ip oxaliplatin . because of the increasing use of crs with hipec as an option to treat selected patients with pc , it is important to report those postoperative complications . this article reports an unprecedented case of seizure in a previously healthy patient who underwent crs with ip oxaliplatin due to metastatic colon adenocarcinoma . despite this being a rare case , the corresponding author acknowledges that she is responsible for complying with ethical requirements and declares that the patient was correctly informed , and written informed consent was obtained ; the confidentiality of the patient was strictly preserved . the patient was informed about the submission of the manuscript and will be acquainted when the article is published .

cytoreductive surgery ( crs ) with hyperthermic intraperitoneal ( ip ) chemotherapy ( hipec ) is believed to improve outcomes in well - selected patients with peritoneal carcinomatosis . however , morbidity and mortality rates associated with this procedure are substantial . here , we describe the case of a previously healthy young man who underwent crs with hyperthermic ip oxaliplatin and developed one episode of tonic - clonic seizure on the second postoperative day .

caesarean section may sometimes be the only means to save the life of the mother and/or foetus . current estimates in cameroon put the national caesarean section rate at about 2% , with the lowest rate of 0.4% being reported in the far north region . this is lower than the national rate of 515% of the estimated live births , currently recommended by the united nations . a stillbirth rate of 7% to 12% was reported at the university hospital , cameroon [ 3 , 4 ] . a recent study reported poor foetal outcome of ftus delivered through caesarean section in far north cameroon region and revealed that one of three caesarean deliveries ended up in foetal death . a study in nigeria revealed a higher perinatal mortality of 34% in women who refused elective caesarean delivery compared to 5% for those who accepted the procedure . refusal of caesarean delivery might be due to the lack of detailed information about the procedure . in order to perform caesarean section at right time for safety of the mother and her child , counseling on cesarean delivery the ultimate decision is based on the woman 's obstetric history and the anticipated mode of delivery . the aim of this study therefore was to identify the possible risk factors of caesarean delivery that can be used as content of prenatal counselling in cameroon . we conducted this retrospective case - control study to identify possible risk factors of delivery through caesarean section that can be used as content of prenatal counselling in cameroon . we conducted a retrospective case - control study of caesarean delivery outcomes in the provincial hospital , maroua , cameroon . data was collected retrospectively from delivery and operating room registers between 01/01/2003 and 31/12/2004 inclusive . there were 144 cases of caesarean deliveries during the study period . after excluding cases of multiple pregnancies ( n = 10 ) and cases where complete information was lacking ( n = 9 ) , we were left with 125 cases for analysis . after identification of each case of cs , data from the next successive 2 vaginal deliveries that followed were collected . since it was possible to have consecutive cs before two vaginal deliveries , we finally compared 125 cases of cs with 244 cases of vaginal deliveries . the maternal variables studied included marital status , number of prenatal visits , route of delivery , and maternal death . exact gestational age was not studied because it was not recorded in such cases . in maroua , women are admitted for delivery irrespective of whether they have had prior antenatal care . the chi - square test was used to compare the distribution of the various variables in the two study populations . the odds ratio and the corresponding 95% confidence intervals were used to measure the relationship between mothers ' characteristic and risk of delivery by caesarean section . the sociodemographic characteristics of women delivered by cs ( n = 125 ) and those delivered vaginally ( n = 244 ) are presented in table 1 . the marital status is similar among the two study populations . compared to women delivered vaginally , women delivered by cs were more likely to be adolescents less than 17 years ( 6% versus 12% ) or to be 40 years old and above ( 3.7% versus 7.2% ) . women delivered by caesarean section are more likely to be nulliparous ( 26% versus 40% ) , to be grand multiparous ( 13% versus 25% ) , and to have less than 4 prenatal visits ( 23.2% versus 31.6% ) or no prenatal visit at all ( 2% versus 8% ) . the foetal weight at index delivery is presented in table 3 . women delivered by caesarean section are more likely to have a foetus of 4000 grams and above ( 2% versus 8.8% ) . risk factors associated with cesarean section were maternal age less than 17 years ( or : 3.55 , 95%ci : 1.468.64 ) , maternal age over 39 years ( or : 3.55 , 95%ci : 1.1710.75 ) , nulliparity ( or : 2.72 , 95%ci : 1.594.66 ) , grand multiparty ( or : 3.43 , 95%ci : 1.796.57 ) , and macrosomia ( or : 4.82 , 95%ci : 1.4916.44 ) . the marital status in this study is consistent with the previous findings that almost all women who deliver in far north cameroon are married . this study shows that when the parturient 's age is less than 17 years , the rate of delivery by caesarean section is doubled and the risk of caesarean delivery is tripled compared to women aged 2029 . documented a higher rate of caesarean section in teenagers compared to women in their twenties . lao and ho found that incidence of caesarean section was lower in the teenage mothers . they attributed the good results observed to the free and readily available prenatal care and the quality of support from the family or welfare agencies that were involved with the care of teenage mothers . however , these studies had some limitations ; they included all teenagers until 19 years and are therefore a heterogeneous population [ 9 , 10 ] . in the present study the proportion of teenagers aged 1719 is similar in both study populations ( 19% versus 20% ) . we found that women delivered by cs were more likely to be aged 40 and above ( 3.7% versus 7.2% ) and the risk of caesarean delivery is more than threefold when compared to women aged 2029 years . in a previous study at the university centre hospital , we found that the risk of delivery by caesarean section for women in their forties ( 16.1% ) was significantly higher compared to women in their twenties ( 10.0% versus 16.1% ) hr : 1.7 ; 95%ci : 1.12.8 ; p = 0.027 ) . other studies found an increased risk of caesarean delivery in nulliparous and multiparous women aged 40 years and above [ 12 , 13 ] . the grand multiparity observed in caesarean delivery group of our study ( 13% versus 25% ) could have contributed to the risk of caesarean delivery . women delivered by caesarean section are more likely to be nulliparous ( 26% versus 40% ) and the risk of caesarean delivery is about 3 times for nulliparous women compared to those with parity of 15 . grand multiparous women were mostly in the caesarean delivery group ( 13% versus 25% ) , with the caesarean delivery risk increased by 3 times among grand multiparous women . nulliparous women in maroua are more likely to be teenagers aged less than 17 years and multiparous women are more likely to be 40 and above . women delivered by caesarean section were more likely to have had less than 4 prenatal visits ( 23.2% versus 31.6% ) or no prenatal visit at all ( 2% versus 8% ) . however , only a weak association was found between the antenatal care and the risk of caesarean delivery . this observation underscores that the anc is not effective as such in preventing cesarean delivery in the far north region of cameroon . our findings corroborate with results from the moma group who failed to identify a strong anc parameter to predict the occurrence of dystocia in west africa . however , in spite of the weakness of the positive predictive value , there is a close relationship between severe maternal morbidity and many sociodemographic and obstetric characteristics of the patients . in the present study the frequency of anc care was about one visit per month this could lead to overload of work which in turn limits the possibility of health workers to correctly identify potential risk factors and ensure the continuity of the care till the delivery . however , studies from west africa revealed a high incidence rate of dystocia ( 18% ) and severe maternal morbidity ( 6.7% ) and underscore the need to be alert during anc and delivery . ten risk factors for severe maternal morbidity were identified , and health workers need to be aware of them during delivering anc . these risk factors are hemorrhage during pregnancy , antecedent of cs , high blood pressure , antecedent of multiple pregnancy , height less than 150 cm , lack of fetal movements , history of stillborn , age over 35 years , nulliparity , and presence of disease during pregnancy . the antenatal visit is the right moment to provide the counselling related to this mode of delivery . the basic component of the new world health organization ( who ) antenatal care model prescribes reduced number of clinic visits at 4 and limited investigations for low - risk pregnant women [ 16 , 17 ] . the inadequate antenatal care in our population could have influenced the caesarean delivery because the anc is also the opportunity to anticipate macrosomia and induce labour before the expected delivery date and avoid excessive foetal growth . first , this is a retrospective study and is therefore subjected to some information bias . in addition , due to the limited number of cases in different categories , we did not perform adjusted analysis . however , this study provides baseline information on the topic and gives the opportunity for further prospective studies . caesarean delivery in cameroon is associated with extreme ages of reproductive life , macrosomia , nulliparous and grand multiparous status . these findings suggest that some of the caesarean section cases could be avoided while others could be anticipated . the study provides relevant information for counselling of pregnant women on timely caesarean section when indicated . we strongly recommend that these issues be taken into consideration in order to strengthen the mother and child health programs in cameroon .

we conducted this retrospective case - control study to identify possible risk factors of delivery through caesarean section in the far north region of cameroon . data was collected retrospectively from delivery room registers at the provincial hospital , maroua , cameroon from 01/01/2003 to 31/12/2004 . the overall 125 eligible caesarean deliveries were compared with 244 women who delivered vaginally during the study period . the odds ratio as well as the 95% confidence interval was used to measure the relationship between maternal characteristic and risk of delivery by caesarean section . we found that the marital status is similar in the two study populations . risk factors associated with cesarean section were : maternal age less than 17 years ( or 3.55 , 95%ci : 1.468.64 ) , maternal age over 39 years ( or 3.55 , 95% ci : 1.1710.75 ) , nulliparity ( or 2.72 , 95% ci : 1.594.66 ) , grand multiparty ( or 3.43 , 95% ci : 1.796.57 ) , and macrosomia ( or 4.82 , 95% ci : 1.4916.44 ) . there was a weak association with absent or poor . caesarean delivery is associated with extreme ages of reproductive life , macrosomia , nulliparous and grand multiparous status . we strongly recommend that these factors be taken into consideration to strengthen the mother and child health programs in cameroon and countries with similar socioeconomic profiles .

the adenoid cystic carcinoma ( acc ) is a relatively rare epithelial tumor of the salivary glands . it accounts for about 5 - 10% of all salivary gland neoplasms , representing 2 - 4% of malignant occurrences of the head and neck area . approximately , 31% of lesions affect minor salivary glands , particularly the palate , although they can also be observed in the sub - mandibular and parotid glands . the frequency reported in the tongue is 19.8% , with 85% observed at the base of the tongue . we report one such rare case of tongue neoplasm which turned out to be acc in a middle aged lady . a 45-year - old - female patient presented with an asymptomatic growth of the tongue , which was perceived just 2 weeks before consultation . the intra - oral examination at that time revealed a mass in the dorsum of the tongue with light pain to pressure , without any evidence of cervical lymphadenopathy . the mass was firm , same color as that of the surrounding mucosa and asymptomatic otherwise [ figure 1 ] . as a pre - operative assessment of the lesion , a fine needle aspiration was done and the smear revealed a salivary neoplasm consisting of well delineated , tightly cohesive clusters of basaloid cells surrounding mucoid , hyaline globules , or clear spaces also forming honeycomb ( cribriform ) pattern [ figure 2 ] . at places dense aggregates of monomorphic small cells with uniform round to oval hyperchromatic nuclei and scanty cytoplasm were seen . smears also showed individual tumor cells with high n : c ratio and nuclear moulding . macroscopically , the mass had firm consistency with an irregular form and surface , brown color and measured 2.5 1.5 1.0 cm . the histopathologic study revealed multiple pseudocystic spaces of variable sizes surrounded by cuboidal cells with scarce cytoplasm and oval nuclei , filled with eosinophilic material and hence was consistent with the diagnosis of acc [ figure 3 ] . however , there was no evidence of perineural infiltration on serial sections . clinical photograph showing a swelling on the dorsum of the tongue cytological smears show well - delineated clusters of basaloid cells surrounding hyaline globules with uniform round to oval hyperchromatic nuclei and scanty cytoplasm ( papanicolaou stain , 400 ) histopathological section showing multiple pseudocystic cavities of variable size composed of cuboidal cells with scarce cytoplasm and oval nuclei ( h and e , 400 ) minor salivary gland neoplasms occur less commonly than the major salivary gland tumors and tongue is a relatively uncommon site for salivary gland neoplasms acc is a malignant neoplasm that originates in both the minor and major salivary glands , characterized by slow growth , diffuse invasion , and potential to produce distant metastases , mainly to the lungs and bones . it is an infrequent lesion , as it represents approximately 1 - 2% of all malignant neoplasms of the head and neck , and up to 10 - 15% of all malignant salivary gland neoplasms . the most common intra - oral site for minor salivary gland tumors is the hard palate , followed by the base of the tongue where up to 96% of all tumors are malignant , and acc represents 30% of them . on the other hand , one of the least frequent sites of presentation for acc is the mobile tongue , as several authors have reported an incidence of only approximately 3% of the cases . , analyzed 178 cases of salivary gland tumors , out of which only six cases were located on the tongue . cytologically , cribriform variety of acc can be diagnosed by hypercellular smears composed of clusters of small , relatively monomorphic epithelial cells with hyperchromatic nuclei . these appear bright magenta in may - grunwald giemsa mgg stains and pale blue with papanicolaou stain . finger - like process of similar material can also be found in between the groups of cells in tubular variety . the solid variant of acc also exhibits the same material and the cells resemble that of small cells of anaplastic carcinoma . the globules of amorphous material surrounded by the monomorphic hyperchromatic cells was a clue to the diagnosis of acc in our case , but since the hyaline globules are also found in other tumors like basal cell adenoma , pleomorphic adenoma , polymorphous low grade adenocarcinoma , epithelial myoepithelial carcinoma etc . it is important to distinguish the adenomas from acc because of the conservative mode of management in case of adenomas . we ruled out the adenomas because of the nature of the globules and the cytological morphology . unlike the adenomas , the hyaline globules were dense and stained intensely with mgg and the cells were relatively monomorphic , hyperchromatic with coarse chromatin and irregular nuclear membrane like that of acc . of three histologic variants - tubular , cribriform and solid ; in our case cribriform pattern was the dominant one without any evidence of perineural infiltration . the main factors associated with patient survival were tumor location , clinical stage , and the observed histologic variable . conversely , spiro et al . , have not found histologic classification to be of any benefit , and deny a correlation between microscopic appearance and prognosis . due to the slow growth pattern of the tumor however , due to local recurrence and late metastasis , surgery remains the mainstay of management with or without radiotherapy .

adenoid cystic carcinoma is a relatively rare epithelial tumor of the salivary glands accounting for about 5 - 10% of all salivary gland neoplasms . approximately , 31% of salivary gland neoplasms affect minor salivary glands particularly the palate . it involves tongue in only 19.8% of cases and even rarely the dorsum of the tongue . we report such a rare case that affected dorsum of the tongue in a 45-year - old - female patient .

premature ovarian aging ( poa ) is defined by elevated age - specific basal follicle stimulating hormone ( fsh ) cut - off levels with menstruation . the age - specific cut - off level under the age of 33 years ( our patient 's age ) is 7.0 miu / ml . there are four main causes for pof , namely , idiopathic , genetic , autoimmune , and viral causes . can we extend causes of pof to poa ? if causes are similar and we evaluate women for poa , can we delay the process of pof ? with these thoughts , we evaluated this patient and we are reporting the case . a study by gleicher et al . titled concluded that presumed underlying etiologies of poa follow a similar distribution pattern as reported for pof . they proved the hypotheses that poa is a precursor stage of pof and hence requires similar evaluation . she had an elevated basal fsh level of 28 miu / ml 3 months back . her height was 1.68 m and she weighed 50 kg with a body mass index of 19 kg / m . a transvaginal ultrasound scan showed a normal - sized uterus but ovaries were not visualized . the repeat basal fsh level ( after 6 weeks ) was 27 miu / ml . since the basal fsh level was above the age - specific cut - off level ( 7 miu / ml for 33 years of age ) , diagnosis of poa was considered and karyotype was requested . jacobs et al . described the first association of triple x syndrome with pof in 1959 . a total of 21 cases of pof with triple x syndrome have been reported in the literature , but to the best of our knowledge , this is the first case report of poa with triple x syndrome . genetic causes comprised approximately 16% of the total in the study conducted by gleicher et al . both autosomes and x chromosomal involvement they are turner mosaicism , partial x chromosome deletion , x chromosome mosaicism , x chromosome inactivation , and fmr 1 ( fragile site mental retardation x gene ) . x chromosome partial deletions are more common , while balanced x chromosome to autosome translocation of xq13q26 is rare , but documented . autosomes involved are at the following gene loci : 3q , 13q , 14q , 17q , 15q , and 11p . genetic defects are proposed to cause poa and pof by increasing atresia of ovarian follicles due to apoptosis or failure of follicle maturation and thus decreasing the pool of primordial follicles . triple x syndrome women also suffer from psychiatric disorders like schizophrenia , eeg abnormalities , scoliosis , and genitourinary malformations . poa with triple x syndrome and primary infertility is treated with ovulation induction by gonadotrophins , because of elevated basal fsh values . prenatal diagnosis for pregnant women with triple x syndrome is definitely required as there will be 25% chance of x chromosomal abnormalities in the offspring . hence , we conclude that it is essential to consider karyotyping for all cases of poa , and age - specific basal fsh values will help us detect cases of poa .

genetic aberrations comprise one - third of women with premature ovarian aging ( poa ) . x chromosome abnormalities are seen in these women . we report a case of a 29-year - old lady with primary infertility and poa . she was phenotypically normal and her basal follicle stimulating hormone level was above the age - specific cut - off . karyotype was triple x syndrome .

the clinical effects of toxic alcohols are mainly due to depression of both the central nervous system and myocardial function.1 there are very few published reports of the magnetic resonance imaging ( mri ) features of central nervous system involvement in various types of toxic alcohol poisoning.15 all reports mention cerebral and/or cerebellar toxicity , also known as toxic encephalopathy . there have been no published reports of brain and spinal cord injury secondary to isopropanol ( isopropyl alcohol ) intoxication.15 a 60-year - old caucasian merchant ship captain presented to the emergency department of our hospital with headache , dizziness , and disorientation one day after ingestion of isopropanol along with ethanol ( ethyl alcohol , drinking alcohol ) . he had celebrated his 60th birthday aboard ship by consuming ethanol and then isopropanol , because ethanol was not available later . his colleague accompanying him to the hospital brought an empty bottle of rubbing alcohol labelled as 70% volume per volume isopropyl alcohol with a capacity of 473 ml ( one pint ) . the label on the bottle mentioned its indication for use as a first aid antiseptic and for rubbing and massaging . the patient s colleague mentioned that the patient had consumed most of the liquid in the bottle and that he ( the colleague ) had consumed a small amount . the patient s colleague did not give any recent or past history of substance abuse . an unenhanced computed tomography ( ct ) scan of the brain was performed immediately and was unremarkable . the patient collapsed in the hospital just 30 minutes after the ct scan was done . he became deeply comatose with a score of 3/15 on the glasgow coma scale ( a scale to assess central nervous system status where a total score of 15 indicates best and normal outcome ) . he also developed hypotension , with a blood pressure of 80/40 mmhg , a pulse rate of 65 per minute , and a respiratory rate of 8 per minute . after administration of intravenous fluids and endotracheal intubation , the patient was admitted to the intensive care department . blood investigations revealed leukocytosis of 26,000 ( normal 4,00010,000 ) white blood cells per l , a hemoglobin of 15.7 ( normal 1317 ) g / dl , and a mean corpuscular volume of 93 ( normal 83101 ) fl . the initial arterial blood gas report indicated a ph of 6.731 ( normal 7.357.45 ) , a pco2 level of 28.2 ( normal 3548 ) mmhg , a po2 level of 141 ( normal 83108 ) mmhg , a bicarbonate level of 3.5 ( normal 2128 ) mmol / l , and oxygen saturation of 95% ( normal 95%98% ) . other blood investigations revealed a blood urea nitrogen of 9.5 ( normal 1.78.3 ) mmol / l , serum creatinine of 157 ( normal 62124 ) mol / l , na of 141 ( normal 134146 ) mmol / l , cl of 107 ( normal 96110 ) mmol / l , hco3 of 5 ( normal 2430 ) mmol / l , and capillary blood glucose of 7.2 ( normal 3.35.5 ) mmol / l . his blood lactic acid level was significantly elevated at 8.7 ( normal 0.52.2 ) mmol / ml . blood levels of isopropanol and other toxic alcohols ( like methanol , ethylene glycol , propylene glycol , and diethylene glycol ) could not be obtained because the necessary investigations were not available . investigations repeated at 2-hour intervals revealed increasing renal impairment , hyperglycemia , and electrolyte imbalance ( low bicarbonate levels and hyperkalemia ) . no growth was detected on cultures of urine and blood , and no crystals were found in the urine on microscopic examination . the patient received hemodialysis because of severe acidosis and hyperkalemia , which led to gradual improvement in his blood ph and lactic acid levels . he was also treated with norepinephrine ( as a vasopressor ) and intravenous fluids , but there was no improvement in blood pressure . he received 500 ml of fractionated plasma protein stat over 30 minutes , 2,000 ml of normal saline over 2 hours , and was then kept on 200 ml / hour of normal saline . he did not respond to intravenous fluids and within the first 2 hours was started on norepinephrine 10 g per minute , which was gradually increased up to a maximum dose of 90 g per minute . unenhanced mri scans of the brain and spine performed 6 days after hospital admission showed bilaterally symmetrical hyperintensities on t2-weighted , t1-weighted , t2 * -weighted , fluid attenuated inversion recovery ( flair ) , and diffusion - weighted images in the cerebral and cerebellar cortex and white matter , basal ganglia , thalami , and brainstem ( figures 14 ) . a swollen and edematous cervical spinal cord was noted with t2-weighted and flair hyperintensities within it ( figures 2 and 4 ) . cerebellar tonsillar herniation of 17 mm was noted to be compressing the proximal cervical cord ( figures 2 and 4 ) . petechial hemorrhages were noted in the brainstem and the gangliocapsular regions bilaterally ( figure 1 ) . all the features described above are compatible with toxic brain and cervical spinal cord damage . finally , the patient expired ten days after hospital admission , despite his improving blood picture . chemicals involved in alcohol intoxication are ethanol , methanol , isopropanol , ethylene glycol , diethylene glycol , and propylene glycol . ethanol is considered as a drinking alcohol and the rest as toxic alcohols . among the toxic alcohols , isopropanol itself is more toxic than its metabolite ( acetone ) , while the metabolites of the rest of the toxic alcohols are more toxic than the parent alcohol . isopropanol poisoning is characterized by an increased osmolal gap in the setting of positive serum and urine ketones and does not cause metabolic acidosis , while the rest of the toxic alcohols cause mild to severe metabolic acidosis.1 when not mixed with ethanol or other intoxicants , the signs and symptoms of intoxication may start earlier ( few hours ) after isopropanol ingestion and may be delayed by up to a day or more after ingestion of other toxic alcohols . unlike methanol and ethylene glycol , isopropanol is more toxic than its metabolites ; hence , alcohol dehydrogenase inhibitors are not given.1 hemodialysis removes both isopropanol and its metabolite.1 in our case , the clinical and biochemical features were atypical for isopropanol toxicity likely due to coconsumption of ethanol and delayed patient presentation . ingestion of approximately 200 ml of pure isopropanol can be lethal.1 the patient was managed symptomatically , mainly with hemodialysis . we wish to emphasize the atypical mri features in this case that have not been reported before in cases of toxic alcohol ingestion . in our case , these were likely due to reduced brain perfusion.6 these lesions were bilaterally symmetrical , and the distribution of the affected areas was highly indicative of a toxic injury.6 bilateral involvement of the basal ganglia and thalami has been reported in a few cases of ethylene glycol intoxication . however , cervical spinal cord involvement and cerebellar tonsillar herniation has not been reported before in cases of toxic alcohol ingestion and even in cases of other substance abuse . in humans , the opioid receptors are predominantly present in the cerebellum and the limbic systems , thus causing cerebellar - predominant toxicity in cases of opioid intoxication.6 involvement of the basal ganglia is also noticed in a few of these cases.6 a similar explanation may be given in cases of alcohol intoxication . to the best of our knowledge , this is the first reported case of cerebral , cerebellar , brainstem , and cervical spinal cord involvement on mri secondary to isopropanol intoxication . here we present a very rare case of involvement of cerebrum , cerebellum , brainstem , and cervical spinal cord demonstrated on mri after coconsumption of isopropanol and ethanol . to our knowledge , involvement of the cervical spinal cord and cerebellar tonsillar herniation after toxic alcohol ingestion have not been reported before in the published literature .

a 60-year - old man presented with headache , dizziness , and disorientation one day after consumption of isopropanol along with ethanol . computed tomography ( ct ) of the brain performed immediately was unremarkable . the patient collapsed within the hospital 30 minutes after the ct scan was done , and remained comatose until death , showing no improvement with symptomatic treatment . magnetic resonance imaging of the brain and spine done 6 days after admission revealed bilaterally symmetrical hyperintensities involving the cerebral and cerebellar cortex and white matter , basal ganglia , thalami , and brainstem on t2-weighted , fluid attenuated inversion recovery and diffusion weighted images ; similar hyperintensities were seen involving the swollen and edematous cervical spinal cord and cerebellar tonsillar herniation compressing the proximal cervical cord . petechial hemorrhages were also noted within the brainstem . these features are compatible with toxic injury to the brain and cervical spinal cord . to our knowledge , the magnetic resonance imaging features of brain and spinal cord injury and cerebellar tonsillar herniation , secondary to isopropanol intoxication have not been reported in the published literature before .

chronic unreduced anterior dislocations of the shoulder are not very common . neurological and vascular complications may occur as a result of an acute anterior dislocation of the shoulder or after a while in chronic unreduced shoulder dislocation . we report a case of neglected bilateral anterior shoulder dislocation with bilateral displaced greater tuberosity fracture . to the best of our knowledge , only a handful cases have been reported in literature with bilateral anterior shoulder dislocation with bilateral fractures . delayed diagnosis / reporting is a scenario which makes the list even slimmer and management all the more challenging . we report a case of a 35-year - old male who had bilateral anterior shoulder dislocation and bilateral greater tuberosity fracture post seizure and failed to report it for a period of 30 days . one side was managed conservatively with closed reduction and immobilization and the other side with open reduction . shoulder dislocations should always be suspected post seizures and if found should be treated promptly . the most common bilateral shoulder dislocation is posterior resulting from seizure or convulsion due to epilepsy , electric shock or other reasons [ 2 , 3 ] . simultaneous bilateral anterior shoulder dislocation is usually of traumatic origin and occurs rarely [ 4 , 5 ] . isolated displaced greater tuberosity fractures are thought to occur in less than 2% of proximal humeral fractures , and are normally associated with anterior shoulder dislocations . the greater tuberosity fragment is pulled superiorly by the supraspinatus and posteriorly by infraspinatus and teres minor [ 8 , 9 ] . neglected anterior dislocations are less frequent than neglected posterior dislocations , because anterior shoulder dislocations are more familiar to the orthopaedic surgeon , and their radiological diagnosis is relatively easy . the purpose of this paper is to report a case of neglected bilateral anterior shoulder dislocation with bilateral fracture , its occurrence , diagnosis and management . a 35-year - old male presented to the casualty 30 days after his first episode of seizure with pain in both shoulders and difficulty in movements . patient had not taken any consultations with any doctor post the episode of seizure . past history failed to shed any light as to the cause of the seizure with no history indicating earlier episodes involving head trauma , substance abuse / withdrawl or any pre - existing neurological cause . on initial evaluation , the patient complained of decreased bilateral shoulder function and motion . on examination , normal shoulder contour was lost , shoulder movement was restricted especially abduction with arm in attitude of external rotation and pain was elicited with movement . x - rays showed bilateral anterior shoulder dislocations with displaced greater tuberosity fractures ( fig . 1 ) . pre - operative x - ray both shoulders at the time of presentation . left side closed reduction was done under general anesthesia followed by 3 weeks of immobilization and intermittent physiotherapy ( fig.2 ) . closed reduction of the right shoulder had been attempted but the shoulder was locked and attempts proved futile . during second sitting , open reduction was performed . even after complete release the gt was freed , repositioned and fixed with ethibond after repositioning , the joint was reduced , capsule repaired , joint was stable and was put in an immobilizer . after subscapularis was released , still there had been difficulty in reduction due to capsular adhesions . after releasing the capsule , adhesions were released and greater tuberosity was brought back into position fixed with ethibond and joint was reduced . joint found stable and greater tuberosity was well reduced and in position under c arm . patient was put in a shoulder immobilizer and continued for 3 weeks . for rehabilitation , gentle pendulum exercises were started post operatively , but the patient was non compliant and moved his shoulder joint inspite of instructions . patient came for follow up after 3 weeks and x- ray showed partial displacement of greater tuberosity . however , for displaced greater tuberosity , we decided to continue rehabilitation and monitor progress . at 6 weeks follow up , patient had active assisted forward flexion of 110 degrees , active abduction of 60 degrees . at 12 weeks we report post - operative follow up of 3 weeks and 3 months wherein at the end of 3 weeks the side that underwent closed reduction had full abduction and the side that underwent open reduction had no wound complications , had started pendular movements and was under physiotherapist care for gradual mobilization ( fig . 4 , 5 ) . at the end of 3 months , the side reduced closed had full abduction , no complaints of pain and the side reduced open had 150 - 160 degrees range of motion ( fig . 6 , 7 , 8) with 3 month follow up x - ray also posted showing union of greater tuberosity ( figs . 9 , 3 weeks follow up x - ray . 3 weeks follow up clinical picture with left shoulder showing full abduction . 3 months follow up clinical picture with left shoulder showing full abduction and right shoulder showing nearly 150 -160 degrees abduction . posterior shoulder dislocations usually occur following unbalanced muscle contractions ( electric shock , epileptic seizure etc ) . the posterior dislocations are more common after seizure since the contraction of the relatively weak teres minor and infraspinatus and the posterior fibers of the deltoid are overcome by the more powerful subscapularis leading to internal rotation and posterior subluxation . keeping the above in mind , it is difficult to explain why an episode of seizure would lead to bilateral anterior shoulder dislocation . connor- read l et al suggested that anterior dislocation with seizure may occur not during the muscle contractions but from the trauma of the shoulders striking the floor , after the collapse . this could have been so in the case presented here as well but the patient did not report fall post seizure or could not remember but the presence of similar looking fracture dislocation patterns on both sides is perplexing . even with trauma bilateral anterior shoulder fracture dislocation pattern is difficult as almost always one extremity takes the brunt of the impact . they have a unique mechanism of injury and were first described in 1902 in patients in whom excessive muscular contraction occurred as a result of camphor overdose by mynter . associated fracture of the greater tuberosity occurs in 15% of the anterior dislocation cases and indicates an associated rotator cuff tear . this may cause long term instability and functional impairment if the fragment is not anatomically reduced . thus , internal fixation after the reduction must be the ruled out in such cases . however with regards to the demands of the patient , cost constraints , closed reduction on one side and open reduction on the other without any implant aided fixation was done . mri was not done on either side to evaluate rotator cuff but open reduction was done on one side due to block in closed reduction . long head of biceps or subscapularis tendon , or as is more likely in our case a bony block . a displaced glenoid labrum , a bony fragment from the glenoid rim , greater tuberosity fragment or an impacted humeral head into inferior lip of glenoid are the mentioned osseous causes of an irreducible anterior shoulder dislocation [ 25 - 28 ] . in our case , both soft tissue interposition caused by tissue and capsular contractures as well as bony block due to adherent greater tuberosity seen while doing open reduction were the cause causing interference in reduction . we could find only 5 other reports of bilateral anterior fracture dislocation patterns involving the greater tuberosity which were chronic . yadav reported a case ( also caused by seizure ) of a 56- years - old man , which was reported 6 weeks after injury but the patient was treated with benign neglect and , at 6-month follow - up , no union was seen at fracture site but the patient could perform some functional activities and had accepted possibility of future ailments like arthritis and periarthritis . carew - mccoll described a case that had been caused by electrocution and treated with open reduction and no fixation . at final follow - up , the 78-year- old female patient had abduction 80 bilaterally and no external rotation . thomas and graham treated a similar injury in a 65- year -old woman who had fallen from a bus but not been diagnosed until 8 months later . salem reported the same injury in a 37- year - old man who had been electrocuted at work . diagnosis was delayed 9 weeks , and he was treated with bilateral open reductions without fixation . seth d. dodds published a report involving a 27-year - old man having similar injury post seizure 2 month late presentation treated bilaterally by open reduction internal fixation . closed reduction of a neglected anterior shoulder dislocation can be performed only up to six weeks post injury . after this period the danger of an iatrogenic fracture or neurovascular damage rises too high and operative procedures shall be followed . given the delay in presentation in our patient the possibility of avascular necrosis must also be considered and should be followed up at intervals to assess the same . closed reduction without hampering the soft tissue attachments and blood vessels which would otherwise be in jeopardy in open surgery could be construed as naive as incidence of avn is more common in fracture dislocations involving surgical or anatomical humeral neck . shoulder fracture dislocations best treated acutely . in old cases special attention needs to be given to patient expectations , functional motion , risk of avn and management given taking into account all relevant factors . shoulder dislocation is extremely common after episodes of seizure and should be evaluated clinically as well as radiologically . fractures of greater tuberosity may also be associated with it , hence any attempt at closed reduction should be made only after radiological evaluation with preparedness for open reduction if the need arises .

introduction : shoulder dislocations are a very common entity in routine orthopaedic practice . chronic unreduced anterior dislocations of the shoulder are not very common . neurological and vascular complications may occur as a result of an acute anterior dislocation of the shoulder or after a while in chronic unreduced shoulder dislocation . open reduction is indicated for most chronic shoulder dislocations.we report a case of neglected bilateral anterior shoulder dislocation with bilateral displaced greater tuberosity fracture . to the best of our knowledge , only a handful cases have been reported in literature with bilateral anterior shoulder dislocation with bilateral fractures . delayed diagnosis / reporting is a scenario which makes the list even slimmer and management all the more challenging.case report : we report a case of a 35-year - old male who had bilateral anterior shoulder dislocation and bilateral greater tuberosity fracture post seizure and failed to report it for a period of 30 days . one side was managed conservatively with closed reduction and immobilization and the other side with open reduction . no neurovascular complications pre or post reduction of shoulder were seen.conclusion:shoulder dislocations should always be suspected post seizures and if found should be treated promptly . treatment becomes difficult for any shoulder dislocation that goes untreated for considerable period of time

fine - needle aspiration ( fna ) is the standard method used to determine treatment plans for thyroid nodules . based on the bethesda system , the most generally accepted system for reporting thyroid cytology , the benign category implies a less than 3% risk of malignancy . follow - up ultrasound ( us ) is recommended when the nodule has benign cytologic result . repeat fna is recommended when a nodule shows significant growth or morphologic transformation with suspicious however , the practical risk of malignancy in nodules with benign cytology varies in each institute , ranging from 2% to 18% , and it has even been reported to have gone up to 62% . therefore , some investigators recommend routine repeat fna for thyroid nodules with benign cytology [ 7 , 8 ] . considering cost - effectiveness and diagnostic value , repeat fna has been considered when the nodule shows any suspicious feature on the initial us [ 9 , 10 ] . however , known us features associated with malignancy show an extremely variable probability of malignancy . microcalcifications , marked hypoechogenicity , and irregular or spiculated margin show a high risk of malignancy , while solid composition and hypoechogenicity show a relatively low positive predictive value ( ppv ) [ 1113 ] . based on these results , each suspicious us feature may not be considered as an equal risk factor for malignancy . radiologists specialized in breast imaging have been confronted with the same problem in the core needle biopsy of a breast lesion . correlation of pathologic results with sonographic findings has been used in some institutions to verify that the lesion was adequately sampled . this approach was suggested owing to the wide range of false negative rates of this category . based on different malignancy rates of suspicious us features in thyroid nodules and considering approaching steps in management of breast lesions , we conjecture that an imaging - cytology correlation can be a better diagnostic approach for patient management than initial us features in a thyroid nodule with benign cytology . therefore , we investigated the role of imaging - cytology correlation to reduce the false negative rates of cytology at thyroid nodules as compared with the use of initial us features . the institutional review board of the severance hospital approved of this retrospective study and required neither patient approval nor informed consent for our review of patients ' images and records . however , written informed consents were obtained from all patients for us - guided fnas ( us - fnas ) prior to each procedure as a daily practice . our institutional registry for the thyroid nodule was settled since 2006 including all patients with thyroid nodules who underwent us examinations and us - fnas at our institution . from march 2006 to december 2006 , 3119 consecutive thyroid nodules in 2866 patients underwent us - fnas . among them , we included 667 nodules in 649 patients ( men , 83 ; women , 566 ) , which fulfilled the following criteria : ( a ) they had no history of prior fna on the same nodule ; ( b ) they were reported as benign ( category ii ) in the initial fna . nondiagnostic , atypia or follicular lesions of undetermined significance , follicular neoplasm or suspicious for a follicular neoplasm , suspicious for malignancy , and malignant were excluded ; ( c ) they were equal to or larger than 1 cm ; ( d ) they underwent further evaluation such as follow - up us , follow - up fna , or thyroid surgery . in nodules which had not underwent operation , determinative cytologic reports ( category ii or category vi ) on follow - up us were used as standard reference . if a nodule decreased in size on follow - up us , the nodule was also included as a benign nodule ; ( e ) there were available radiologic reports that included an additional radiologist 's opinion about the concordance or discordance between imaging and cytologic results in postbiopsy correlation ( figure 1 ) . mean lesion size of the thyroid nodules was 20.7 mm ( range , 1070 mm ) . median follow - up of 601 nodules in 584 patients which were included based on followup fna or us without surgical pathology was 1509 days from the date of initial fna to the last followup ( iqr , 1522 days ; range , 1722744 days ) . all us examinations were performed using a 7 to 15 mhz linear array transducer ( hdi 5000 ; philips medical systems , bothell , wash ) or a 5 to 12 mhz linear probe ( iu22 , philips medical systems ) by 1 of 5 board - certified radiologists with 1 to 12 years of experience in thyroid imaging . all us - fnas were performed by the same radiologist who performed the us examinations . us features of all thyroid nodules that underwent us - fnas were prospectively recorded by the previously described methods . us features suspicious for malignancy were determined using previously published criteria from our institution : marked hypoechogenicity , microlobulated or irregular margin , microcalcifications , and taller than wider shape . when overall echogenicity of a nodule was darker than that of the surrounding strap muscle , it was defined as marked hypoechogenicity to differentiate it from hypoechogenicity based on the parenchymal echogenicity of the thyroid gland . only calcifications equal to or less than 1 mm in diameter were indicated . if microcalcifications were detected with macrocalcifications , the lesion was considered to have microcalcifications as a worrisome finding . if hyperechoic foci accompanied comet - tail artifacts on conventional us , they were considered as colloids . an anteroposterior to transverse dimension ratio greater than 1 was defined as taller than wider shape . us - fnas were performed on either thyroid nodules with suspicious assessment or the largest nodule among nodules with probably benign assessment on us . if there were multiple nodules with suspicious us findings in one patient or if the patient or physician requested a biopsy of a benign - looking nodule coexisting with a nodule showing suspicious us features , fnas were performed on multiple nodules in one patient . a free - hand biopsy technique was used with either a 23-gauge needle attached to a 20 ml disposable plastic syringe and an aspirator or a 23-gauge needle attached to a 2 ml disposable plastic syringe , depending on the performing radiologist 's preference . each lesion was aspirated at least twice , and the aspirated materials were expelled onto a slide and immediately placed in 95% alcohol for papanicolaou staining . an inadequate specimen was defined as less than 6 groups of cells containing more than 10 cells . adequate specimens were categorized as benign , indeterminate , suspicious for malignancy , or malignant samples . the radiologist who performed fna routinely reviewed the initial us images within a week of the fna after the cytologic results were reported . for benign cytologic results , radiologists who performed the us - fnas decided and reported whether the cytology was concordant or discordant with the imaging findings . as researchers at our institution always try to assess lesions based on their most worrisome finding , the saved images should represent these worrisome us features . the final conclusion was not derived from the number of suspicious us features but from the subjective decision made by the radiologist who performed the us - fna . in our institution , concordant lesions included some nodules which had suspicious us features on the initial us but were acceptable for the benign cytology in postbiopsy image review as well as the nodules without features suspicious for malignancy on the initial us . concordant benign thyroid nodules were recommended for follow - up by us after one year . in contrast , discordant lesions included nodules which were initially suspected for malignancy on us and were still thought to be suspicious for cancer even after obtaining benign cytology . repeat fnas were usually recommended for discordant benign thyroid nodules after 612 months . among the 667 nodules that met all the inclusion criteria , 586 nodules ( 87.9% , 586 of 667 ) were reviewed by radiologists who had more than three years of experience in thyroid imaging and fna whereas the remaining nodules were managed by less experienced radiologist . we compared the clinical characteristics of patients between benign and malignant nodules by using the test for categorical variables and independent t - test for continuous variables . we also compared the risk of malignancy as well as the clinical characteristics between concordant and discordant nodules by using or fisher 's exact test for categorical variables and independent t - test for continuous variables . the baseline characteristics were also compared between patients with included and excluded nodules among thyroid nodules equal to or larger than 1 cm with the same methods . the risk of malignancy was calculated for several subgroups classified according to initial us features and imaging - cytology concordance . using the generalized estimating equation , we compared the risk of malignancy in thyroid nodules with initially benign cytologic results with those of the remaining subgroups and also compared the risk of malignancy of thyroid nodules among subgroups . logistic regression analysis was performed to assess the odds ratio for the risk of malignancy . statistical analysis was performed using commercial statistical software ( sas version 9.1 , sas inc . , cary , nc , usa ) . among 667 nodules with initially benign cytologic results , 656 nodules were benign ( 98.4% ) and 11 nodules were malignant ( 1.6% ) based on cytopathology ( table 1 ) . the mean age of patients with malignant nodules was not significantly different from that of patients with benign nodules ( p = 0.277 ) . the mean size of malignant nodules ( 17.6 12.5 mm ) was not significantly different from that of benign nodules ( 20.7 10.1 mm , p = 0.315 ) . there were 70 nodules with initial suspicious us features and 597 nodules without initial suspicious us features . the risk of malignancy was higher in nodules with initial suspicious us features ( 11.4% , 8 of 70 ) than in nodules without initial suspicious us features ( 0.5% , 3 of 597 ; p < 0.001 , table 1 ) . when reviewing us images after initial fna results were reported , 40 out of 70 nodules which had suspicious features on initial us evaluation were finally concluded as concordant with benign cytology ( figures 2 and 3 ) . therefore , in 667 nodules with benign cytology , 637 nodules were concordant with cytology , whereas 30 nodules were discordant with benign cytology . the reasons that 40 nodules with revised radiologic diagnosis after imaging - cytologic correlation were initially classified as suspicious nodules were microcalcifications ( n = 16 ) , microlobulated or irregular margin ( n = 9 ) , taller than wider shape ( n = 3 ) , or marked hypoechogenicity ( n = 1 ) in order of frequency , respectively , and more than one characteristic of the above features in 11 nodules . between the concordant and discordant group , gender of the patients was not significantly different ( p = 0.159 ) . the patients with discordant nodules were significantly older than other patients with concordant nodules ( 53.5 10.5 years versus 48.9 12.0 years ; p = 0.038 ) . the mean size of discordant nodules was significantly smaller than that of concordant nodules ( 16.0 6.6 mm versus 20.9 10.2 mm ; p < 0.001 ) . 3% ; 7 of 30 ) than in the concordant group ( 0.6% , 4 of 637 ; p about 44.5% ( 534 of 1201 ) of 1 cm or larger nodules with benign cytology in initial fna were excluded because they had neither standard reference , such as follow - up us , follow - up fna , or thyroid surgery , nor available radiologist 's additional reports regarding imaging - cytologic correlation . the mean age of patients with included nodules was statistically different from the other patients ( 49.1 12.0 versus 50.7 13.1 years ; p = 0.033 ) . patient gender ( p = 0.392 ) and mean nodule size ( p = 0.601 ) were not significantly different between included and excluded nodules . there were 60 nodules with suspicious findings in the initial us evaluation of excluded nodules ( 11.2% , 60 of 534 ) , and the proportion was not significantly different from that of included nodules ( 10.5% , 70 of 667 ; table 2 ) . when comparing the risk of malignancy between benign cytology alone and each subgroup by a combination of benign cytology with initial us findings or postbiopsy concordance , all combinations had significantly different risk values from cytology alone ( table 3 , figure 4 ) . also , when comparing the risk of malignancy between discordant lesions and lesions with suspicious features on initial us , the former ( 23.3% , 7 of 30 ) was significantly higher than the latter ( 11.4% , 8 of 70 ) . however , there was no significant difference in the risk of malignancy between concordant lesions ( 0.6% , 4 of 637 ) and lesions without suspicious features on initial us ( 0.5% , 3 of 597 ; p = 0.438 ) ( figure 4 ) . although fna is a widely used tool for the diagnosis of thyroid nodules , the most significant problem it has is false negative results which bring out misses and delays in treatment of the cancer . errors in cytologic reports have arisen from the overinterpretation of nondiagnostic specimens as diagnostic ones [ 23 , 24 ] . therefore , many reports discussed the differentiation of a nondiagnostic specimen from a diagnostic one in the cytologic interpretation of thyroid fna [ 1 , 3 ] . diagnostic errors of thyroid fna can also be caused by the mistakes of cytopathologists and the inherent nature of thyroid nodules due to overlapping cytologic criteria among hyperplastic adenomatoid nodule in goiter , follicular adenoma , well - differentiated follicular carcinoma , and follicular variant of papillary carcinoma . moreover , reported false negative rates are variable among institutions and operators due to variable sampling skills [ 5 , 6 ] . several guidelines recommend follow - up us in thyroid nodules with benign cytology unless the nodule shows significant growth or morphologic change in follow - up us [ 1 , 3 , 4 ] . however , it has been argued that follow - up might be not enough in some nodules because of the inevitable false negative diagnosis and the possible risk of delayed treatment [ 6 , 22 ] . to reduce false negative results of thyroid fnas , there have been two suggested approaches ; first , routine repeat fna in thyroid nodules with benign cytology [ 26 , 27 ] and , second , selective repeat fna [ 8 , 10 , 28 , 29 ] . in the aspect of cost - effectiveness , it is more rational to consider performing follow - up fna selectively for nodules with a high - risk of malignancy rather than performing a total inspection of cytologically benign nodules in initial fna . based on several reports , the rate of malignancy in benign thyroid nodules with suspicious us features was 3.747.1% which was significantly higher than that of benign thyroid nodules without suspicious us features ( table 4 ) [ 9 , 1518 ] . although the us criteria applied to each study had subtle differences , initial us features may be reliable factors in determining whether to repeat fna or not . going one step further from simply matching cytologic results against imaging findings evaluated before biopsy , the postbiopsy correlation of us features with cytologic results could be an alternative in determining whether the nodule should be reaspirated to confirm its cytology or not . imaging - pathologic correlation after biopsy has been found to be useful in validating biopsy results of breast lesions , and discordance has been suggested as an indication for excision because of its higher upgrade rate than that of concordant lesions [ 3032 ] . however , there has been no organized study that applies imaging - cytology correlation to patient management and considers how to accept results of postbiopsy correlation in regard to reducing false negative diagnosis in thyroid nodules . in this study , 1.6% of nodules with benign cytology in initial fna were finally proven to be malignant . as expected , the malignancy rate of thyroid nodules ( 11.4% ) with suspicious features on initial us was significantly higher than that of nodules ( 0.5% ) without suspicious features on initial us , and the malignancy rate of nodules ( 23.3% ) with discordant imaging findings was also significantly higher compared to concordant nodules ( 0.6% ) in postbiopsy imaging - cytologic correlations . furthermore , the rate of malignancy was higher in the nodules showing imaging - cytology discordance compared to nodules showing suspicious feature on the initial us . however , there was no significant difference in the risk of malignancy between concordant nodules in postbiopsy correlation and nodules without suspicious features on initial us . this result lets us conclude that imaging - cytology correlation is a more effective approach than using initial us features alone when deciding follow - up management in patients with cytologically benign thyroid nodules without a statistical increase in missing malignancy . in this study , 40 of 70 nodules with suspicious features on initial us were determined as concordant with benign cytology after postbiopsy imaging - cytology correlation . although many descriptions of each suspicious us feature are present , interobserver and intraobserver variability still exist for the us assessment of thyroid nodules . among us characteristics , margin and calcification showed relatively less consistency between observers and nodules in most patients whose radiologic assessments were changed after obtaining benign cytology were initially assumed as suspicious nodule due to calcification ( 16 of 40 ) , margin ( 9 of 40 ) , or multiple features ( 11 of 40 ) including them in our study . also , there have been difficulties in deciding whether a thyroid nodule shows echogenic spots on us . echogenic spots can be due to microcalcifications related to cancer or crystals related to colloids . therefore , postbiopsy imaging - cytology correlation can be a good diagnostic approach in deciding whether to repeat fna or not at a thyroid nodule with benign cytology . first , some nodules were excluded in analysis despite having benign cytologic results due to loss of follow - up and absence of additional reports . however , the initial us assessment was not significantly different between included nodules and excluded nodules which were 1 cm or larger with benign cytology in the initial fna . second , interobserver and intraobserver variability among radiologists are possible in the interpretation of us images and among cytologists , especially when reviewing follicular lesions . third , there might be a bias arising from the postbiopsy review process itself which was based on saved images instead of on a review in real - time us . although we always tried to save any images showing worrisome us findings and the postbiopsy review was preferably done within a week of biopsy by the performer , an observer bias might not have been completely removed from the final results . fourth , suspicious us features such as calcification , margin , vascularity , and echogenicity have been differently applied to thyroid nodules by various guidelines and different institutions . fifth , most ( 87.9% ) of the nodules in this study were reviewed by highly experienced radiologists in thyroid imaging . repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging - cytology correlation , which reduces unnecessary repeat aspirations as well as decreasing false negative results .

objective . to determine the role of imaging - cytology correlation in reducing false negative results of fine - needle aspiration ( fna ) at thyroid nodules . methods . this retrospective study included 667 nodules 1 cm or larger in 649 patients diagnosed as benign at initial cytologic evaluation and that underwent follow - up ultrasound ( us ) or fna following a radiologist 's opinion on concordance between imaging and cytologic results . we compared the risk of malignancy of nodules classified into subgroups according to the initial us features and imaging - cytology correlation . results . among included nodules , 11 nodules were proven to be malignant ( 1.6% ) in follow - up fna or surgery . the malignancy rate was higher in nodules with suspicious us features ( 11.4% ) than in nodules without suspicious us features ( 0.5% , p < 0.001 ) . when a thyroid nodule had discordant us findings on image review after having benign fna results , malignancy rate increased to 23.3% , significantly higher than that of nodules with suspicious us features ( p < 0.001 ) . however , no significant difference was found in the risk of malignancy between the nodules without suspicious us features ( 0.5% ) and imaging - cytology concordant nodules ( 0.6% , p = 0.438 ) . conclusions . repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging - cytology correlation after initial biopsy , which reduces unnecessary repeat aspirations .

continuous care delivery demands that health care providers lead patients smoothly through a chain of care that includes a number of professionals , departments and organizations , each of which are necessary for the provision of quality care at the right time and in the right place for each and every patient [ 15 ] . experience has taught us that this process is complex . indeed , in countries like sweden , england , the netherlands and austria , hospitals struggle with a bed - blocking problem . a bed - blocker is a patient who has completed treatment in one part of the care chain ( e.g. a hospital ) and is waiting for admittance to the next part of the chain ( e.g. a nursing home or home care ) . , it impacts health care costs as an occupied bed in a hospital is more expensive than an occupied bed in a nursing home or , alternatively , an occupied bed at home . hospitals have attempted to tackle the bed - blocking problem by setting up departments or areas that handle bed - blockers . in austria , these are called after care areas . in the netherlands , where in 2006 6.1% of all hospital days were bed - blocking days an icd is a nursing department usually situated in the hospital building that functions as a buffer when the hospital is over its capacity . initially , the establishment of icds appeared to be a promising and effective solution to bed - blocking . in fact , between 1999 and 2002 , the total number of bed blocking days in hospitals with an icd decreased by 30% while hospitals without an icd saw a decrease of only 15% . however , with the establishment of icds , another problem arose , namely queues for admission to the icds . although the average waiting time and the size of the queue had decreased between 2003 and 2006 from 11 to 8 days and from 8 to 7 patients , respectively , the queue remained [ 7 , 9 ] . in essence , the solution ( i.e. icds ) generated the same problem it was meant to solve ( i.e. waiting times and bed - blocking ) . previously , we explained that this was likely caused by a lack of buffer management ( bm ) , a tool that endeavors to balance patient flow in the hospital to nursing home chain of care . we hypothesized that bm was not applied by health care professionals and management because of a number of negative beliefs regarding bm . we further concluded that bm is indeed the most promising way for increasing the effectiveness of buffering . however , we must question whether our assumption that providers and managers hold negative beliefs about bm is correct . we are also interested in what can be done to make bm more feasible in health care settings . with this in mind , this paper seeks to firstly explain the theory of bm and then discuss the tenability of our previous assumptions given the results of ( explorative ) research recently conducted . bm builds upon the theory of constraints drum - buffer - rope [ 1117 ] . it aims to maximize the throughput in the chain by firstly identifying impeding factors or constraints and then taking measures to manage them properly . constraints can be identified by analyzing the sub - processes of the ( patient ) flow in the chain . the most impeding factor is called the system s bottleneck . because all sub - processes are linked to one other , the bottleneck determines the flow of the entire process and thus requires immediate and appropriate attention . in identifying the bottleneck , when the bottleneck is found , the drum - buffer - rope method is used to balance the patient flow in the whole chain . in the dutch hospital to nursing home or home care chain of care , the patient flow into the nursing home is the bottleneck [ 7 , 9 ] . the drum - buffer - rope method prescribes determining how many patients should be admitted to the hospital as well as the throughput from the hospital to the nursing home via the icd using a so - called rope . when the buffer overflows , the rope , which is an information stream , closes the hospital doors for potential ( elective ) buffer patients , which are mainly patients with mobility problems , rehabilitation needs or cardiovascular problems [ 7 , 9 ] . this procedure is illustrated in figure 1 , which displays three situations : the chain with no buffer ( situation a ) , the chain with a buffer but without bm ( situation b ) and the chain with a buffer and bm ( situation c ) . correct execution of bm requires that the following prerequisites for successful bm be taken into account [ 13 , 18 ] : in order to balance the flow , the entire process including its sub - processes must be considered.total flow can only be effectively increased by increasing the flow in the bottleneck.in order to prevent queues , the buffer needs free space to absorb fluctuations which are most frequently caused by non - elective patients . a maximum occupancy of 70% is considered acceptable.the use of a rope to adjust the input rate at the beginning of the process is necessary to prevent overflow in the sub - processes . in order to balance the flow total flow can only be effectively increased by increasing the flow in the bottleneck . in order to prevent queues , the buffer needs free space to absorb fluctuations which are most frequently caused by non - elective patients . the use of a rope to adjust the input rate at the beginning of the process is necessary to prevent overflow in the sub - processes . in practice the abovementioned prerequisites for effective bm are infrequently considered and the rope is rarely used . another misconception is that if the sub - processes are optimized , the flow of the entire process will increase . in fact , this leads to an unbalanced flow because the flow is determined by the bottleneck . with respect to bed - blocking , what will happen is the following : because patient flow in nursing homes is lower than patient flow in hospitals and because fluctuations at the entrance can not be absorbed , in the absence of bm , the icd will overflow . as mentioned earlier , a queue will form . this will result in bed blockers in the hospital ( bb ) who are waiting to be transferred to the icd . although this appears to be pretty straightforward , the problem of bed - blocking nonetheless remains . it is thus important to better understand why care providers involved fail to fulfill the abovementioned prerequisites . one possible explanation is that management and professionals in hospitals and/or nursing homes hold one or more negative beliefs about bm . we have previously provided objections to these beliefs . the beliefs and our objections are : buffer management is unnecessary.the argument is that bm is unnecessary when discharge management is a sufficient and more manageable alternative [ cf . however , discharge management does not balance patient flow nor does it establish a figurative tap to prevent additional patients per time unit from entering the chain when upstream capacity is limited . although discharge management , when used effectively , is a helpful coordination tool , research has indicated that discharge management has many problems with respect to , among other things , communication and information exchange .patient arrivals are unpredictable.the argument is that fluctuations in patient flow with respect to the frequency and intensity of patient arrivals can not be predicted . patient flow is predictable when elective and non - elective admissions are distinguished from one another [ cf . additionally , although it might not be possible to predict the exact number of emergent arrivals per day , the size of emergent demand generally appears to be quite stable and predictable over time . as such , we contend that the required size of the departments and buffer can be defined.buffer management is unethical.the argument is that putting a figurative tap on patient flows is ethically unacceptable as it implies the refusal of some patients [ cf . we ask whether it is safe and ethically acceptable to treat ( elective ) patients without knowing if they can be nursed and cared for afterwards [ cf . apparently , it is impossible to provide accessible high quality care that is tailored to the needs of the patient . we contend that we must be aware of this dilemma and subsequently take conscious clinical and political decisions.care organizations are unwilling to cooperate.cooperation between care providers using a chain perspective is necessary in order to maintain the delicate balance in the chain . previous research has indicated that care organizations are often unwilling to cooperate [ 5 , 32 ] thus supporting this argument against bm . however , we contend that the current trend in many european countries actually demonstrates an increased awareness of the importance of cooperation among most care providers [ 5 , 33 , 34 ] . the argument is that bm is unnecessary when discharge management is a sufficient and more manageable alternative [ cf . . however , discharge management does not balance patient flow nor does it establish a figurative tap to prevent additional patients per time unit from entering the chain when upstream capacity is limited . although discharge management , when used effectively , is a helpful coordination tool , research has indicated that discharge management has many problems with respect to , among other things , communication and information exchange . the argument is that fluctuations in patient flow with respect to the frequency and intensity of patient arrivals can not be predicted . patient flow is predictable when elective and non - elective admissions are distinguished from one another [ cf . although it might not be possible to predict the exact number of emergent arrivals per day , the size of emergent demand generally appears to be quite stable and predictable over time . as such , we contend that the required size of the departments and buffer can be defined . the argument is that putting a figurative tap on patient flows is ethically unacceptable as it implies the refusal of some patients [ cf . we ask whether it is safe and ethically acceptable to treat ( elective ) patients without knowing if they can be nursed and cared for afterwards [ cf . apparently , it is impossible to provide accessible high quality care that is tailored to the needs of the patient . we contend that we must be aware of this dilemma and subsequently take conscious clinical and political decisions . cooperation between care providers using a chain perspective is necessary in order to maintain the delicate balance in the chain . previous research has indicated that care organizations are often unwilling to cooperate [ 5 , 32 ] thus supporting this argument against bm . however , we contend that the current trend in many european countries actually demonstrates an increased awareness of the importance of cooperation among most care providers [ 5 , 33 , 34 ] . van hartingsveldt conducted three small case studies to further explore care providers beliefs about bm ( regarding both the sub - processes and the chain as a whole ) and to investigate whether or not providers held the four negative beliefs about bm as we assumed . the case studies were derived from qualitative data gathered in a larger mixed methods research project , using questionnaires and interviews with top - managers that addressed the availability of icds in all dutch hospitals and their functioning in terms of patient flows , waiting times , manpower , task division and responsibilities . the data gathered in 2006 were compared with data from a similar study conducted in 2003 in order to determine if things had changed over the years . one case study was conducted with an icd that had an average outcome , another with an icd that had a lower than average outcome and yet another with an icd that had a higher than average outcome . in each case study , an in - depth interview was conducted with icd executives using a structured interview protocol . the managers of the selected icds were interviewed and asked to mention two additional interview candidates , one that is responsible for referring patients to the icd and one that is responsible for admitting patients to nursing homes . consequently , in each case study , three people with different work positions were interviewed . in total , nine interviews were conducted . hermeneutic and narrative approaches were applied in the analyses . based on the assumption that respondents answers derive meaning from the whole and the whole derives meaning from the parts , the interviews were read and reread in an effort to develop a comprehensive understanding of the data . the interview data showed the following : all respondents considered the hospital , the icd and the nursing home to be separate links in the care chain . in their view , the organizations are independent . meetings that aimed to enable optimal patient flow and that included representatives of the three organizations failed to discuss whether equal numbers of patients were being admitted and discharged from the icd.because icds are financed according to how many beds are occupied , it is not surprising that icd respondents emphasized striving for a 100% occupancy rate in order to gain sufficient funding . this was also illustrated by the following citations : we celebrated the moment that our department was fully occupied ; currently we have an occupancy rate of 70% . i am therefore searching for patients to stay in the icd so that i can reach my production target.all icd and nursing home respondents said that they did not have any information regarding planned patient referrals from nursing departments and icds , respectively . this was confirmed by respondents from nursing departments referring to icds and respondents from icds referring to nursing homes.one nursing home respondent said that she did not benefit from investing energy into the facilitation of patient flow from the icd to the nursing home as the number of patients being admitted to nursing homes is already high enough to fill all the nursing home beds.all respondents shared objections to the contention that bm is unnecessary . more specifically , they indicated that insurers do not reimburse empty beds.all respondents did not agree with the contention that patient flows are unpredictable and that this serves as an inhibitor to bm . they , in fact , claimed that health care providers always have to deal with unpredictability.none of the respondents felt that bm is unethical . they indicated that patient refusal is simply part of daily practice in health care . according to one icd respondent : also here at the icd . then you have to refuse patients . that s just how it is in practice.all respondents indicated that cooperation is necessary in order to enable optimal patient flow and in order to reduce the number of bed - blockers . all respondents considered the hospital , the icd and the nursing home to be separate links in the care chain . in their view , the organizations are independent . meetings that aimed to enable optimal patient flow and that included representatives of the three organizations failed to discuss whether equal numbers of patients were being admitted and discharged from the icd . because icds are financed according to how many beds are occupied , it is not surprising that icd respondents emphasized striving for a 100% occupancy rate in order to gain sufficient funding . this was also illustrated by the following citations : we celebrated the moment that our department was fully occupied ; currently we have an occupancy rate of 70% . i am therefore searching for patients to stay in the icd so that i can reach my production target . all icd and nursing home respondents said that they did not have any information regarding planned patient referrals from nursing departments and icds , respectively . this was confirmed by respondents from nursing departments referring to icds and respondents from icds referring to nursing homes . one nursing home respondent said that she did not benefit from investing energy into the facilitation of patient flow from the icd to the nursing home as the number of patients being admitted to nursing homes is already high enough to fill all the nursing home beds . all respondents did not agree with the contention that patient flows are unpredictable and that this serves as an inhibitor to bm . they , in fact , claimed that health care providers always have to deal with unpredictability . they indicated that patient refusal is simply part of daily practice in health care . according to one icd respondent : also here at the icd , we are sometimes fully occupied . . then you have to refuse patients . that s just how it is in practice . all respondents indicated that cooperation is necessary in order to enable optimal patient flow and in order to reduce the number of bed - blockers . at first sight , the interview data suggest that our previous assumption that health care providers and management hold negative beliefs about bm is incorrect . for , they broadly supported the necessity and feasibility of bm [ cf . 57 ] . in practice , however , they do not apply the bm solution and they do not cooperate as is needed for bm application . for , although respondents said that cooperation is most valuable and necessary [ cf . they failed to share information with one another ; they failed to provide feedback about patient flows to one another ; and they sought to meet the interests of their own department or organization ( e.g. acquiring maximum funding ) before considering the interests of the total chain and its patient flow [ cf . they quite simply failed to cooperate in ways that are imperative to the success of bm . such a paradox between words and actions has previously been referred to as skilled incompetence by argyris . in order to shed light on why , when it comes to cooperation , respondents actions did not coincide with their words , we will further explore the inhibiting and promoting conditions for cooperation below . in seeking to understand cooperation between organizations , organizational units or professionals in health care , we have opted to borrow from organizational theories and literature on integrated care . organizational theories claim that organizational operations , like cooperation , communication , governance and knowledge transfer , are , in the end , determined by actions . actions are , in turn , dependent on the willingness and ability of those involved [ 3639 ] . one of the basic conditions for willingness to cooperate is the presence of interdependency between the organizations involved [ 3941 ] . in health care , interdependency among providers is rapidly increasing as pressure to coordinate services and deliver integrated care is increasing [ 2 , 4 , 42 , 43 ] . a second basic condition for willingness to cooperate is the presence of at least one common goal [ 5 , 39 , 44 ] . in health care , common goals are often construed in one s mind but not manifested in one s actions . a third basic condition is that the cooperation contributes to the department or organization s own goals [ 4447 ] . with the resource dependency theory in mind , we assume that interdependency increases when the number and weight of common goals and the benefits of cooperation for the organization s involved increases . we also assume that greater willingness generates more opportunities to act . in order to act means include money , personnel , time , equipment and accommodation . in health care , , cooperation always requires transaction costs in order to generate revenues later . this combination of scarce resources and transaction costs for cooperation does not promote providers abilities to cooperate . furthermore , limited ability is likely to reduce willingness . in most health care systems , the availability of means is dependent on legal and financial structures and regulations [ 3 , 34 , 49 ] . most countries have some laws and regulations fostering cooperation and others that hinder cooperation [ 5 , 33 , 49 ] . the availability of means is also dependent on managerial capabilities [ 38 , 50 ] , especially political and negotiating skills but also certain leadership characteristics , such as transformational leadership ( vision , ability to motivate and convince ) and intrinsic personal leadership features , such as charisma , charm and attractiveness [ 38 , 5053 ] . our previous research on integrated care revealed that managerial capabilities , especially leadership features and behavior , were strongly related to success or failure of integrated care networks [ 44 , 46 , 47 ] . cooperation is not simply determined by promoting and inhibiting conditions . in the real world , the willingness and ability to act in cooperative ways is also dependent on the answers to two questions . the first is , how do those involved perceive the abovementioned conditions? [ cf . according to the thomas theoremif men perceive things as real , they are real in their consequences the perception of the situation affects the actions taken . the second question concerns the extent they are able to change the routines , principles and beliefs ( rpbs ) employed in their daily work in order to achieve successful cooperation . we must ask if they are able to shift their focus from their own organization or department to the chain as a whole . are they able to find solutions for financial problems and develop trust in their partners instead of harboring distrust and prejudice ? are they able to replace their belief in their own power with a belief in shared power ? it is likely that the ability to change rpbs is in part dependent on perceptions while rpbs also impact perceptions . as such , perceptions and this is a vicious cycle and experience has taught us that breaking vicious cycles is often a laborious endeavor . one of the basic conditions for willingness to cooperate is the presence of interdependency between the organizations involved [ 3941 ] . in health care , interdependency among providers is rapidly increasing as pressure to coordinate services and deliver integrated care is increasing [ 2 , 4 , 42 , 43 ] . a second basic condition for willingness to cooperate is the presence of at least one common goal [ 5 , 39 , 44 ] . in health care , common goals are often construed in one s mind but not manifested in one s actions . a third basic condition is that the cooperation contributes to the department or organization s own goals [ 4447 ] . with the resource dependency theory in mind , we assume that interdependency increases when the number and weight of common goals and the benefits of cooperation for the organization s involved increases . means include money , personnel , time , equipment and accommodation . in health care , means are always scarce . to make matters worse , cooperation always requires transaction costs in order to generate revenues later . this combination of scarce resources and transaction costs for cooperation does not promote providers abilities to cooperate . furthermore , limited ability is likely to reduce willingness . in most health care systems , the availability of means is dependent on legal and financial structures and regulations [ 3 , 34 , 49 ] . most countries have some laws and regulations fostering cooperation and others that hinder cooperation [ 5 , 33 , 49 ] . the availability of means is also dependent on managerial capabilities [ 38 , 50 ] , especially political and negotiating skills but also certain leadership characteristics , such as transformational leadership ( vision , ability to motivate and convince ) and intrinsic personal leadership features , such as charisma , charm and attractiveness [ 38 , 5053 ] . our previous research on integrated care revealed that managerial capabilities , especially leadership features and behavior , were strongly related to success or failure of integrated care networks [ 44 , 46 , 47 ] . cooperation is not simply determined by promoting and inhibiting conditions . in the real world , the willingness and ability to act in cooperative ways is also dependent on the answers to two questions . the first is , how do those involved perceive the abovementioned conditions? [ cf . according to the thomas theoremif men perceive things as real , they are real in their consequences the perception of the situation affects the actions taken . the second question concerns the extent they are able to change the routines , principles and beliefs ( rpbs ) employed in their daily work in order to achieve successful cooperation . we must ask if they are able to shift their focus from their own organization or department to the chain as a whole . are they able to find solutions for financial problems and develop trust in their partners instead of harboring distrust and prejudice ? are they able to replace their belief in their own power with a belief in shared power ? it is likely that the ability to change rpbs is in part dependent on perceptions while rpbs also impact perceptions . this is a vicious cycle and experience has taught us that breaking vicious cycles is often a laborious endeavor . with respect to the icd case , we must question whether the dutch hospitals and nursing homes are aware of their interdependency on a daily basis . it is quite likely that hospital staff members ( doctors , interns , administrative personnel ) are unaware of their dependency on nursing home capacities when admitting potential buffer patients . at the same time , nursing home staff members probably do not realize that their hampered patient flow causes waiting lists for hospitals . furthermore , we must question whether the hospitals and nursing homes are truly aware of a common goal . most hospitals and nursing homes do formulate common goals to improve patient flow in a formal agreement [ 7 , 9 ] but are they really making decisions on the basis of these goals in their daily work ? it is likely that some parties in some parts of the chain do not even feel that improvement of the patient flow is necessary . this seems to be the case in nursing homes where the patient flow into the nursing home is sufficient to fill the available beds . we must also question whether hospitals and nursing homes are really aware of bm and how it can contribute to the attainment of an organization or department s own goals . instead , they most often consider bm to be a threat to their own goals . they may believe that bm threatens their autonomy and the acquisition of sufficient funding . at the same time , they fail to tackle the impeding factors that make bm a threat . for instance , with respect to funding , they often do not realize that it may be worthwhile fighting the prevailing system for the provision of funding . it may also be worthwhile to seek other resources or work more efficiently in order to enlarge their ability to cooperate . as such , it appears that the advantages of the icd buffer and bm ; namely improved patient flow and increased nursing home capacity due to additional beds in hospitals , is not perceived , or is at the very least , not perceived as important enough to change the rpbs of those involved . our conclusion is that , in health care practice , three of the four negative beliefs regarding bm can be refuted . although the providers involved in the case studies perceived a need for cooperation , they were not able or willing to translate this into action . in addition , a shortage of means caused by inappropriate incentives from financiers hampered concrete efforts to improve cooperation . in fact , the financial structure may very well be used as an argument against cooperation . we must realize that the current behavior is difficult to change as it is part of a vicious cycle of rpbs and perceptions , both of which are not in favor of cooperation . more importantly , a major reason for not favoring cooperation has institutional roots [ cf . 55 ] , more specifically , the health care system s financial structures , rules and regulations as well as its professional values ( i.e. professional autonomy ) . in essence , providers skilled incompetence with respect to cooperation is in part an institutionalized skilled incompetence that is very difficult to tackle . in order to make bm not only theoretically feasible , but also practically applicable , essentially , it boils down to a shift of focus from parts ( i.e. each individual organization ) to the whole ( i.e. the flow between participating organizations ) . only then theoretical acceptance of bm will be followed by consistent bm actions . for this to be achieved , we recommend the promotion of providers willingness and ability to take actions which foster cooperation ( i.e. the heart of our cooperation model figure 2 ) . two ( change ) strategies could be followed simultaneously : first , a negotiating strategy , to create win - win - situations , and second , a learning strategy , on how to adopt cooperation in the daily routines , anchored in people principles and beliefs [ cf . several interventions at the system s macro , meso or micro level are possible . examples of interventions related to the negotiating strategy are : convincing politicians and other powerful stakeholders to change the system such that incentives for cooperation are present as this is a macro - level intervention , it is an important job for top managers , but also for occupational organizations . making care providers and professionals aware of the chain perspective . convincing them that adopting this perspective is needed to solve their bed - blocking problems and , by doing that , improve quality and save costs . more concrete : an icd manager could stress the chain perspective and its advantages on the meso and micro levels , in discussions with medical professionals , hospital governors and nursing home managers . negotiating with care insurers or health authorities in order to enlarge financial means for cooperation . it is important to stress cost savings on the macro level , related to successful cooperation . striving for funding based on cooperation between organizations ( i.e. flow funding ) instead of funding the participating organizations individually ( as is the case now ) . appointing a chain manager or director that can oversee the entire care process and consider the prerequisites for bm in this process . appointment of a chain manager should be a common action on meso / micro level , i.e. the ( top ) managers of the organizations involved in the chain , with commitment of professionals . the chain director should be provided with the right competences , like transformational leadership and have appropriate negotiating skills as this can promote change in providers rpbs . drafting and signing agreements that ensure provider compliance with the bm prerequisites , including obligations and potentially even sanctions . the chain manager ( at meso level ) could prepare this in cooperation with the organizational managers and professionals involved . examples of interventions related to the learning strategy are : learning to find and use other ( local ) sources at macro and meso levels to finance a care process that allows for the regulation of hospital intakes based on icd bed occupancy . this learning can be stimulated in brainstorm sessions of professionals and providers involved ( meso and micro level ) . motivating and teaching professionals and providers involved to develop common goals and to come to common adherence based on those common goals . higher management and the chain manager could take initiatives in this direction by first mobilizing professionals who understand the chain perspective and are motivated to convince their colleagues of the advantages of cooperation . setting up a virtual network organization to manage cooperation and learn how to make use of the virtual organization ( meso level ) . ict is a helpful tool to execute cooperative actions , but it takes time to become familiar with using it . use of ict experts can be helpful , but more important is that ict becomes part of providers daily routines . table 1 gives an overview of the two strategies , and the related interventions on macro , meso and micro level of the health and social system . it depends of the individual situation what interventions to choose and when to apply them . in addition , we realize that the chain manager s task is not a simple one . breaking down is that the chain manager is or makes him / herself accepted by those involved . if not , it will be difficult to realize the conditions for cooperation , as mentioned in figure 2 . so , the interventions require investments , and the return on investment in terms of quality and savings should be analyzed in advance . our conclusion is that , in health care , the failure to cooperate is a serious inhibitor of bm . continuous persistent and purposeful efforts to improve cooperation between care providers are therefore imperative , as people often relapse into old routines , principles and beliefs . maren sogstad , associate professor , phd , centre for care research , gjvik university college , post box 191 , 2802 gjvik , norway birthe dinesen , phd , associate professor , department of health science and technology , aalborg university , fredrik bajersvej 7 d1 , dk-9220 aalborg , denmark jason ch yap , mbbs , mmed ( public health ) , fams , mba(is ) , chief ( health information & innovation ) , agency for integrated care , singapore

introductionbed - blocking problems in hospitals reflect how difficult and complex it is to move patients smoothly through the chain of care . in the netherlands , during the first decade of the 21st century , some hospitals attempted to tackle this problem by using an intermediate care department ( icd ) as a buffer for bed - blockers . however , research has shown that icds do not sufficiently solve the bed - blocking problem and that bed - blocking is often caused by a lack of buffer management.toolbuffer management ( bm ) is a tool that endeavors to balance patient flow in the hospital to nursing home chain of care.resultsadditional research has indicated that the absence of bm is not the result of providers thinking that bm is unnecessary , unethical or impossible because of unpredictable patient flows . instead , bm is hampered by a lack of cooperation between care providers.conclusionalthough stakeholders recognize that cooperation is imperative , they often fail to take the actions necessary to realize cooperation . our assumption is that this lack of willingness and ability to cooperate is the result of several impeding conditions as well as stakeholders perceptions of these conditions and the persistence of their current routines , principles and beliefs ( rpbs).discussionwe recommend simultaneously working on improving the conditions and changing stakeholders perceptions and rpbs .

stroke is still a major cause of death and long - term disability worldwide and it is associated with significant clinical and socioeconomical problems . despite the continuous efforts to develop the new pharmacological strategies , there is no effective neuroprotective therapy so far for ischemic stroke . novel approaches are needed to improve the recovery and quality of life of stroke patients . development of tissue damage after ischemic insult is dependent not only on duration and intensity of the blood flow reduction , but also on flow independent mechanisms , especially in the peri - infarct brain area . the blood flow dependent mechanisms of tissue damage develop in brain ischemic focus in the short time after onset of blood flow reduction . at that time cell death is a consequence of the acute energy failure and permanent anoxic cells depolarization is induced by loss of ionic gradients . a few hours later , the infarct expands into the adjacent penumbra , and cellular damage is mainly triggered by excitotoxicity , mitochondrial disturbances , reactive oxygen species production , and programmed cell death [ 1 , 2 ] . excitotoxicity is a pathologic process based on massive activation of ampa and nmda receptors in the brain . inappropriate activation of ampa and nmda receptors is a trigger for subsequent dysregulation of calcium ions homeostasis in the neurons and finally results in neuronal loss . massive activation of those receptors is observed in many cns disorders including stroke , epilepsy , multiple sclerosis , amyotrophic lateral sclerosis , parkinson , alzheimer , and huntington diseases . the most important factor leading to ampa and nmda upregulation is glutamate . glutamate is an important neurotransmitter in physiological concentration , but in pathologically high concentration it is neurotoxic [ 35 ] . lately , it is suggested that stroke triggers immune responses leading to inflammatory cell activation and infiltration of cerebral parenchyma . in the stroke brain upregulation of a variety of cytotoxic agents like cytokines , matrix metalloproteinases ( mmps ) , nitric oxide ( no ) , and more ros can be detected [ 68 ] . there is also upregulation of expression of some chemokines like ccl2 in the csf [ 9 , 10 ] and serum of patients with stroke . studies in experimental stroke ( middle cerebral artery occlusion model ( mcao ) ) confirmed involvement of chemokine ccl2 and its receptors ccr2 in stroke development . reported no difference in the level of ccl5 but montecucco and colleagues detected increased expression of plasma ccl5 in symptomatic as compared with asymptomatic patients . moreover , canou - poitrine confirmed that , higher systemic levels of ccl5 and cxcl10 in asymptomatic men are independent predictors of ischemic stroke . there is also a recent report from tokami et al . supporting the concept that ccl5 may be neuroprotective during stroke development . they showed upregulation of ccl5 but not ccl2 , ccl3 , and ccl4 on day 0 in stroke patients . this upregulation correlated with plasma concentrations of neuroprotective factors bdnf , egf , and vegf . other data from mcao model also showed upregulation of several chemokines and their receptors including ccl7 , cxcl10 , ccl20 , and chemokine receptors cxcr4 and ccr6 . et-1 induced model of stroke has been previously described by anthony et al . who induced the acute rat cerebral blood volume changes after intravenous and intracranial injections of this vasoconstrictor [ 21 , 22 ] . after microinjection of et-1 into selected brain regions they observed using magnetic resonance imaging ( mri ) an acute reduction of local perfusion in the injected hemisphere , loss of neurons in the grey matter and a macrophage / microglia and astrocyte response . after injection of et-1 into the cortical white matter , those authors observed amyloid precursor protein - positive immunostaining ( indicative of axonal disruption ) and an increase in tau-1 immunostaining in oligodendrocytes . similar to the grey matter lesions , no neutrophils were present and macrophage / microglia response did not occur . additionally , no breakdown in the blood - brain barrier was detected in the white and grey matter . in this study expression of several chemokines including : ccl2 , ccl3 , ccl5 , and cxcl2 as well as expression of markers of neuroinflammation like cd3 , f4/80 , and il-1 beta was studied . correlation of this expression with intensity of early neurodegeneration detected in the brain during the et-1 induced model of stroke was also analyzed . in all experiments , 8- to 12-week - old female sjl / j mice ( n = five for each time point ) were used . all animals were housed at the animal facility of the medical university of lodz , lodz , poland , under standard conditions . all experiments in this study have been approved by the local ethics committee for affairs experiments on animals . animal stroke model was induced by stereotactic , intracerebral injection of endothelin-1 ( et-1 , sigma - aldrich , poznan , poland , ) ( 20 pmol in 1 l of pbs per mouse ) into the left hemisphere of the brain . prior injection mice were anaesthetized with mixture of ketamine ( 1,15 mg , biowet , pulawy , poland ) and ksylazine ( 0,1 mg , biowet , pulawy , poland ) per mouse . after complete anaesthetization mice were placed in stereotactic frame ( david kopf instruments , ca , usa ) , skin on the head was cut , and a small hole in the skull was made using surgical drill . et-1 was administered with a hamilton syringe ( 32 g needle ) ( hamilton company , bonaduz , gr , switzerland ) . site of injection ( a-2 mm , l-1 , 2 mm , d-2 , 5 mm ) was selected using the stereotactic atlas tissue samples were collected 24 and 72 hours after the model induction . as a controls , brains from uninjected mice and from mice injected in the same way with pbs were used . to obtain rna animals , were perfused with a saline solution . tissues were weighed and then homogenized using a mechanical homogenizer ultra turrax ( ika , staufen , germany ) . tissues were homogenized in a volume of 1ml of trizol ls reagent ( gibco brl , invitrogen , carlsbad , ca , usa ) . rna was isolated from the homogenates with trizol ls reagent using phenol - chloroform method described by chomczynski and sacchi ( chomczynski and sacchi , 1987 ) . after rna isolation , its concentration was estimated using the photometric method ( biophotometr plus , eppendorf company , wien , austria ) . to obtain the proteins for the elisa assay harvested organs were weighed using a laboratory balance ( radwag radom , poland ) and homogenized using a mechanical homogenizer ultra turrax ( ika ) . homogenisation was performed in a volume of 1 ml hepes buffer ph 7.4 containing : hepes 20 mm ; edta 1.5 mm ; benzamidyn 0.5 mm ; chicken egg owoinhibitor 10 ug / ml pmsf ( phenylmetylsulfonyl fluoride ) 0.1 mm ( sigma - aldrich , poznan , poland ) . supernatants were obtained after centrifugation ( 20 000 g , time 30 minutes at 4c mpw , warsaw , poland ) . analysis of the rna expression was performed using the corbett real - time pcr machine rotor gene 3000 apparatus ( corbett research , sydney , australia ) . the key enzyme used in this reaction was taq polymerase with activity of 5 u / ml . additional reaction components were buffer for polymerase , 25 mm mgcl2 , 10 mm dntps , fluorescent dye evagreen ( biomibo , warsaw , poland ) , 10 m primers specific to the duplicated sequences , and rnase / dnase free water . for each reaction 2 l of cdna derived from the reverse transcription reaction was used and the total volume was 20 l . as a control histone h3 gene and reference rna ( qpcr mouse reference total cellular rna , stratagene , la jolla , ca , usa ) were used . quantitative analysis of gene expression at the protein level was performed using elisa method with commercially available immunoenzymatic quantikine kits ( r & d systems , mn , usa ) . each set consisted of 96 well plates coated by manufacturer , standard proteins used to prepare the calibration , secondary and tertiary antibodies combined with the horseradish peroxidase enzyme , and washing buffer and color substrate for peroxidase . the assay procedure was performed according to the protocol provided by the manufacturer . after stopping the color reaction protein concentration was evaluated using a photometric reader victor2 wallac 1420 ( perkinelmer , waltham , ma , usa ) with for 450 nm filters , corrected at 595 nm . all samples were analyzed in duplicates . quantitative assessment of the intensity of neurodegeneration was performed using elisa method with primary antibodies directed against phosphorylated neurofilaments . the first step was the coating of 96 well maxisorb microtitre plate ( nunc , roskilde , denmark ) with monoclonal anti - nfh antibodies ( smi35r , sternberger monoclonals , convance princeton , nj , usa ) and overnight incubation at 4c . the next day tested samples and standard curve samples ( neurofilament 200 kd , progen , heidelberg , germany ) were added . as a secondary antibody rabbit polyclonal antineurofilament 200 antibody ( sigma - aldrich , poznan , poland ) was used . as a tertiary antibody swine antibodies against rabbit immunoglobulin conjugated with horseradish peroxidase ( dako , glostrup , denmark ) were used . the final step was the addition of color substrate for horseradish peroxidase , which was 3,35,5-tetramethylbenzidine ( sigma - aldrich , poznan , poland ) . inhibition of the reaction was performed with 1 m hcl and finally color photometric assessment was done using victor2 reader wallac 1420 ( perkinelmer , waltham , ma , usa ) . the analysis was performed using 450 nm filter with correction at 595 nm . all samples were analyzed in duplicates and the concentration of nfh was determined by referring to the standard curve . assessment of the localization and severity of neurodegeneration at the level of protein was performed using the fluorescent fluoro - jade c dye ( chemicon , millipore , warsaw , poland ) . animals were perfused with 4% buffered formalin solution and tissues samples were embedded in paraffin blocks . 10 m thick sections were applied to a polished super frost slides ( menzel - glaser braunschweig , germany ) . before final staining paraffin fluoro - jade c staining was performed according to the protocol provided by the manufacturer ( chemicon ) . for staining of nuclei sections were counterstained using the blue fluorescent dye dapi ( sigma - aldrich , poznan , poland ) . then the tissue was mounted and coverslipped using dpx ( sigma - aldrich , poznan , poland ) . for the analysis and acquisition of images an inverted microscope axioobserver a1 ( carl zeiss inc . , the following lenses made by carl zeiss inc . were used : plan - achromat : 4x/0.10 , a- plan 10x/0.25 ph1 ; ld a - plan 20x/0.3 ; ld plan - neofluar 40x/0.6 , ph2 korr . the images were obtained with a digital camera , axiocam mrc5 ( carl zeiss group , goettingen , germany ) attached to the microscope . for image acquisition we used axio - vision rel . nonparametric kruskal - wallis and mann - whitney tests were used . for correlation analyses kendal significant upregulation of expression of t cell line marker - cd3 was observed in the et-1-injected hemisphere at 72 h after injection ( p = 0.03 , mann - whitney test ) ( figure 1(a ) ) . at that time a significant difference in expression of cd3 was detected between ipsilateral and contralateral hemispheres ( p = 0.019 , mann - whitney test ) ( figure 1(a ) ) . expression of cd3 in ipsilateral hemisphere was also increased when compared to pbs injected hemisphere ( p = 0.28 , mann - whitney test ) . the expression of monocyte / macrophage lineage marker f4/80 was significantly elevated only in et-1 injected hemisphere at 72 hours after injection . at that time significant difference was observed in expression of f4/80 between et-1 injected hemisphere and untreated control group ( p = 0.022 ; mann - whitney test ) ( figure 1(b ) ) . we detected also a significant difference in expression of f4/80 between ipsilateral and contralateral hemispheres of et-1 injected mice at 72 hours after injection ( p = 0.035 , mann - whitney test ) ( figure 1(b ) ) . upregulation of cytokine il-1 expression was observed in et-1 injected hemispheres only at 24 h after injection . significant difference in expression of il-1 was observed between contralateral hemispheres and normal control group at 72 hours after injection ( p = 0.03 and 0.019 , resp . ; we detected also a significant difference in expression of il-1 between ipsilateral and contralateral hemispheres of et-1 injected mice 72 hours after injection ( p = 0.019 , p = 0.019 , resp . ; upregulation of chemokine ccl2 expression was observed in ipsilateral hemispheres at 24 and 72 h after injection of et-1 ( p = 0.005 , p = 0.005 , resp . ; mann - whitney test ) ( figure 2(a ) ) . at 24 h ccl2 expression in ipsilateral hemisphere was significantly higher than at 72 h ( p = 0.019 ; mann - whitney test ) . significant difference in ccl2 expression after et-1 injection was also observed between ipsilateral and contralateral hemispheres at 24 h and 72 h ( p = 0.012 and 0.036 , resp . ; we also showed that during early stage of et-1-injection stroke model expression of ccl3 is significantly upregulated at 24 and 72 h after model induction ( figure 2(b ) ) . there was significant upregulation of ccl3 expression in ipsilateral hemispheres of et-1 injected mice in comparison to normal controls and contralateral hemispheres at 24 h after injection ( p = 0.008 and 0.012 , resp . ; mann - whitney test ) . at 72 h after model induction we observed significant upregulation of ccl3 expression in et-1-injected hemispheres in comparison to normal brains and contralateral hemispheres ( p = 0.014 and 0.019 , resp . ; mann - whitney test ) ( figure 2(b ) ) . ccl5 was observed in et-1-injected hemispheres in comparison to uninjected animals at 24 and 72 h ( p = 0.008 , and 0.008 resp . ; significant difference was also observed in ccl5 expression between ipsilateral and contralateral hemispheres at 24 h and 72 h after injection of et-1 ( p = 0.008 and 0.012 , resp . ; mann - whitney test ) ( figure 2(c ) ) . initially increasing expression of cxcl2 in et-1 injected hemispheres was detected with the peak at 24 h and subsequent decrease at 72 h but still significantly higher than in normal controls ( p = 0.036 , and 0.036 , resp . ; mann - whitney test ) ( figure 2(d ) ) . at 24 h cxcl2 expression in ipsilateral hemisphere was significantly higher than at 72 h ( p = 0.012 ; mann - whitney test ) and then at 24 h after et-1 injection in contralateral hemispheres ( p = 0.012 ; mann - whitney test ) ( figure 2(d ) ) . the expression of cxcl12 in the et-1 and pbs - injected brains and normal controls did not show a significant difference between analysed groups ( data not shown ) . the most severe neurodegeneration was observed in et-1-injected hemispheres at 24 and 72 h after model induction ( p = 0.019 and 0.029 , resp . ; there was also increased neurodegeneration in contralateral hemispheres of et-1 injected mice at 24 and 72 h , but it was significantly lower than in ipsilateral hemispheres ( p = 0.03 , and 0.03 , resp . ; the localization of ischemic lesion was detected in et-1 injected ipsilateral hemispheres using cresyl violet staining ( figure 3(b ) , large box ) . inside the ischemic focus injured neurons were abundant ( detected by fluoro - jade and marked by arrows ) cells nuclei counterstained with dapi are marked on the picture by arrowheads ( figure 3(c ) ) . we observed the positive correlation between expression of lymphocyte lineage marker cd3 ( kendall tau = 0,62 ; p = 0.0004 ) ( figure 4(a ) ) as well as monocyte / macrophage lineage marker f4/80 ( kendall tau = 0,56 ; p = 0.0007 ) ( figure 4(b ) ) and the severity of neurodegeneration in et-1 injected brain hemispheres . although the expression of several studied chemotactic inflammatory mediators ( chemokines ccl2 , ccl3 , ccl5 , and cxcl2 ) was significantly increased in the early stage of this stroke model , there was no clear correlation between this expression and intensity of neurodegeneration ( data not shown ) . in this study we analyzed potential relationship between neuroinflammation and neurodegeneration in experimental model of ischemic stroke induced by intracerebral et-1 injection . we focused on a group of proinflammatory chemokines , especially the classical representatives of ccl subfamily . the reason for selecting these chemokines was their confirmed participation in pathogenesis of many central nervous system diseases . during the first few days of the experimental brain ischemia we observed increasing neurodegeneration in et-1 injected hemisphere . there are several studies showing that neurodegeneration occurs early during brain ischaemia [ 23 , 24 ] . at that time also the relationship between those two processes is complex and still requires further studies . to measure the intensity of neuroinflammation the expression of inflammatory cells markers ( cd3 for t cells and f4/80 for monocytes / macrophages ) has been measured at the same time . this analysis showed increased lymphocyte migration to ischemic brain hemisphere at 72 h after model induction . similarly , infiltration of ipsilateral hemisphere by monocytes / macrophages was significantly increased at 72 h after initiation of brain ischemia . comparable observation was reported by others who showed the presence of macrophages / activated microglia at 72 h after intracerebral injection of et-1 to rat brain . in another study using mcao stroke model , the influx of mononuclear cells to the site of brain ischemia was recorded between 2 and 15 days after model induction . the presence of neuroinflammation during early brain ischemia was also confirmed in our study by elevated expression of inflammatory mediator - cytokine il-1. its presence was observed as early as 24 h after model induction . in several cell types including astrocytes , microglia , neurons , and endothelium . in another study increased production of il-1 was reported even at 36 hours after induction of brain ischemia . the peak of this expression was observed at 12 h , and it returned to baseline level after 5 days . other studies confirmed also that inflammatory mediators , such as il-1 and tnf , are important contributors to cns neural tissue damage induced by ischemia [ 28 , 29 ] . in our stroke model analysis of the relationship between the infiltration of ischemic hemisphere by mononuclear inflammatory cells and this may suggest that there is close connection between neuroinflammation and neurodegeneration in ischemic stroke . migration of inflammatory cells from the blood to the ischemic brain may be at least partially induced by chemotactic cytokines - chemokines . to study this concept the highest expression of ccl2 was observed in our model at 24 h after initiation of brain ischemia . increased expression of ccl2 was still observed at 72 h but was at that time significantly lower . increased expression of ccl2 in mcao model in the ipsilateral hemisphere was observed on neurons at 12 h and on astrocytes at 24 h after cardiac arrest , suggesting that these cells are the potential source of ccl2 during ischemic stroke . minami and satoh using double in situ hybridization method pointed to microglia as the cellular source of ccl2 during mcao . it was shown in another mcao study that ccl2 leads to infiltration of the cns by monocytes and thus enhances brain damage induced by ischemia . also in human stroke patients elevated level of ccl2 was detected in cerebrospinal fluid and serum [ 9 , 11 ] . the highest ccl3 expression was detected at 24 h after et-1 injection . at 72 h this expression was still increased but it was much lower than at 24 h. also at the protein level we observed significant increase in ccl3 production in the ischemic hemisphere . our results are in line with the report by gourmala et al . who observed an increase in ccl3 expression at mrna level already at 1 h after mcao in rats , with peak expression at 816 h . in addition , they observed higher expression of ccl3 during temporary mcao than in permanent mcao , suggesting the impact of reperfusion on the neuroinflammation in the damaged tissue . gourmala et al . using in situ hybridization localized the expression of ccl3 on microglial cells / macrophages during brain ischemia . in addition , another studies have concluded that ccl3 application to the brain ventricles after complete mcao enhances mcao harmful effects . we observed almost 46-fold and 30-fold increase in ccl5 expression at 24 h and 72 h , respectively , after induction of et-1 induced stroke model . there are only a few reports concerning the role of ccl5 in the development of ischemic stroke . it was suggested that ccl5 mediates blood - brain - barrier ( bbb ) disruption and cns tissue damage as well as inflammation after reperfusion during mcao model . these data were not confirmed by tokami and colleagues who observed neuroprotective effect of ccl5 in ischemic stroke suggesting that ccl5 is expressed during stroke mostly in neurons . in et-1 induced experimental stroke a significant increase in cxcl2 expression at 24 h after brain ischemia induction was also observed . observed increased expression of cxcl2 in the brain of rats with the permanent mcao as well as in the brain and spleen during temporary mcao in mice . in other study increased cxcl2 expression was observed at 6 h of reperfusion and decreased by almost half at 22 h after reperfusion . vikman et al . showed increased cxcl2 expression in the brain vessels in the model of subarachnoid haemorrhage and in organotypic cultures . cxcl2 involvement in the inflammatory process in the cns during mcao was also confirmed in a scid mice . mcao induced in scid mice led to development of significantly reduced area of brain damage and lower inflammatory infiltration in ipsilateral hemispheres . reduced expression of many inflammatory mediators including cxcl2 was also observed in t- and b - cell - deficient mice mcao study . unfortunately , therapy of ischemic stroke with cxcl2 receptor - cxcr2 antagonists sb225002 was not successful . in report presented by copin et al . the cxcl1/cxcl2 chemokine - binding protein evasin-3 treatment was associated with reduction in neutrophilic inflammation in mice mcao model . although in our study the expression of several studied chemokines was significantly increased at the early phase of et-1 induced stroke model , no clear correlation of this expression with neurodegeneration was observed . that they suggest that inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration . upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke .

neurodegeneration is a hallmark of most of the central nervous system ( cns ) disorders including stroke . recently inflammation has been implicated in pathogenesis of neurodegeneration and neurodegenerative diseases . the aim of this study was analysis of expression of several inflammatory markers and its correlation with development of neurodegeneration during the early stage of experimental stroke . ischemic stroke model was induced by stereotaxic intracerebral injection of vasoconstricting agent endothelin-1 ( et-1 ) . it was observed that neurodegeneration appears very early in that model and correlates well with migration of inflammatory lymphocytes and macrophages to the brain . although the expression of several studied chemotactic cytokines ( chemokines ) was significantly increased at the early phase of et-1 induced stroke model , no clear correlation of this expression with neurodegeneration was observed . these data may indicate that chemokines do not induce neurodegeneration directly . upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke . inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration .

the app is a type i transmembrane protein with characteristics of an orphan receptor , which shares with other members of its class a particular signaling mechanism termed regulated intramembrane proteolysis ( rip ) . the first occurs outside the transmembrane domain , usually in response to ligand binding , inducing the release of the extracellular domain . this first cleavage event elicits a conformational change that triggers the second proteolytic cleavage which takes place on the transmembrane segment . this mechanism controls several cellular processes , such as the unfolded protein response , cholesterol synthesis , and cell fate instruction . rip of the app is mediated by three different proteases . while - and -secretases catalyze extracellular cleavage , the -secretase complex cuts at the intramembrane domain and leads to the generation of two peptides : an app active fragment , termed aicd and the a . in contrast , two independent groups indicate that aicd is produced mainly from the 695 aminoacids isoform of app through the amyloidogenic pathway ( dependent on -secretase activity ) [ 6 , 7 ] and is therefore generated in equimolar quantities with a . the last one accumulation and the formation of various aggregates and deposits in the brain have been the main hypothesis to explain the neuropathological development of ad for almost 20 years . initially , the study of the functions associated with the aicd was limited by the hindrance in its detection . however , recent studies showing that the levels of the aicd are increased in brains of ad patients and murine models reproducing the disease , open up the possibility that this fragment participates in the molecular mechanisms contributing to ad . the aicd is the most evolutionarily - conserved region of the app , accounting for its functional importance . despite its relatively small size ( 59 aminoacids or less ) , it acts as a docking site for a particularly large group of intracellular proteins . amongst this group of proteins are pin1 , the x11 protein family , disabled ( dab)-1 , shc , jnk - interacting protein ( jip)-1 , and the fe65 protein family [ 1719 ] , which includes fe65 itself and two closely related homologues , fe65l1 and fe65l2 . fe65 family members contain three protein - protein interaction domains : a ww domain at the n - terminal involved in interactions with proline - rich sequences and two phosphotyrosine binding domains ( ptb1 and ptb2 ) located at the c - terminal . the second ptb domain ( ptb2 ) is responsible for the interaction between fe65 and the sequence 682yenpty687 of the app ( following the numbering of the app695 isoform ) . the possibility of aicd to form multiprotein complexes through its association with fe65 and its multiple ligands ( table 1 ) has unexpectedly expanded the potential roles of aicd . aicd binds to fe65 in a region that is essential for a production , making fe65 a good candidate for regulating app processing . this could occur via two mutually - exclusive pathways : the amyloidogenic pathway , leading to a production mediated by the -secretase and the nonamyloidogenic pathway leading to the production of a large extracellular fragment ( sapp ) , which is mediated by the -secretase and prevents the generation of a. fe65 acts as a potent modulator by altering the balance between the two pathways . the overexpression of fe65 in cell lines induces a dramatic increase in a secretion , whereas a secretion was decreased in fe65 knockdown cells and in hippocampal neurons of fe65/fe65l1 knockout ( ko ) mice . the effect on the a secretion appears to be dependent on the interaction between fe65 and app , because the knock - in mice carrying the y682 g mutation , that inhibits aicd binding to fe65 , show decreased levels of a and a massive increase in sapp , as a consequence of the nonamyloidogenic pathway this is in agreement with a study showing that fe65 is a potent suppressor of the nonamyloidogenic pathway in primate cells . the mechanism by which fe65 modulates a secretion is related to its interaction with the apolipoprotein e ( apoe ) receptors : the low density lipoprotein receptor - related protein ( lrp ) and apoe receptor 2 ( apoer2 ) . related to the participation of the aforementioned receptors , the effect of fe65 in the secretion of soluble app fragments is lost in cells lacking lrp . the functional relation with apoer2 is more complex and depends on the presence of its extracellular ligand , reelin , and its intracellular adapter , dab-1 . reelin reduces a secretion by promoting the binding of dab1 to the app and displacing fe65 , because they share the same binding region . a decrease in reelin expression in the entorhinal cortex ( the first region of the brain where a deposits can be observed ) , displayed in pdapp transgenic mice ( which carry human app with mutations swedish ( swe ) and indiana ) and in ad patients , could seriously affect the balance of dab1 and fe65 in their binding to aicd , increasing a secretion . this has been observed in transgenic mice which lack reelin expression ( reeler ) and carry the mutations swedish and arctic in app . a decade ago , a possible role for the rip of app was first suggested . since app processing seems to be similar to notch processing , it has been suggested that rip of app could be involved in transcriptional regulation . in fact , the fusion of the dna binding domain of yeast gal4 ( gal4db ) to the c - terminal of app induced a strong transactivation of a luciferase reporter dependent on the formation of a trimeric complex with the adapter protein fe65 and the histone acetyltransferase tip60 . a reciprocal experiment using tip60 or fe65 fused to the gal4db gave rise to some contradictory results [ 49 , 50 ] . nevertheless , a consensus model can be generated including the vast majority of observations derived from these studies ( figure 1 ) . the app acts as an anchor for fe65 and fe65-associated proteins that is , : tip60 , inducing its association with membrane compartments . membrane recruitment seems to be essential for the activation of the complex , since the overexpression of soluble aicd has no effect on transactivation . the autoinhibited conformation of fe65 , produced by the association of the ww domain with a region flanked by the ptb1 and ptb2 domains . the association with the plasma membrane allows the activation of the complex , induced by the phosphorylation of tip60 by cyclin - dependent kinases ( cdks ) . an excellent prospective candidate is cdk-5 , that can be found associated with plasma membranes through its activator p35 and displays high activity in the brain . the release of the complex from the plasma membrane may be produced by the app cleavage by -secretase or additionally by the app phosphorylation at thr668 which induces a conformational change in the region recognized by fe65 , decreasing the affinity for each other . although some groups have observed aicd in the nucleus , particularly in nuclear domains such as transcriptional factories , the splicing factor compartment or directly at promoters of some genes [ 5961 ] , apparently in the artificial transactivation system , the nuclear translocation of aicd is not essential to enhance luciferase expression . the n - terminal region of fe65 that includes the ww domain is necessary for nuclear translocation and therefore for its activity as a transactivating protein . although this region lacks a nuclear localization sequence ( nls ) , it could be directed to the nuclei by association with a protein carrying a functional nls . a good candidate to perform this function would be the nucleosome assembly protein set that binds the ww domain and is required for transactivation mediated by the fe65gal4db fusion protein . the phosphorylation of tyr547 in the fe65 ptb2 domain mediated by the abl kinase stimulates its transactivational activity , possibly preventing the association of fe65 with dexras , a ras family gtpase , that acts as an inhibitor of the complex . the search for target genes regulated by the aicd has been complex and has yielded conflicting findings . it has been reported that the aicd / fe65 complex regulates the app expression itself , glycogen synthase kinase ( gsk)-3 [ 63 , 64 ] , tip60 , the -secretase ( bace1 ) , the primate - specific caspase 4 , the a degrading enzyme neprylisin [ 61 , 65 , 66 ] , the tetraspanin kai1 [ 26 , 63 ] , the lipoprotein receptor - related protein ( lrp1 ) , the epidermal growth factor receptor ( egfr ) , and the tumor suppressor p53 . nevertheless , many of these studies have been refuted by others , which using different strategies for modulating the aicd / fe65 complex did not produce changes in the expression of the aforementioned genes [ 6973 ] . the possible origin of the reported differences is unclear , but regarding the most intensively discussed target , neprilysin , recent data may shed light on the controversy . it was shown that the aicd - binding to neprilysin gene promoter is cell type - dependent [ 61 , 74 ] . furthermore , aicd - dependent gene regulation is influenced by the passage number and cell density , providing two likely experimental explanations for this disagreement . the majority of the evidence pointing to a role of aicd in transcriptional responses derives from the use of artificial reporter systems that in fact measure the release of components from the membrane , without monitoring endogenous transcriptional activity . besides the potential participation of fe65 in promoting the expression of several genes described above , fe65 has been also proposed to perform other nuclear functions such as the repair of dna damage . fe65 ko mice are more sensitive to dna damage , and this can be overcome by increasing the availability of nuclear fe65 . moreover , genotoxic damage produces a rapid translocation of fe65 to the nuclear matrix and stimulates app processing by the -secretase complex and app phosphorylation in thr668 , two mechanisms that allow translocation to the nucleus of the complexes associated with aicd . fe65 is required for efficient repair of dna double strand breaks ( dsb ) , a function that depends on its interaction with tip60 and aicd . the fe65-dependent recruitment of tip60 to dsb sites is essential because the histone acetyltransferase activity leads to chromatin opening at the injury site , enabling the access of the complexes involved in repair . on the other hand , tip60 acetylates and activates the ataxia telangiectasia mutated ( atm ) kinase which in turn phosphorylates a histone h2a variant , called h2ax , which acts as a mark for the recruitment of the reparation machinery . changes in h2ax phosphorylation could be also dependent on the stability of p53 in a mechanism that requires the accumulation of fe65 in the nuclei [ 81 , 82 ] . however , the fact that phosphorylated h2ax may be also increased in fe65 ko cells under genotoxic damage suggests that complementary mechanisms may regulate this behavior . fe65 is highly enriched in the brain where it is expressed as two isoforms produced by the alternative splicing of a 6 bp miniexon . the isoform that includes this exon ( which encodes arg - glu inserted in the ptb1 domain ) is expressed exclusively in neurons , whereas the isoform lacking these two aminoacids is expressed only in nonneuronal cells . fe65 protein expression may change during development and also in pathological conditions such as ad , opening up the possibility that it participates in plastic processes in neurons , which is reflected in the phenotype of fe65 and fe65l1 double ko mouse . these mice exhibit defects in the positioning of cortical neurons characterized by the presence of ectopic neurons that break the pialmeningeal basement membrane and displace cajal - retzius neurons and also have serious defects in axonal projections . many of these phenotypical features are shared by mice lacking some of the fe65-binding partners such as the app family and the mammalian homolog of drosophila enabled ( mena ) . mena belongs to a family of proteins that regulate actin dynamics and thereby modulate cell motility and morphology . mena is located in areas of dynamic actin remodeling such as lamellipodia and growth cones and interacts with the actin - binding protein , profilin . mena interacts with the fe65 ww domain , assembling a macromolecular complex with app that regulates axonal branching , cell motility , and possibly the dynamics of actin at the growth cone and synapsis . in a previous attempt to generate a fe65 ko , it was expressed a truncated protein lacking the n - terminal domain and translated from met261 . this 60 kda variant does not contain the ww domain and does not display the transactivation activity of the larger isoform . in spite of the expression of this smaller protein , the animal shows defects in hippocampal - dependent learning and long - term potentiation ( ltp ) [ 93 , 94 ] . however , it is difficult to assess whether these defects are due to the 97 kda isoform loss or the appearance of this new 60 kda isoform acting as a dominant negative protein . although the amyloid cascade hypothesis has become the mainstream in the study of ad neurobiological mechanisms , several groups have recently suggested that this should be at least reevaluated in the light of new findings [ 9597 ] . transgenic mice that overexpress the aicd and the adapter fe65 in the forebrain ( under the control of the camkii promoter ) display several neuropathological features observed in various transgenic models and in the ad patients brains , with the exception that they do not show a accumulation in the brain . the expression of aicd together with fe65 seems to be essential to induce an ad - like phenotype in the transgenic model , since a single aicd transgenic mouse developed by an independent group does not present the characteristics of the double transgenic , indicating that the functional relationship between both proteins , discussed in the previous sections , is indeed essential . as in the brain of patients with ad and several other transgenic models used to study ad , the aicd / fe65 mice show an increase in gsk-3 activity . interestingly , the double aicd / fe65 transgenic does not affect the gsk-3 mrna or protein levels , as would be expected from a previous study which suggests that the kinase should be transcriptionally regulated by the aicd / fe65 complex . kinase activation in the double transgenic is indeed correlated with an increase in the tyr216 activating phosphorylation and a decrease in the ser9 inhibitory phosphorylation . a molecular explanation for this may be related with the fact that fe65 , through its ww domain , interacts and promotes gsk-3 phosphorylation on tyr216 . increased gsk-3 activity in the aicd / fe65 mice produces hyperphosphorylation of two direct targets : the microtubule - binding proteins , collapsin - response mediator protein ( crmp)-2 and tau [ 11 , 98 ] . increased crmp-2 phosphorylation is also found in transgenic mice expressing mutated forms of app and presenilin ( ps)-1 and also in the cerebral cortex of ad patients . increased crmp-2 phosphorylation is an early event that precedes the formation of amyloid plaques and neurofibrillary tangles . interestingly , this posttranslational modification seems to be specific for ad , since it has not been reported in other neurodegenerative conditions like the frontotemporal dementia and pick 's disease [ 100 , 101 ] . hyperphosphorylation of tau is the initial event in the pathway to tau self - aggregation , forming the paired helical filaments ( phfs ) . phfs are found at the core of the highly insoluble intraneuronal neurofibrillary tangles , one of the two neuropathological lesions ( another is the senile plaques ) that characterize the ad patients brains . the aicd / fe65 mouse shares with 3xtg mice the capacity to promote the formation of tau insoluble aggregates , which are not observed in most mouse models for ad . the aicd / fe65 double transgenic mouse has nonconvulsive seizures with aging , abnormal electroencephalogram ( eeg ) spiking , and a greater sensitivity to seizures induced by kainic acid ( ka ) in young animals . it also presents several alterations in hippocampal neural circuits , characterized by abnormal sprouting of the mossy fiber terminals with increased neuropeptide y ( npy ) expression and loss of calbindin - positive neurons . alterations in the eegs and seizures have been observed in ad patients and in mouse models for this pathology , such as mice r1.40 ( with appswe ) , appps1 , and pdapp [ 105 , 106 ] . aged aicd / fe65 animals ( > 18 months ) show neurodegeneration in the ca3 hippocampal area , although the defects in working memory ( evaluated by the y maze paradigm ) start at a young age ( 8 months ) . since most of the mouse models for ad are based on the expression of mutant variants of the human app or presenilin found in cases of familiar ad , the identity of neurotoxic app fragments has not been clearly discerned yet . several studies have shown that a deposition in senile plaques does not correlate with neuronal death and cognitive deficits present in different transgenic models [ 107 , 108 ] . for example , the overexpression of wild type happ in mice produces memory deficits , tau hyperphosphorylation , synaptic loss , and neurodegeneration without inducing an increase in a levels . surprisingly , overexpression of happ together with -secretase in mice induces a decrease in a levels and plaque deposition , but the animals suffer severe neurodegenerative disorders and learning defects . in both models , an accumulation of c - terminal fragments of app including the aicd is it therefore possible that this fragment generated along with the a may be responsible for the alterations in transgenic models of ad ? interestingly , the ad model termed pdapp , when combined with a mutated form of the aicd ( d664a ) , shows a complete reversion of the neuropathological hallmarks of the disease , including synaptic loss , the dentate gyrus atrophy , the astrogliosis , the deficits in synaptic transmission and memory , and the behavioral abnormalities without affecting the a levels or the plaque accumulation [ 111114 ] . these results strongly suggest that the causal relationship between the a accumulation and the neuropathological defects usually associated with ad may be challenged and position the aicd as a good candidate to explain the effects observed in various transgenic models based on mutations in app and ps1 . the two hallmarks of ad , the amyloid plaques , and neurofibrillary tangles , which are elegantly related through the amyloid cascade hypothesis , are the main components in the current research on the molecular mechanisms leading to this pathology . since its origin , the amyloid cascade hypothesis has accumulated substantial evidence in its support , which has virtually overshadowed the fact that clinical trials based on this hypothesis have been shown to be unsuccessful . one of many possibilities to explain the failure of clinical trials could be related with the fact that several mouse models express the human - mutated app found in familial ad , so it is unclear which abnormalities detected in these models are product of specific a species ( like oligomers ) or another toxic metabolites of app ( like aicd ) or simply due to effects of overexpression of happ . however , the evidence collected from the transgenic models here reviewed could help to discern whether the a species or the aicd are the key elements triggering neurodegeneration . three independent transgenic mice lines ( a single transgenic of happ , a double aicd / fe65 transgenic , and the double happ/-secretase transgenic ) recapitulate the neuropathological alterations of the disease without any increase in a secretion . all of these models have an accumulation of the app c - terminal fragments . moreover , the introduction of a point mutation in the aicd in transgenic mice expressing the happ with the swe and indiana mutations , the ad - like phenotype is reversed , in spite of increased a production . all of these evidences suggest that the aicd could be acting as the bona fide toxic intermediate in the ad progression and could become a target for future therapeutic interventions against this devastating disease .

since its proposal in 1994 , the amyloid cascade hypothesis has prevailed as the mainstream research subject on the molecular mechanisms leading to the alzheimer 's disease ( ad ) . most of the field had been historically based on the role of the different forms of aggregation of -amyloid peptide ( a ) . however , a soluble intracellular fragment termed amyloid precursor protein ( app ) intracellular domain ( aicd ) is produced in conjunction with a fragments . this peptide had been shown to be highly toxic in both culture neurons and transgenic mice models . with the advent of this new toxic fragment , the centerpiece for the ethiology of the disease may be changed . this paper discusses the potential role of multiprotein complexes between the aicd and its adapter protein fe65 and how this could be a potentially important new agent in the neurodegeneration observed in the ad .

the most recent national hospital discharge survey reported over 400,000 patients underwent cardiac surgery in the united states in 2010 . in cardiac patients , respiratory problems following extubation are the most significant cause of postoperative comorbidity , despite considerable improvement in postoperative care and monitoring . in a recent study of 7,105 cardiac patients , 216 were readmitted to the intensive care unit ( icu ) due to respiratory complications . this represented 39% of all icu readmissions in that study . in recent study of the 7.8% of patients readmitted to the icu following cardiac surgery , respiratory failure was reported to be the cause for 39% of them [ 2 , 4 ] . current trends in cardiac anesthesia recommend early extubation , but that is not always feasible and prolonged endotracheal intubation and mechanical ventilation place cardiac patients at an increased risk of respiratory complications . previous data show that the airway can constrict immediately after extubation as a result of mechanical irritation to the upper airway and prolonged supine position . cardiopulmonary bypass patients are at an even greater risk since they can develop temporary pulmonary ischemia that decreases the production of surfactant and stiffens respiratory tissue . timely detection and prevention of respiratory compromise in this population has become a priority . despite the inherent dangers caused by respiratory complications , current in - hospital monitoring is still lacking . a non - invasive technology that could reliably measure respiratory function after extubation and give early warning signs of respiratory compromise before harmful events occurred could markedly improve patient care and safety . subjective clinical assessment and respiratory rate ( rr ) monitoring are both inconsistent and unreliable indicators of respiratory sufficiency , and current clinical practice relies heavily on continuous pulse oximetry or capnography . though used in almost every clinical setting , pulse oximetry can be unreliable and studies have shown nearly two - thirds of alarms triggered by the oximeter to be false [ 6 , 7 ] . furthermore , oxygen saturation is a lagging indicator , as it decreases only after a significant drop in ventilatory adequacy and readings are often confounded by the use of supplemental oxygen leading to possible delay in interventions and risk to patient safety . one of the difficulties with capnography in the non - intubated patients is patient compliance and maintaining proper placement of the sampling cannula . both oximetry and capnography are indirect measurements of respiratory performance , as they do not directly monitor the adequacy of respiration , but are rather surrogate measures of respiratory status . while a skilled clinician may measure a patient s rr by counting chest wall movements , an obstructed airway or chest muscle contraction could result in this same movement rather than true respiratory effort . in the obese population , observing a chest rise and fall is increasingly challenging and even if one can accurately count the rr , one would not be able to easily distinguish between adequate and inadequate breathing . the currently available measures are limited and often do not provide enough clinically relevant information to trigger healthcare provider response and early intervention to prevent respiratory compromise . recent development of a non - invasive respiratory volume monitor ( rvm ) that provides quantitative , continuous , real - time measurements of mv , tv and rr in non - intubated patients has addressed a number of these challenges . with the recommended electrode placement and calibration algorithms , strong correlations ( 0.96 0.16 , mean 95% ci for regular and erratic breathing ) between rvm and spirometric measurements the rvm can quantify ventilation in non - intubated patients and help identify patients at risk for respiratory complications following cardiac surgery and in other clinical settings [ 11 - 15 ] . the goal of this study was to demonstrate that an alternate electrode placement , to accommodate the sternal dressing , would not affect the rvm correlations to the ventilator and to show the utility of using rvm to monitor mv , tv , and rr in cardiac surgery patients after median sternotomy . it was hypothesized rvm would provide accurate , continuous measurements of mv , tv , and rr in this cohort before and after extubation . fourteen cardiothoracic patients ( four females and 10 males , average age 69 11.8 years , average bmi 29 6.0 kg / m , eight coronary artery bypasses , four valve replacements , and two myomectomies , table 1 ) were enrolled in an irb approved protocol for monitoring respiratory status in the tufts cardiothoracic unit ( ctu ) . respiratory traces were recorded from thoracic electrodes using an impedance - based rvm system ( exspiron , respiratory motion , inc . , all cardiothoracic procedures were performed under general anesthesia and all patients arrived in the ctu intubated and sedated . upon arrival in the ctu , the exspiron electrode padset was positioned to the right of the sternal dressing , approximately at the right mid - clavicular line , instead of the standard midsternal placement . the device was calibrated while the patient was on the ventilator ( puritan - bennett 840 , covidien , mansfield , ma ) during synchronous intermittent mandatory ventilation mode ( simv ) with setting at 6 - 8 ml / kg ibw as determined by the anesthesiologist . respiratory data ( mv , tv and rr ) were acquired only every 15 s from the ventilator , as the ventilator did not support continuous respiratory trace streaming . rvm - based mv , tv , and rr were calculated from continuous 30-s segments . rvm data were recorded continuously from ctu arrival until 24 h after extubation or ctu discharge , whichever occurred first . arterial blood gases were collected before extubation and again approximately 1 - 2 h after extubation in a subset of patients ( n = 12 ) as deemed necessary by the surgical care team . rvm traces were continuously recorded in the ctu for 27 3.7 h ( 6 - 49 h ) . after extubation , patients remained in the ctu and were monitored by the rvm for another 18 2.5 h ( 2 - 29 h ) . twelve of 14 of the patients had ventilator reading for analysis . during mechanical ventilation , tvs measured by the rvm strongly correlated with tvs reported by the ventilator ( r = 0.97 ) . this correlation was maintained during both mandatory ventilation ( simv ) and during spontaneous breathing with pressure support ( ps ) modes . patient average error in tv measurements between rvm and the ventilator was -2.6% , the average precision was 23.9% and average accuracy was 26.2% . an example of simultaneous rvm and ventilator recordings for 30 min is shown in figure 1 . during this time , the ventilator measured an average mv of 6.3 l / min , a tv of 520 ml , and a rate of 12 breaths / min , while rvm measured an average mv of 5.9 l / min ( 7% error ) , an average tv of 490 ml ( 6% error ) , and an average rr of 12.2 breaths / min ( 3% error ) . measured mv , tv , and rr over a 30-min period using rvm ( red ) and the puritan bennett ventilator in simv mode ( black ) for one example patient . average errors between rvm and the ventilator for mv , tv , and rr are less than 10% . before and after extubation , the rvm recorded respiratory traces continuously , in real - time , and captured a distinct pattern of post - extubation respiratory variation . based on body surface area , the average predicted mv was calculated to be 7.6 0.6 l / min for the 14 patients . during 2 min of breathing on simv mode on the ventilator , the average baseline ventilation was slightly above the predicted at 8.2 0.4 l / min ( 1095% of predicted ) . analysis of the exspiron data from the 14 studied patients showed mv was significantly reduced 30 min after extubation to 779% of baseline ( 8.2 0.4 to 6.4 0.9 l / min , p < 0.05 ) and tv was significantly reduced to 719% of baseline ( 520 40 to 370 40 ml , p < 0.01 ) relative to the pre - extubation baseline mv ( table 2 , fig . mv began to increase and by 60 min after extubation , average mv and tv had returned to baseline levels ( mv : 11816% of baseline ( 9.6 1.2 l / min ) ; tv : 10516% of baseline ( 490 50 ml ) ; p > 0.4 ) . thirty minutes after extubation , rr was 1138% of baseline ( 17.1 1.5 to 17.5 1.0 breaths / min , p = 0.4 ) . it is important to note that rr is the only direct and continuous respiratory parameter currently monitored after extubation . sao2 monitoring was recorded every hour by the nursing staff and revealed no significant drops in blood oxygen saturation after extubation ( fig . ( a ) mv , tv , and rr as percent of baseline before and after extubation . a nadir in ventilation occurs 30 min after extubation with mv significantly reduced to 779% of baseline ( * p < 0.05 ) and tv significantly reduced to 719% of baseline ( * p < 0.01 ) , while rr is not significantly different at 1138% of baseline . sixty minutes after extubation , mv has returned to 11816% of baseline and tv has returned to 10516% of baseline . ( b ) absolute values of mv , tv , and rr measured before and after extubation . mv falls significantly from 8.2 0.4 l / min at baseline to 6.4 0.9 l / min 30 min after extubation and tv was significantly reduced from 520 40 ml at baseline to 370 40 ml after extubation . the minimum spo2 value recorded at each time point among all subjects is shown as a blood gas measurements were taken on average 56 25 min before extubation and 106 27 min after extubation . spo2 remained unchanged from before to after extubation ( 98 0.4 vs. 98 0.8 , p = 0.21 , table 3 ) . there was a slight significant increase in bicarbonate from before to after extubation , but was of no clinical importance ( 21.3 0.5 vs. 22.3 0.4 , p = 0.04 ) . example respiratory traces from a representative patient ( 60-year - old male , bmi 29 ) . as the patient awakes from anesthesia and progresses toward extubation , faster and shallower spontaneous breaths predominate with an occasional ventilator breath ( b ) . just after extubation , the patient s breathing pattern is more irregular ( c , d ) and tvs and mvs decrease ( d , e ) . tv and mv measurements increase over time after successful extubation ( f , g , h ) . while intubated , rvm - based mv , tv and rr correlated well with ctu ventilator measurements in patients after median sternotomy . after extubation , rvm continuously reported mv , tv and rr , providing a continuous , non - invasive , quantitative assessment of respiratory competence . a pattern of mv and tv decrease during the first 30 min after extubation was noted in all patients . subsequent recovery to pre - extubation levels within 1 h as confirmed by paco2 from arterial blood gas was noted in all patients . rvm showed distinct changes in mv and tv after extubation , while rr showed no significant variations . although no patients in this study required reintubation , based on data from other studies collected after extubation in premature infants , we propose that patients who continue to have a decrease in mv with no rise to baseline within 1 h after extubation will require intervention such as non - invasive ventilation or reintubation . further studies to evaluate the use of rvm in postoperative cardiac surgical patients for early detection of respiratory failure for development of reintubation protocols are ongoing . this study demonstrated that , while intubated , rvm - based mv , tv and rr correlated well with ventilator measurements ( r = 0.97 ) . in addition , the change in the padset placement to accommodate the sternal wound and dressing did not affect the device measurements . this confirms that the rvm can provide accurate measurements of mv , tv and rr in patient s post - median sternotomy using the modified positioning of the electrode padset . after extubation , rvm continuously reported mv , tv and rr , providing a real - time , non - invasive , quantitative assessment of respiratory competence . a pattern of mv and tv decrease during the first 30 min after extubation subsequent recovery to pre - extubation levels within 1 h as confirmed by paco2 from arterial blood gas was noted in all patients . this shows that rvm is capable of providing patterns and trends in ventilation status not available to healthcare providers with other technology . respiratory complications are one of the leading causes of readmission to the icu for cardiac surgery patients . this study shows the benefits of this new technology and its application in the ctu and possibly other locations . pulse oximetry is the leading technology used to assess respiratory status in non - intubated patients ; however , spo2 levels do not begin to decline until well after respiratory decompensation has begun and is known for being prone to inaccuracies [ 13 , 17 ] . intermittent blood gas measurements were taken intermittently and on average over 1 h after extubation , well after rvm confirmed a recovery in mv . rvm showed distinct changes in mv and tv after extubation , while rr , generally measured with cardiac monitoring , showed no significant variations . the time it takes a patient to return to baseline mv is inversely proportional to successful extubation , therefore demonstrating the importance of obtaining mv measurements . additionally , determination of mv after extubation using rvm could potentially aid in quicker assessment of the necessity and timing of reintubation , or provide an early indication of when institute non - invasive ventilation ( niv ) , in the form of continuous positive airway pressure or biphasic positive airway pressure . although no patients in this study required reintubation , based on data from other studies collected after extubation in premature infants , it is hypothesized that patients who continue to have a decrease in mv with no rise to baseline within 1 h after extubation will require intervention such as non - invasive ventilation or reintubation . recently , the american society of anesthesiology has voiced the opinion that quantitative monitoring of respiratory status is superior over qualitative methods . qualitative clinical signs such as chest excursion and auscultation of breath sounds are useful , but they can only be obtained periodically and are often inaccurate . carbon dioxide monitoring is also a qualitative sign of respiration in non - intubated patients and its accuracy is dependent on many factors . however , non - invasive mv and tv have been previously unavailable in non - intubated patients . the rvm permits a more comprehensive , quantitative assessment of respiratory status . with this functionality , rvm - derived respiratory data can bring forth reproducible and comparable data across patients over a wide range of breathing patterns . this can promote better evaluation of respiratory status in the ctu and other healthcare settings . while intubated , rvm - based mv , tv and rr correlated well with ctu ventilator measurements in patients after median sternotomy . after extubation , rvm continuously reported mv , tv and rr , providing a continuous , non - invasive , quantitative assessment of respiratory competence . a pattern of mv and tv decrease during the first 30 min after extubation was noted in all patients . subsequent recovery to pre - extubation levels within 1 h as confirmed by paco2 from arterial blood gas was noted in all patients . rvm showed distinct changes in mv and tv after extubation , while rr showed no significant variations . although no patients in this study required reintubation , based on data from other studies collected after extubation in premature infants , we propose that patients who continue to have a decrease in mv with no rise to baseline within 1 h after extubation will require intervention such as non - invasive ventilation or reintubation . further studies to evaluate the use of rvm in postoperative cardiac surgical patients for early detection of respiratory failure for development of reintubation protocols are ongoing . this study demonstrated that , while intubated , rvm - based mv , tv and rr correlated well with ventilator measurements ( r = 0.97 ) . in addition , the change in the padset placement to accommodate the sternal wound and dressing did not affect the device measurements . this confirms that the rvm can provide accurate measurements of mv , tv and rr in patient s post - median sternotomy using the modified positioning of the electrode padset . after extubation , rvm continuously reported mv , tv and rr , providing a real - time , non - invasive , quantitative assessment of respiratory competence . a pattern of mv and tv decrease during the first 30 min after extubation was noted . subsequent recovery to pre - extubation levels within 1 h as confirmed by paco2 from arterial blood gas was noted in all patients . this shows that rvm is capable of providing patterns and trends in ventilation status not available to healthcare providers with other technology . respiratory complications are one of the leading causes of readmission to the icu for cardiac surgery patients . this study shows the benefits of this new technology and its application in the ctu and possibly other locations . pulse oximetry is the leading technology used to assess respiratory status in non - intubated patients ; however , spo2 levels do not begin to decline until well after respiratory decompensation has begun and is known for being prone to inaccuracies [ 13 , 17 ] . intermittent blood gas measurements were taken intermittently and on average over 1 h after extubation , well after rvm confirmed a recovery in mv . rvm showed distinct changes in mv and tv after extubation , while rr , generally measured with cardiac monitoring , showed no significant variations . the time it takes a patient to return to baseline mv is inversely proportional to successful extubation , therefore demonstrating the importance of obtaining mv measurements . additionally , determination of mv after extubation using rvm could potentially aid in quicker assessment of the necessity and timing of reintubation , or provide an early indication of when institute non - invasive ventilation ( niv ) , in the form of continuous positive airway pressure or biphasic positive airway pressure . although no patients in this study required reintubation , based on data from other studies collected after extubation in premature infants , it is hypothesized that patients who continue to have a decrease in mv with no rise to baseline within 1 h after extubation will require intervention such as non - invasive ventilation or reintubation . recently , the american society of anesthesiology has voiced the opinion that quantitative monitoring of respiratory status is superior over qualitative methods . qualitative clinical signs such as chest excursion and auscultation of breath sounds are useful , but they can only be obtained periodically and are often inaccurate . carbon dioxide monitoring is also a qualitative sign of respiration in non - intubated patients and its accuracy is dependent on many factors . however , non - invasive mv and tv have been previously unavailable in non - intubated patients . , the rvm permits a more comprehensive , quantitative assessment of respiratory status . with this functionality , rvm - derived respiratory data can bring forth reproducible and comparable data across patients over a wide range of breathing patterns . this can promote better evaluation of respiratory status in the ctu and other healthcare settings .

backgroundpatients who have undergone cardiac surgery are generally mechanically ventilated postoperatively . early postoperative extubation is currently recommended in anesthesia guidelines . no current technology can accurately , non - invasively , measure respiratory competence after extubation . pulse oximetry has been helpful , but this is a late indicator of respiratory compromise . a novel , non - invasive , respiratory volume monitor ( rvm ) has been shown to deliver accurate continuous , real - time minute ventilation ( mv ) , tidal volume ( tv ) and respiratory rate ( rr ) measurements and provide an objective measure of respiratory competence . the rvm will accurately reflect mv , tv and rr in cardiac surgery patients before and after extubation.methodsrvm traces were recorded from patients before and after cardiac surgery . continuous monitoring began on admission to the unit and was ended at 24 h after extubation . rvm - based mv , tv and rr were calculated from 30-s segments . mv , tv and rr were also continuously recorded from the ventilator prior to extubation . the rvm was calibrated to each patient using the readings from the ventilator.resultsduring mechanical ventilation , the rvm measured tvs strongly correlated with the ventilator tvs ( r = 0.97 ) . following extubation , the patient s breathing became more erratic and tvs and mvs decreased . within 1 h , all patients studied showed a marked recovery of mv and tv.conclusionsrvm-based mv , tv and rr correlated well with similar data collected from ventilators . after extubation , rvm shows promise as a means to monitor respiratory competence of non - intubated patients , and has implications for use in other settings and improving patient safety .

to report an unusual case of multifocal bacterial keratitis that despite success - ful treatment caused chronic ocular hypertension . a 67-year - old woman with unilateral multifocal keratitis and no previous ocular pathology was admitted to our hospital . corneal scrapings and conjunctival samples were obtained for culture and the patient received intensive therapy with fortified vancomycin and tobramycin eye drops . the cultures demonstrated two strains of staphylococcus epidermidis , one resistant to ciprofloxacin and both sensitive to vancomycin . treatment was effective and gradually discontinued after total cessation of the inflammatory activity . during the follow - up period , the patient developed late and persistent ocular hypertension of unknown etiology , in absence of any detectable inflammation or complication , and received permanent antiglaucoma therapy . patients with multifocal bacterial keratitis may need intraocular pressure monitoring , even after complete infection healing . staphylococcus is a common gram - positive bacterium causing keratitis that tends to present with a focal and well - defined white or yellow - white infiltrate . during the infection , corneal inflammation can lead to neovascularization and scarring , while internal ocular inflammation can cause synechia formation , elevated intraocular pressure and cataract [ 1 , 2 ] . several staphylococcal strains have been reported resistant to topical quinolone therapy [ 3 , 4 ] . we report an unusual case of quinolone - resistant multifocal staphylococcal keratitis without a history of previous ocular pathology , which caused chronic intraocular pressure elevation despite successful keratitis treatment and complete resolution of external and internal ocular inflammation . a 67-year - old woman presented in the emergency section of our department with gradual onset of redness , tearing , photophobia and a 4-day history of blurred vision in her right eye . there was no history of contact lens wear , previous ocular disease , trauma or surgery . examination of the right eye revealed visual acuity of counting fingers , intense conjunctival injection , mild corneal edema and three corneal ulcers of different extension ( 15 mm in diameter ) . a moderate aqueous flare was noted in the anterior chamber without presence of hypopyon or pupillary synechiae ( fig . 1 ) . intraocular pressure and fundus were normal , and there were no pathological findings in her left eye . corneal scrapings and conjunctival samples were obtained from the right eye and sent to the laboratory for culture . the patient was hospitalized and received empirical treatment with fortified vancomycin + tobramycin ( voncon , lilly + tobrex , alcon ) eye drops every 30 min the first day , every hour the second day and afterwards every 2 h. topical cyclopentolate ( cyclogyl , alcon ) 3 times daily was also administered . the cultures demonstrated the presence of two strains of coagulase - negative staphylococcus epidermidis , one resistant to ciprofloxacin and both sensitive to vancomycin . one week after treatment implementation , the patient reported partial relief from her initial symptoms . a considerable reduction of the extension of corneal ulcers was noted , though aqueous flare and intraocular pressure increased ( 32 mm hg ) and corneal neovascularization appeared at the limbus adjacent to the corneal ulcers . treatment was modified to topical vancomycin , dexamethasone ( maxidex , alcon ) and cyclopentolate initially 3 times daily , with a progressive tapering scheme during the following 3 weeks . after the 3-week - period , the corneal ulcers were completely healed ; there was no conjunctival injection , no visible aqueous flare but only a mild scarring at the superior limbus ( fig . two months later , a persistent ocular hypertension ( 2628 mm hg ) of unknown etiology and without any external or internal ocular inflammation was noted in the right eye at three consecutive visits . a goldmann contact lens was used to perform a 360 gonioscopy that did not reveal any pathological findings . intraocular pressure was normal in the left eye and corneal pachymetry was normal in both eyes . topical brinzolamide was restarted and follow - up was continued monthly . for the next 6 months , intraocular pressure in the right eye fluctuated within normal limits and no inflammatory recurrence was observed . occasionally , staphylococcal keratitis can present with predominantly multifocal epithelial infiltration , especially in the setting of hydrophilic contact lens wear or after laser in situ keratomileusis . multiple foci of abscesses resemble fungal satellite lesions [ 1 , 5 ] . in the present case , furthermore , we did not detect any predisposing factors for chronic intraocular pressure elevation , such as history of glaucoma or ocular hypertension , trauma , relapsing inflammation and anterior or posterior synechiae . the existence of aqueous flare in the anterior chamber could explain the temporary intraocular pressure rise diagnosed in the second week , though after total cessation of the inflammatory activity the intraocular pressure returned to normal values as expected . steroid - induced ocular hypertension does not seem to be relevant since steroid treatment was of limited duration , low dosed and gradually discontinued . a possible explanation for the late and persistent intraocular pressure elevation may be a permanent damage of the trabecular meshwork caused by staphylococcal toxins . despite normal appearance of the iridocorneal angle on gonioscopy , invisible microscopic changes could not be excluded . the resistance of one s. epidermidis strain to ciprofloxacin is in accordance with previous reports and indicates that microbial culture is necessary in cases of extensive bacterial corneal ulcers [ 3 , 4 ] . furthermore , it also shows that an initial empirical treatment with a combination of fortified topical antibiotics may be superior to topical quinolone monotherapy in these cases . however , further evidence is required to support the above hypothesis since there are studies showing equivalence of fluoroquinolone monotherapy to fortified medications in microbial keratitis [ 6 , 7 ] . unfortunately , it was not possible for our laboratory to test the sensitivity of the ciprofloxacin - resistant strain to newer - generation quinolones , which could have had better activity against these gram - positive organisms . in conclusion , the present case demonstrates that in patients with multifocal keratitis , differential diagnosis between fungal and bacterial infection is critical for successful treatment . besides treatment , patient follow - up and intraocular pressure monitoring may be needed , even after complete infection healing and resolution of inflammatory signs .

purposeto report an unusual case of multifocal bacterial keratitis that despite success - ful treatment caused chronic ocular hypertension.methodsa 67-year - old woman with unilateral multifocal keratitis and no previous ocular pathology was admitted to our hospital . corneal scrapings and conjunctival samples were obtained for culture and the patient received intensive therapy with fortified vancomycin and tobramycin eye drops.resultsthe cultures demonstrated two strains of staphylococcus epidermidis , one resistant to ciprofloxacin and both sensitive to vancomycin . treatment was effective and gradually discontinued after total cessation of the inflammatory activity . during the follow - up period , the patient developed late and persistent ocular hypertension of unknown etiology , in absence of any detectable inflammation or complication , and received permanent antiglaucoma therapy.conclusiondifferential diagnosis between fungal and bacterial infection is critical in cases of multifocal keratitis . patients with multifocal bacterial keratitis may need intraocular pressure monitoring , even after complete infection healing .

although s. pneumoniae is the most common cause of community - acquired pneumonia ( cap ) in many countries , there are considerable geographic differences in the incidence of other pathogens . tuberculosis ( tb ) is often overlooked as a cause of cap , particularly when it presents as an acute illness ( 2 ) . although the presentation of pulmonary tb as acute pneumonia is not unusual , its manifestation as acute respiratory distress syndrome ( ards ) is rare . ards is a severe form of diffuse alveolar injury . the american - european consensus conference ( aecc ) ( 1994 ) defined ards as an acute condition characterized by bilateral pulmonary infiltrates and severe hypoxemia in the absence of evidence for cardiogenic pulmonary edema . the severity of hypoxemia necessary to make the diagnosis of ards was defined by the ratio of the partial pressure of oxygen in the patient s arterial blood ( pao2 ) to the fraction of oxygen in the inspired air ( fio2 ) . ards was defined by a pao2/fio2 ratio of less than 200 , and , in acute lung injury ( ali ) , by a ratio less than 300 ( 3 ) . recently , a panel of experts redefined the definition ( berlin definition ) ( 2011 ) and categorized ards into three levels : mild ( pao2/fio2 200300 ) , moderate ( pao2/fio2 100200 ) , and severe ( pao2/fio2 100 ) ( 4 ) . even though ali and ards are well recognized complications in patients with severe tb , such as miliary tb or tb bronchopneumonia , sparse epidemiological data are available on this entity . most patients who develop ali / ards have underlying chronic medical conditions or hiv infections , and their diagnoses were delayed ( 5 ) . herein , we present a rare case of a previously healthy young patient with acute pulmonary tuberculosis in the form of tb pneumonia that was complicated by ards . a 21-year - old woman , a radiology technician student , presented with acute respiratory failure due to progressive respiratory symptoms , including productive cough and dyspnea that started seven days before presentation . at the time of her admission to the hospital her initial vital signs were as follows : bp = 110/70 mmhg , hr = 122/min , rr = 40/min , and temperature = 40 c . lung examination revealed bilateral coarse crackles . because she presented during the influenza season and because the symptoms were compatible with severe pneumonia , a broad spectrum of antibiotics and oseltamivir were started , and she was intubated and received mechanical ventilation . there was no history of previous illness or recent travel and no family history of note . arterial blood gas revealed hypoxemia ( pao2 = 24.3 mmhg ) , hypercapnia ( paco2 = 55.1 mmhg ) , and respiratory acidosis ( ph = 7.25 , hco3 : 26 ) . the hiv test was negative . based on an initial pao2/fio2 ratio of less than 100 on ventilator setting that included peep and characteristic diffuse bilateral infiltrates on chest x - ray ( figure 1 ) , acute respiratory distress syndrome ( ards ) bacterial and fungal cultures of respiratory secretions showed no growth after seven days . a real - time pcr test on respiratory specimens for influenza a and b was negative . accordingly , diagnosis of ards secondary to pulmonary tuberculosis was made , and the patient received anti - tuberculous drugs and intravenous hydrocortisone 100 mg three times per day . the patient continued to improve gradually during the first week , and she was successfully weaned and extubated on her seventh day in the hospital . her chest infiltrates gradually resolved during the first week , but left lower- and mid - zone consolidation remained ( figure 3 ) . she remained hospitalized in the infectious ward and received o2 therapy plus anti - tuberculous drugs for another 15 days . corticosteroids were stopped . during this period she remained mildly tachypnic and hypoxic . on the 24 day after presentation , she was discharged with stable vital signs and no hypoxia in the room air . a 21-year - old woman , a radiology technician student , presented with acute respiratory failure due to progressive respiratory symptoms , including productive cough and dyspnea that started seven days before presentation . at the time of her admission to the hospital her initial vital signs were as follows : bp = 110/70 mmhg , hr = 122/min , rr = 40/min , and temperature = 40 c . lung examination revealed bilateral coarse crackles . because she presented during the influenza season and because the symptoms were compatible with severe pneumonia , a broad spectrum of antibiotics and oseltamivir were started , and she was intubated and received mechanical ventilation . there was no history of previous illness or recent travel and no family history of note . arterial blood gas revealed hypoxemia ( pao2 = 24.3 mmhg ) , hypercapnia ( paco2 = 55.1 mmhg ) , and respiratory acidosis ( ph = 7.25 , hco3 : 26 ) . the hiv test was negative . based on an initial pao2/fio2 ratio of less than 100 on ventilator setting that included peep and characteristic diffuse bilateral infiltrates on chest x - ray ( figure 1 ) , acute respiratory distress syndrome ( ards ) bacterial and fungal cultures of respiratory secretions showed no growth after seven days . a real - time pcr test on respiratory specimens for influenza a and b was negative . accordingly , diagnosis of ards secondary to pulmonary tuberculosis was made , and the patient received anti - tuberculous drugs and intravenous hydrocortisone 100 mg three times per day . the patient continued to improve gradually during the first week , and she was successfully weaned and extubated on her seventh day in the hospital . her chest infiltrates gradually resolved during the first week , but left lower- and mid - zone consolidation remained ( figure 3 ) . she remained hospitalized in the infectious ward and received o2 therapy plus anti - tuberculous drugs for another 15 days . corticosteroids were stopped . during this period she remained mildly tachypnic and hypoxic . on the 24 day after presentation , she was discharged with stable vital signs and no hypoxia in the room air . this case features a rare manifestation of pulmonary tuberculosis in a previously healthy young woman with acute presentation of tuberculous pneumonia complicated by ards . tuberculous pneumonia is defined as nodular lesions resulting from air - space consolidation due to endobronchial spread to lobar or multilobar locations ( 5 ) . m. tuberculosis is an important cause of community - acquired pneumonia ( cap ) in developing countries . tb can present as an acute process and should be included in the differential diagnosis of cap . it may mimic classic bacterial pneumonia or masquerade as an atypical pneumonia , with non - productive cough and systemic symptoms . missed diagnosis is common , as illustrated in report from baltimore in which 16 of 33 patients ( 48% ) with culture - confirmed pulmonary tb were treated initially for presumed cap ( 1 ) . the incidence of tb being diagnosed among patients presenting with clinical and radiological signs of a cap has varied in different series . previously , studies from china , japan , kenya , and sub saharan africa also demonstrated high prevalence of pulmonary tb ( 920.5% ) in patients who presented with cap ( 611 ) . m. tuberculosis was the second most common pathogen ( 12% ) identified in the study performed by levy et al . , only patients who presented with features of acute pneumonia were included ; those with chronic illness or typical radiologic changes suggestive of tb were excluded . a similar high incidence of tb ( 10% ) among subjects presenting with acute pneumonia was reported in france ( 7 ) . although presentation of pulmonary tb as acute pneumonia is not unusual , its manifestation as ards is rare . this complication is associated with a very high mortality ( 4080% ) despite treatment ( 5 ) . even in a large study from india that was conducted by agarwal et al , tb accounted for only 4.9% of patients ( 9 of 187 ) who were admitted with a diagnosis of ards over a 7-year period , of whom two patients died . the lower mortality rate in this study could be attributed to the fact that all subjects were started empirically on anti - tb therapy with the median time to initiation of therapy being three days . tracheal aspirate sent for ziehl - neelsen staining did not indicate that there were acid - fast bacilli in any of the patients of this study ( 13 ) . in another study by sharma et al . in india , of 2,733 tb patients treated during 19802003 , 29 ( 1.06% , i.e. , 1.21 patients / year ) developed ards ( 14 ) . another large retrospective study conducted by deng et al . in china over a five - year period ( 20062010 ) showed that , among 16,238 patients who were admitted to the respiratory departments with a diagnosis of pulmonary tb , 471 patients were diagnosed as having miliary tb , of whom 85 developed ards and were admitted to the icu during the study period . the mortality rate in this study was 47.1% , and the mean duration of mechanical ventilation was 8.5 3.0 days in all patients ( 15 ) . another study from south korea described 67 patients with ards caused by miliary tb admitted to the icu during 19992008 . all - cause mortalities in the icu and the hospital were 58.2 and 61.2% , respectively . in their study , 28.4% of the patients were older than 71 , but clinical outcomes in these patients did not differ significantly from outcomes in younger patients ( 16 ) . ( 17 ) found that higher psi scores were independently associated with the presence of concomitant pulmonary tb in both health care associated pneumonia ( hcap ) and cap patients . in the study performed by levy et al . ( 18 ) , the incidence rate of 1.5% was reported for acute respiratory failure among hospitalized patients who presented with tuberculous cap . although ards invariably occurs in patients with severe tb , such as miliary tb or tb bronchopneumonia ( 5 ) , its occurrence in tuberculous pneumonia and cavitary pulmonary tb also was described ( 5 ) . in the study of agarwal et al . , radiography of eight of nine patients with tb and ards revealed miliary nodules , and consolidation was observed in only one patient ( 13 ) . seven out of 29 patients with tb and ards in the study of sharma et al . had pulmonary tb , and 22 had miliary tb ( 14 ) . several predisposing factors for development of ards in tb patients and several prognostic factors in these patients have been described . according to the study of deng et al . , the duration of time to diagnosis , time from diagnosis to mechanical ventilation , and time to anti - tuberculosis therapy were significantly shorter in survivors than for non - survivors . diabetes mellitus , increased ast , alt , and d - dimer , decreased hemoglobin , and albumin were found to be independent predictors of the development of ards in the setting of miliary tb ( 15 ) . in the study reported by sharma et al . , the presence of miliary tb , duration of illness beyond 30 days at presentation , absolute lymphocyte count less than 1625/mm and serum alt greater than 100 iu / l were independent predictors of the development of ards . patients with apache ii scores > 18 , those with apache ii scores < 18 in the presence of hyponatraemia , and those with pao2/fio2 ratios < 108.5 were likely to die ( 14 ) . showed that the sequential organ failure assessment ( sofa ) score on the day that ards is diagnosed is a valuable prognostic indicator ( 16 ) . the case presented is special in its manifestation from several clinical perspectives , including lack of underlying medical condition or immune defect and development of ards in non - miliary and non - disseminated tuberculosis . in conclusion , the diagnosis of tb should be considered in all patients who present with cap in our region . it has been suggested that , in a patient with unexplained ards , a history of fever of more than 15 days duration and elevation of serum alkaline phosphatase should arouse the suspicion of disseminated tb as the underlying cause ( 5 ) . it also has been suggested that , in patients with ards of obscure etiology where the clinical features suggest tb as the inciting cause , anti - tb therapy should be started empirically , and the diagnosis actively pursued later ( 13 ) . both primary and reactivation tb may present as an acute illness and mimic cap . as a result of the impressive mimicry of tb , the diagnosis must be considered , at least initially , in all cases of acute pneumonitis , especially in regions where tuberculosis is endemic .

the presented case features a rare manifestation of pulmonary tuberculosis in a previously healthy young woman who had acute presentation of tuberculous pneumonia complicated by acute respiratory distress syndrome . in developing countries , mycobacterium tuberculosis is an important cause of community - acquired pneumonia ( cap ) . tb can present as an acute process and should be included in the differential diagnosis of cap . this case is special in its manifestation from several clinical perspectives , including the lack of an underlying medical condition or immune defect and the development of acute respiratory distress syndrome ( ards ) in non - miliary and non - disseminated tuberculosis . in conclusion , the diagnosis of tb should be considered in all patients who present with cap in endemic regions .

in may 2014 , a pig fattening farm in southern germany ( federal state of baden - wuerttemberg ) that continuously houses 1,400 fattening pigs reported watery diarrhea in pigs in all age groups ( feeders to slaughter animals ) . the first cases occurred after new feeder pigs from a large piglet producer were brought onto the farm . fecal samples from diseased pigs were submitted to the regional laboratory for diagnosis , and coronaviruses were detected by electron microscopy ( figure 1 ) . additionally , 3 pigs with catarrhal enteritis were euthanized ; postmortem examination at the regional laboratory confirmed coronavirus infection in all 3 animals . porcine epidemic diarrhea virus particles seen by negative - stain electron microscopy of fecal samples . subsequently , ped was diagnosed in a private laboratory ( ivd gmbh , hannover , germany ) by use of a published multiplex reverse transcription quantitative pcr ( 7 ) . selected positive samples were submitted to the friedrich - loeffler - institut , isle of riems , germany , for confirmation and further virus characterization . at this institution , 2 fecal samples with high genome load , as determined by 2 independent , published ( 8,9 ) reverse transcription quantitative pcrs selective for pedv nucleocapsid ( n ) and spike ( s ) genes , were chosen for routine virologic testing and next - generation sequencing . the phylogenetic tree , based on full - length genomes , was constructed by using phyml ( 11 ) in the geneious software suite ( http://www.geneious.com/ ) with a generalized time reversible substitution model , and the tree was supported by 1,000 bootstrapping replicates . virus isolates were obtained after inoculation of cells of different permanent cell lines ( pig kidney [ pk]-15 and vero ) with clinical material from the pigs . sequencing of nucleic acids isolated directly from diagnostic samples pedv / ger / l00719/2014 and pedv / ger / l00721/2014 resulted in 2 viral genomes ( table ) encompassing all typical pedv coding sequences . each sequence encodes a large replicase polyprotein , a spike ( s ) protein , an alphacoronavirus - specific accessory membrane protein , an envelope protein , a membrane protein , and a nucleocapsid ( n ) protein . * kj778615 , kf272920 , kf468753 , kj584361 , kj408801 , kf650374 , kf468752 , kf267450 , kc210147 , jn825712 , kc210145 ) . # aa 632f detected in reference strain kf267450 only . comparative analyses of the full genomes showed that the strains share a very high ( 99.5% ) identity with the new variant oh851 ( genbank accession no . a more comprehensive comparison of 21 full - length pedv genomes from different years and locations revealed lower similarities ( 98.7% ) with currently circulating highly virulent strains from the united states and from china ( technical appendix figure , panel a ) . in contrast , the new isolates from germany , pedv / ger / l00719/2014 and pedv / ger / l00721/2014 , are less similar ( 97.1% ) to the isolate from europe , cv777 , which dates back to the 1970s ( 12 ) . the nucleotide alignment of the obtained pedv genomes and the available references from the database revealed a region with high variability of the first 1,200 nt in the 5 portion of the s protein the n terminal s1 domain of the coronavirus s protein is necessary for virus attachment by interaction with host cell receptors ( 13 ) and might therefore be highly mutable . although in - depth analysis of the deep - sequencing data for pedv / ger / l00721/2014 revealed a genetically homogenous population , this analysis for pedv / ger / l00719/2014 uncovered a mixed viral population with a total of 8 single - nucleotide variants . one nonsynonymous single - nucleotide variant ( variant position g19042u , amino acid substitution s6348i ) was detected in the polyprotein coding sequences . of note , 7 single - nucleotide variants are located in the aforementioned variable region in the s protein coding sequences , 5 of which are nonsynonymous ( table ) , thereby confirming the high variability in the n terminal part of the s protein . because quite extensive differences ( 50 aa ) were found between the recent n terminal s protein region of the isolates from germany and the highly virulent pedv strains from the united states and china , the isolates from germany described in this article seem to not be directly linked to the highly virulent pedv strains circulating in the united states ( figure 2 ) . in contrast , the recent isolates from germany and strain oh851 share not only high identity over the entire genome , including the highly variable 5 region of the s protein coding sequences , but also their clinical phenotype observed under field conditions . phylogenetic analysis based on 21 full - length porcine epidemic diarrhea virus ( pedv ) genomes . the new strains from germany ( pedv / ger / l00719/2014 and pedv / ger / l00721/2014 , in boldface and italics ) and the new 2014 pedv variant from the united states ( oh851 , in boldface ) were included and compared with current circulating strains from the united states and china . numbers above branches indicate proportions calculated from 1,000 bootstrap replicates : the scale bar represents nucleotide substitutions per site . comparative analyses of full - length sequences revealed that the isolates from these pigs in germany show very high nucleotide similarity with strain oh851 found in the united states in 2014 . however , differences exist that distinguish the strains from germany from the highly virulent pedv strains that caused the major losses in the united states . given the fact that pedv surveillance has been lacking in germany , we can not exclude the possibility that the strains described here have already been circulating in europe for a longer time or were indeed recently introduced from the united states or asia to europe . therefore , our report provides useful information about recent pedv strains in europe , but a comprehensive evaluation is still difficult because of a lack of data about additional strains . future studies should therefore concentrate on analysis of additional pedv from different years and locations . pairwise similarity matrix based on full - length genomes and schematic representation of the nucleotide sequence alignment of the complete spike protein coding sequences .

since 2013 , highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the united states . to determine the relation of us strains to those recently causing disease in germany , we compared genomes and found that the strain from germany is closely related to variants in the united states .

intraorally , it presents as a soft - tissue swelling of the maxillary anterior mucolabial fold , lateral to midline leading to obliteration of the nasolabial fold . it is submucosal and extraosseous , spreads in the labial sulcus and expands all the soft tissues outwards . extraorally , it appears as a swelling between the upper lip and nasal aperture causing elevation of nasal ala . the terms nasolabial and nasoalveolar cysts have been used synonymously , as these are similar cysts with the only difference that the former is a lesion located entirely within the soft tissues and the latter causes maxillary alveolar bone erosion . a 69-year - old female reported to the dental college with a chief complaint of an extraoral swelling on the right side of her upper lip and elevation of the alar base [ figure 1a ] since 7 - 8 months . history of present illness revealed that the swelling had increased in size since its initiation but was not associated with any pain , drainage through oral / nasal cavity or difficulty in breathing through nasal obstruction . furthermore , there was no history of dacryocystitis / watery eyes but there was a swelling intraorally corresponding to this extraoral swelling which was obliterating the labial vestibule on that side . intraoral examination revealed bilateral smooth , soft , fluctuant masses lateral to the central incisor teeth in the maxillary labial vestibule . the right - sided mass was well - defined [ figure 1b ] and measured approximately 2 3 cm and left - sided mass measured 1.5 1.5 cm when palpated bimanually . aspiration biopsy from right - sided mass revealed straw - colored fluid [ figure 1c ] . ( a ) clinical picture showing extraoral swelling and elevation of alar base on the right side . ( b ) clinical picture showing intraoral swelling in the labial vestibule on the right side . ( c ) straw - colored fluid on aspiration from the right - sided cyst occlusal radiograph [ figure 2a ] showed a large scooped - out radiolucent area with cupping surrounding upper left central incisor ( ul1 ) in the middle and coronal thirds of its root , but not in the periapical area . the cysts were enucleated [ figure 2b and c ] using intraoral approach under local anesthesia and adrenaline ( 1:80,000 ) . a full - thickness incision was reflected along the crest of maxillary alveolar ridge in the canine to canine region and blunt dissection by periosteal elevator exposed two smooth , well - circumscribed cystic swellings just below the nasal floor . there had been scooping of the anterior maxilla on the left side due to expansion of the cyst . in contrast , morphology of alveolar bone on right side was not affected . in the same appointment , extractions of hopeless teeth were also done and the wound was closed with loop sutures using 3 - 0 non - absorbable black mersilk after debridement and irrigation with normal saline . ( c ) enucleation of left cyst histopathology of the right - sided cyst revealed a cystic cavity lined by variable thickness of epithelium predominantly made up of many layered stratified squamous cells along with a few mucosal cells [ figure 3 ] . at few places , stratified squamous and pseudostratified columnar epithelia with many cilia and goblet cells were also evident [ figure 4a and b ] . left - sided cyst specimen showed two or more layered stratified squamous lining epithelium with thin capsule [ figure 5 ] . photomicrograph of a section of right cyst showing epithelial lining predominantly made of many layered stratified squamous cells with few mucosal cells ( h&e stain , 100 ) ( a ) photomicrograph of right cyst : stratified squamous epithelium with mucous cells and cilia ( h&e stain , 400 ) . ( b ) photomicrograph of right cyst : pseudostratified ciliated columnar epithelium with goblet cells ( h&e stain , 400 ) photomicrograph of left cyst showing cystic cavity lined by stratified squamous non - keratinized epithelium with thin capsule ( h&e stain , 40 ) patients seek medical assistance when the cyst becomes infected or cause a cosmetic defect . in the present case , intraoral swellings were present in the labial vestibule on both sides , lateral to the maxillary central incisors , which is the most common symptom associated with this cyst . as the nasolabial cysts arise in soft tissues , in most cases there are no radiographic changes . occasionally , pressure resorption of the underlying bone may occur , in that case , it is known as nasoalveolar cyst , where the cyst being external to the alveolar bone , produces cupping of the underlying cortical plate . in the present report , erosion of bone in a cupping fashion around ul1 was observed as a sequel of pressure from the cyst . routine intraoral radiographs are not diagnostic for nasoalveolar cysts but assist in excluding other odontogenic or non - odontogenic cysts . periapical inflammatory lesions can be excluded by vitality testing of the associated teeth and the absence of radiolucency in the periapical region on intraoral radiographs . globulomaxillary and nasopalatine duct cysts can easily be mistaken as nasolabial cysts ; however , both of these are intraosseous lesions . in the present case , , the rarefaction was observed in the middle and coronal third of the root of ul1 . however , cohen and hertzanu have reported a case of nasolabial cyst with a high growth potential that resulted in the erosion of maxillary alveolus , invaded supporting structures in the region of incisors and caused displacement of these teeth . in the present case also , there were signs of displacement of adjacent teeth . distolabial migration of the crowns of upper central incisors was primarily due to loss of periodontal support caused by periodontitis in addition to pressure from the cyst . 10.6% of nasolabial cysts are bilateral , this finding suggests that these cysts are developmental in origin . etiopathogenesis of this cyst involves two theories suggesting that it arises : as a fissural cyst which develops from embryonic nasal epithelium entrapped between the merging maxillary processes and the medial and lateral nasal processesas a developmental cyst arising from the remnants of the embryonic nasolacrimal ducts , due to histological similarities to the latter . furthermore , this cyst is in the area of the naso - optic furrow and proliferation of the nasolacrimal duct has been seen at this site . although this theory may need some revision , due to the finding of apocrine secretion along the surface of the nasolabial cyst , it seems to be the most accepted one at this stage . the fact that high percentage of linings demonstrate pseudostratified columnar epithelium , often with cilia or numerous goblet cells , is compatible with origin from primitive ductal or glandular structures . so , the most likely origin is from the nasolacrimal duct . in case presented here , the cystic lining was predominantly stratified squamous type , although few areas showed pseudostratified ciliated columnar epithelium . su et al . , in their scanning electron microscopic examination revealed that cilia observed in the light microscope were in fact numerous short globular or irregular microvilli which probably resulted from lack of stimulation by air . as a fissural cyst which develops from embryonic nasal epithelium entrapped between the merging maxillary processes and the medial and lateral nasal processes as a developmental cyst arising from the remnants of the embryonic nasolacrimal ducts , due to histological similarities to the latter . furthermore , this cyst is in the area of the naso - optic furrow and proliferation of the nasolacrimal duct has been seen at this site . although this theory may need some revision , due to the finding of apocrine secretion along the surface of the nasolabial cyst , it seems to be the most accepted one at this stage . the fact that high percentage of linings demonstrate pseudostratified columnar epithelium , often with cilia or numerous goblet cells , is compatible with origin from primitive ductal or glandular structures . so , the most likely origin is from the nasolacrimal duct . in case presented here , the cystic lining was predominantly stratified squamous type , although few areas showed pseudostratified ciliated columnar epithelium . , in their scanning electron microscopic examination revealed that cilia observed in the light microscope were in fact numerous short globular or irregular microvilli which probably resulted from lack of stimulation by air . in the present case , bimanual palpation ( best method of diagnosing this cyst by placing one finger in the labial sulcus and the other on the nasal floor ) revealed soft , fluctuant masses bilaterally . dimension of the cyst in the soft tissues of the labial sulcus was more ( 2 3 cm ) on the right side which also produced an extraoral swelling because this cyst was exclusively an extraosseous one . in contrast , the left - sided cyst measured relatively less ( 1.5 1.5 cm ) in the soft tissues with no corresponding extraoral swelling because the cyst was occupying space between the vestibular sulcus and alveolar bone as was evident on surgical exploration . the preferred method of treatment of this cyst is surgical excision by intraoral sub - labial approach , although care must be exercised to prevent perforation and collapse of the lesion . the surgical outcome of this method is generally successful ; however , complications associated with the procedure include facial swelling , gingival numbness , decreased sensation of the teeth , and wound infection . an alternative transnasal approach has been suggested that allows endoscopic marsupialization of the cystic cavity . in the present case , full thickness incision was given on the crest of the ridge instead of sub - labial incision ( and a mucoperiosteal flap was reflected with a periosteal elevator to expose the cysts ) because the surgical procedure was inclusive of extractions of adjacent mutilated teeth . so , in the presented case ; clinical , radiographic and histological features of the lesions were suggestive of nasolabial cyst on the right side and nasoalveolar cyst ( residing on the alveolar surface ) on the left side , which were successfully enucleated by a modification of intraoral sublingual surgical approach . in 1-year

nasolabial cysts are painless , submucosal , non - odontogenic jaw cysts presenting as soft tissue swellings in the maxillary anterior mucolabial fold lateral to midline , leading to elevation of nasal ala . present case documents bilateral nasolabial cysts in a 69-year - old asian female patient . in the present case , extraoral swelling of maxillary lip and elevation of nasal ala was observed on right side of the face . intraorally , soft and fluctuant bilateral cysts were observed . straw - colored fluid was aspirated from the right cyst . radiographically , erosion of bone in a cupping fashion was observed in the region of left cyst . the cysts were enucleated using intraoral approach . histopathology of the right - sided cyst revealed a cystic cavity lined by stratified squamous cells along with a few mucosal cells . at few places , stratified squamous and pseudostratified columnar epithelia with many cilia and goblet cells were also evident . capsule was loosely arranged with fibrous tissue and chronic inflammatory infiltrate . left - sided cystic specimen showed two or more layered stratified squamous lining epithelium with thin capsule . diagnosis of bilateral nasolabial / nasoalveolar cysts was confirmed .

osteoarthritis ( oa ) of the hip induces regressive changes in the hip joint cartilage and subsequent abnormality in the synovial membrane and articular capsule , leading to the impairment of joint function , and osseous changes also occur . the prevalence of this disease is not high,1 but coxalgia and surrounding pain and limitation of the range of motion occur , and subsequently limit adl with the progression of lesions . oa of the hip is classified into primary and secondary cases based on the presence or absence of causes . primary oa of the hip is defined as coxarthrosis with normal hip joint alignment and acetabular formation.2 in japan , primary cases account for only 0.65% , and many cases are secondary.2 the cause of secondary oa of the hip in which lesions are localized in the hip joint includes congenital hip joint dislocation , acetabular dysplasia , necrosis of the femoral head , and perthes s disease . when acetabular dysplasia remains in the growth period , it may become oa of the hip.3 when the femoral head is impaired with the treatment of congenital hip joint dislocation , deformation of the femoral head and neck occurs . the natural course of this disease is diverse and classified based on the severity of joint deformation into pre- , early , progressive , and end - stage coxarthrosis , but it does not necessarily progress with aging . the natural course is influenced by the lifestyle , and it has been reported that x - ray radiographic findings improved in some cases.4 conservative or surgical treatment is selected in the natural course , and the timing is dependent on individual cases . in japan , kampo has been clinically applied for rheumatic diseases for centuries . in this study , we applied kampo for progressive to end - stage coxarthrosis in a female in her 50 s , and achieved improvement of the hip joint function and qol . we encountered a 52-year - old woman with end - stage oa of the hip accompanied by acetabular dysplasia in whom the quality of life ( qol ) was improved by kampo treatment . she had demonstrated acetabular dysplasia at birth , and dislocation of the hip was treated with a plaster cast . although anterior coxarthropathy was noted by a general practitioner at the age of 31 , she was observed without treatment , due to the absence of joint symptoms . at the age of 42 , she developed pain in the left hip joint , and early - stage oa of the hip was diagnosed . therefore , she took non - steroidal anti - inflammatory drugs ( nsaids ) , and received conservative therapies such as diet and muscle training . however , pain in the hip joint increased and her adl decreased at the age of 50 . nevertheless , she continued to receive conservative therapies , although her symptoms did not change . at the age of 52 , she consulted our department requesting japanese oriental ( kampo ) medicine . hip joint x - ray showed end - stage oa in the left hip joint ( fig . 1 ) . we administered the kampo formulae ; keishikaryojutsubuto ( 12tab / day : kracie co. ltd . treatment for 3 months resulted in a decrease in hip joint pain as well as improvement of her adl . one year later , her joint symptoms have not increased , and both the harris hip score6 and the japanese orthopaedic association ( joa ) hip score7 have improved ( table 1 ) . although kampo treatment continued over one year without discontinuation , there were no adverse effects . osteoarthritis of the hip begins with degeneration or wear of the joint cartilage , and various articular changes progress . it is considered that degeneration and wear of cartilage occur when mechanical loads , such as labor , exercise , and trauma , are added to background factors , such as race , gender , aging , obesity , and heredity . the metabolic disorder of chondrocytes occurs and cartilage destruction progresses , which induces inflammation of the synovial membrane and causes swelling of the joint and bone destruction . it progresses from precoxarthrosis early stage progressive stage end stage in the general natural course , and various clinical symptoms appear in each disease stage . secondary osteoarthritis of the hip , as described above , and the presented case involved acetabular dysplasia - associated secondary coxarthrosis . in japan , most patients with this disease are female , as was this patient . in treatment , conservative treatment is prioritized , such as instruction in daily living and physical and drug therapies , but some physicians consider that surgery in the early stage is desirable , even though symptoms are mild , when progression is apparently predicted . various surgical methods have been proposed corresponding to the condition and stage of acetabular dysplasia and coxarthrosis ( various osteotomy procedures , shelf operation , and total hip replacement ) . the patient showed end - stage coxarthrosis on x - ray radiography , and felt pain not only while going up and down stairs but also when walking , limiting adl . however , pain was rapidly alleviated after the initiation of kampo treatment ( keishikaryojutsubuto and boiougito ) and adl improved . methods to judge therapeutic effects on osteoarthritis of the hip are roughly divided into comprehensive health scales8,9 widely covering physical functions through mental health and disease - specific scales specialized for specific diseases including coxarthrosis . of the latter , it is comprised of pain ( 44 points ) , function ( 47 points ) , deformation ( 4 points ) , and range of motion ( 5 points ) . pain is divided into 6 categories , and function is comprised of the walking ability ( 33 points ) including claudication , support for walking , walking distance , and daily living activities ( 14 points ) including going up and down stairs , wearing shoes and socks , sitting , and the use of public transportation . the clinical evaluation criteria of osteoarthritis of the hip most commonly used in japan are the hip joint function assessment criteria established by the japanese orthopedic association ( joa hip score).7 the current joa hip score was prepared in 1995 based on the old joa hip score established in 1971 , and it is comprised of the following 4 items : pain ( 40 points ) , range of motion ( 20 points ) , walking ability ( 20 points ) , and daily living activities ( 20 points ) . categories proposed by charnley , such as unilateral , bilateral , and multiple joint developments , were adopted . the maximum score of the range of motion is 20 points , accounting for a large proportion of the overall score , compared to that in the harris hip score . in this patient , the joa and harris hip scores improved over the about one - year course , and so we consider that the objective effect of kampo treatment was exhibited . in japan , arthralgia has been treated with kampo for centuries , and many kampo formulations are administered for osteoarthritis,10 rheumatoid arthritis,11 and psoriatic arthritis.12 this kind of remedy is often used with several joints such as a knee joint13 in addition to hip joint caused by some pathogenesis such as degeneration , metabolism or inflammation . although there has been no report on administrations of boiogito and keishikaryojutsubuto for osteoarthritis of the hip , an effect on osteoarthritis of the knee comparable to that of nsaids and synergistic effect with nsaids have been reported.14 the mechanism of the effect of kampo on osteoarthritis has not been clarified . pathologically , cracks and erosion occur in the cartilage surface in excessively loaded regions , loosing joint cartilage , in osteoarthritis of the hip . in the deep layer of joint cartilage , blood vessels invade calcified cartilage , and subchondral bone comes to exhibit a bone effect . on the other hand , an immunomodulatory effect of kampo has been shown,15 and the inhibition of bone destruction in some cases has been reported.16 the inhibition of bone turnover in osteoarthritis is assumed . additionally , there is another problem in this kind of treatment . namely , kampo medicine has the important feature that differ from western medicine ; the diagnostic system in kampo medicine is different from that in western medicine . therefore , it is surely thought that kampo diagnosis may not be easy for readers to understand . when we treat ra patients with kampo medicine , it is necessary to make a kampo diagnosis as well as a diagnosis by western medicine . this issue makes it difficult to perform controlled clinical trials . in this case , we selected the keishikaryojutsubuto and boiogito according to the traditional diagnosis system . the target group for keishikaryojutsubuto and boiogito is follows : easy fatigability , coldness , obesity , sweating with swollen joints.5,17 furthermore , kampo formulae are generally composed of several herbal components , but not purified chemical compound . therefore it is considered that these remedies are safe . however , pseudoaldosteronism by licorice root is well known to be an adverse effects of herbal medicine , and there are also allergic effects , such as skin eruptions and liver injury , that can be induced by crude drugs.1820 in summary , we reported a patient with end - stage osteoarthritis of the hip accompanied by acetabular dysplasia in whom the qol was improved by kampo treatment . although the clinical use of this remedy should be with careful deliberation due to the absence of evidence , the course of this disease varies depending on the lifestyle among patients and kampo formulations may offer safe , potent supplemental treatment . the clinical course of a present patient may open the way to the achievement of randomized controlled trials and the further basic analysis of this remedy in the future .

we report a 52-year - old female with end - stage osteoarthritis of the hip accompanied by acetabular dysplasia in whom quality of life ( qol ) was improved by kampo treatment.when she was 42 years old , she developed pain in the left hip joint , and early - stage oa of the hip was diagnosed by hip joint x - ray . therefore , she took nsaids , and received conservative therapies such as diet and muscle training . however , pain in the hip joint increased and her activity of daily life ( adl ) decreased at the age of 50 , although she continued to receive the conservative therapies . at the age of 52 , she consulted our department requesting japanese oriental ( kampo ) medicine . kampo formulae ; keishikaryojutsubuto ( 12tab / day : kuracie co. ltd . japan ) , and boiougito ( 7.5 g / day : kuracie co. ltd . japan ) , were administered . treatment for 3 months resulted in a decrease in the left hip joint pain using visual analogue scale ( vas ) and improvement of her adl . one year later , her joint symptoms have not increased , and both the harris hip score and the clinical evaluation criteria of osteoarthritis of the hip have improved.the course of this disease varies depending on the lifestyle of the patient , and kampo formulations may offer safe , potent supplemental treatment .

the development of titanium fixtures has brought several benefits for the rehabilitation of edentulous patients . when biological and mechanical principles are respected , this treatment modality may successfully restore the functional and esthetic impairments caused by tooth loss3 . in spite of the significant evolution of a number of implant systems , implant design and features , such as those related to the mechanical behavior of implant - supported prostheses , dental prostheses do fail during function mainly due to abutment and prosthesis screw loosening and/or fracture . in addition , it has been reported that abutment screw loosening is only surpassed by loss of osseointegration as the main cause of failure on implant - supported restorations , as shown in longitudinal follow - up studies5 . when two metal surfaces are in contact , adhesion and friction forces do limit the movement between them . an applied method aimed to reduce this friction and improve adhesion consists of interposing a lubricating film between these surfaces . a metal with low shear strength , such as pure gold , may act as a dry lubricant . when compared to screws without gold coating , it has been found that gold - coated abutment screws subjected to torques of 12 , 20 , and 32 ncm aiming a 0.0064 mm opening of the implant - abutment interface presented 26 , 24 , and 24% of preload increase , respectively7 . in another study , it was found that when a torque force is applied to an abutment screw , a significant part of this force is lost due to friction between the contact points of metal surfaces , inhibiting the rotation of the screw . thus , decreasing of friction between metallic surfaces may increase the screw rotation and , consequently , the preload . the rotation of gold - coated abutment screws placed with torque forces of 12 , 20 , and 32 ncm increased 73 , 76 , and 62% , respectively , when compared to titanium abutment screws2 . the purpose of this study was to evaluate by strain gauges the preload and torque removal values on three abutment screws ( gold , titanium , and titanium with surface treatment screws ) applying an equivalent torque force ( 30.070.28 ncm ) . three machined self - tapping external hex titanium screws with 4 mm in diameter and 15 mm in length were used in this investigation ( master screw , conexo sistemas de prtese ltda . three transmucosal cera one type abutments ( 2.0 mm height , cera one , conexo sistemas de prtese ltda . , reference 045022 , batch # 5080915121 ) , designed for cemented single standing implant - supported restorations were also used . the transmucosal abutments were attached to the fixtures using 10 gold screws ( conexo , sistemas de prtese ltda . , reference 121022 , batch # 5073147 ) ( figure 1 a a ) , 10 titanium screws ( conexo sistemas de prtese ltda . , reference 121024 , batch # 5063223 ) ( figure 1a b ) , and 10 surface treated titanium screws ( ti - tite , conexo sistemas de prtese ltda . , reference 121026 , batch # 5063035 ) ( figure 1a c ) . a load cell adapted from a model described in the literature this load cell presented a central void for fixture fixation in its upper portion and a horizontal plate for fixation of four strain gauges on its lower portion ( figure 1 b ) . the abutments were placed on the upper plate of the cell ( figure 1 c ) in such way that the contact between abutments and fixtures was free of any interference . therefore , when the abutment was fixed to the fixture by the abutment screw , tensions generated by this fixation pulled the unit against the abutment , producing a deformation in the lower plate connected to the strain gauges . next , the force ( preload ) generated on the abutment screws was captured by the strain gauges in volts ( v ) and later converted to newton ( n ) . the aforementioned sample was arranged into 3 different groups according to the characteristics of each abutment screw : group a was formed by gold screws , group b was formed by titanium screws , and group c was formed by surface - treated titanium screws . to determine fixation and removal torques , a digital torquemeter was used ( torqueleader , model tsd150 , type i , class e , part # 117317 ) , previously calibrated according with iso 6789:2003 ( e ) standard . a square hand wrench ( 1.27 mm diameter , conexo , , reference 062300 , batch # 5091632 ) was attached to the torquemeter to allow proper connection between torquemeter and screws ( figure 1 d ) . following the proper assembly of fixture and abutment on group a , the gold screw was then attached to the fixture with an applied torque force of 30.070.28 ncm , being repeated afterwards for groups b and c. then , the initial preload value for the abutment screws was determined ( in volts ) and all screws were kept in their positions for 5 minutes for preload stabilization , according to the literature9 . during this the preload stabilization period , preload values were obtained after 1 , 2 , 3 , 4 , and 5 minutes after fixation , and thereafter a mean value was calculated . after this evaluation , the abutment screw was removed using a digital torquemeter and the maximum value of reverse torque force required for screw removal was recorded in ncm . next , the same screw was fixed again another 4 times , obtaining the preload and torque removal mean values . nine other screws from group a as well as 10 other screws from b and c groups were subjected to the same procedure described above . the values of fixation torque forces of the 3 groups were analyzed statistically by to one - way analysis of variance ( anova ) . comparison of preload values within groups was possible using the mean of 6 measurements ( 0 to 5 minutes ) obtained at the first abutment fixation , using two - way anova . one - way anova was used to compare the mean preload values among the groups . tukey 's test was used for multiple comparisons among the groups . for all statistical tests , all fixations for each screw ( n=5 ) were considered for evaluation of the torque removal to obtain an average value , and the statistical analysis was performed similarly to the preload data . two - way anova was used to evaluate the mean torque removal among the groups , and one - way anova to evaluate the differences between screws . tukey 's multiple comparison test was used for individual comparisons among the groups . for all statistical tests , the mean fixation torque force when all screws were considered was 30.070.28 ncm ( c.i . there were no statistically significant differences ( p=0.1244 ) among the groups regarding the fixation torque . the gold screws presented the highest preload values and the titanium screws the lowest values ( figure 2 ) . the mean preload values of the gold screws was 131.728.98 n , and presented statistically significant differences ( p<0.01 ) compared to groups b and c. screw # 3 and screw # 1 presented the highest ( 140.48 n ) and the lowest ( 117.73 n ) preload values , respectively . the titanium screws presented statistically significant differences ( p<0.01 ) ( mean 37.035.69 n ) . the highest preload value was obtained for screw # 7 ( 49.68 n ) , while screw # 3 presented the lowest value ( 25.30 n ) . in the group of surface - treated titanium screws , the mean preload value was 97.784.68 n. statistically significant differences ( p<0.01 ) were also observed in this group , ( maximum : 104.09 n for screw # 4 ; minimum : 90.28 n for screw # 2 ) . the removal torque presented statistically significant differences among the analyzed materials ( p<0.001 ) ( figure 3 ) . the gold screws presented a mean removal torque of 17.641.12 ncm , i.e. they did not shown statistically significant differences ( p=0.3713 ) . titanium screws presented a mean removal torque of 18.751.89 ncm , and showed statistically significant differences ( p<0.001 ) . the group of surface - treated titanium screws present statistically significant difference ( p=0.004 ) ( mean : 16.431.33 ncm ) . the results of this study are similar to those of previous investigations4,8 , which found statistically higher preload values for gold fixation screws in comparison to other tested materials , in spite of using different methodologies and materials . a maximum preload value of 666.4 n for gold screws and 458.2 n for titanium screws was reported8 . in addition , the finds of the present study suggest that the evaluated surface treatment of titanium screws was effective , since these screws presented higher preload values than conventional titanium screws . most previously published reports in the dental literature do not specify the removal torque of abutment screws . the effect of different cyclic loads has been evaluated using a 32 ncm torque for fixation of the screws , which was repeated after 10 minutes to avoid contact relaxation6 . several manufacturers have suggested this clinical approach to decrease screw loosening . in the present study , to allow a direct comparison between results of preload and torque removal for the studied groups , all screws received a similar fixation torque of30.070.28 ncm , despite the manufacturer 's recommendations of using 35 ncm torque for the ti - tite screws . this was performed to eliminate a possible bias caused by elastic deformation that might occur in titanium screws during fixation , which might not only quantitatively but qualitatively influence the study outcomes and impair comparison among groups . this approach allowed that , in this investigation , only the screw was left as a study variable . another relevant issue is that up to 10% of the initial preload may be lost to smooth contact surfaces ( embedment relaxation ) , rather than elongation stresses . however , it has been previously observed that when the same screw is fixed several times , its preload values increased4 . comparison to different results obtained in other studies might be a difficult task due to variations of tested products and variables that may influence the produced preload value , such as the elastic modulus of the screws , opposing joint surfaces , abutment design , friction coefficient , lubrication , rate of the applied torque and the adaptation between the fixture hexagon and the abutment2 . the findings of the present study suggest a trend for greater preload values verified for gold screws , followed by surface - treated titanium screws and titanium screws , confirming the outcomes of previous investigations4,8 . finite element analysis has been used to evaluate preloading through elongation of the abutment screw . a study evaluating the preload of two different systems of the same implant manufacturer in function , found that the optimal preload produced should be 75% of the screw yield stress , and also that the preload value increases dramatically as the friction coefficient between implant and screw decreases7 . as loosening of implant / abutment joint causes clinical problems , another question that may be raised is whether the torque values recommended by the manufacturers should be increased in order to obtain greater longevity of screw tightening . the preload produced by gold or titanium fixation screws can be evaluated through the measurement of elongation stresses . it has been found that the stresses were 60% lower than the strengths against the torques applied according to the manufacturer 's instructions4 . on the other hand , it has been reported that gold or titanium screws could support higher torques than those indicated by the manufacturers without presenting plastic deformations , although it has been recommended elsewhere that the stresses should not exceed 65% of the screw 's fracture strength1 . although the results of this study showed that gold screws have a clear superiority of the produced preload , application of cyclic loads would be required for a closer simulation of the masticatory function on implanted - supported restorations . it may be concluded : 1 . gold should be the material of choice for abutment fixation screws , since it produced the highest preload values , followed by surface - treated titanium screws and conventional titanium screws ; 2 . titanium screws presented the highest torque removal values , followed by gold screws , and surface - treated titanium screws .

several authors still consider the mechanical problems of fracture and component loosening as the main causes of failure of implant - supported restorations . the purpose of this in vitro study was to compare the preload of three types of screw for transmucosal abutment attachment used in single implant - supported prosthesis through strain gauge and removal torque measurements . three external hex fixtures were used , and each received a transmucosal abutment ( cera one ) , which was fixed to the implant with its respective screw : group a- gold screw , group b- titanium screw and group c- surface - treated titanium screw ( ti - tite ) . ten screws of each type were attached applying a 30.070.28 ncm torque force and maintained in position for 5 minutes . after this , the preload values were measured using strain gauges and a measurement cell . gold screws presented higher preload values ( 131.728.98 n ) , followed by surface - treated titanium screws ( 97.784.68 n ) and titanium screws ( 37.035.69 n ) . anova ( p<0.05 ) and tukey 's test ( p<0.05 ) were applied . statistically significant differences were found among the groups for both preload and removal torque values . in conclusion , gold screws may be indicated to achieve superior longevity of the abutment - implant connection and , consequently , prosthetic restoration due to greater preload values yielded .

ketamine has been used clinically since the 1960s as a general anesthetic.1 a decade after its approval by the us food and drug administration , researchers showed that ketamine reduces the excitatory response to n - methyl - d - aspartic acid ( nmda ) in central neurons , thus revealing its nmda receptor antagonism properties2 and advancing our understanding of the pharmacology of ketamine . currently , clinicians use ketamine as a general anesthetic less frequently , given its emergence psychotomimetic effects and findings of neurotoxic effects shown in the developing brain of animals.3 conversely , over the past decade , researchers have explored the effect of subanesthetic doses of ketamine as an antidepressant4 and as an analgesic.5,6 the use of subanesthetic ketamine as an analgesic is supported by evidence , from both animal and human studies , implicating activation of nmda receptors in the pathobiology of nociceptive , inflammatory , and neuropathic pain and in central sensitization.7 in addition , activation of nmda receptors has also been shown in animals8 and in human experimental studies9 to play a role in settings where acute or chronic use of opioids is associated with tolerance or opioid - induced hyperalgesia . therefore , based on these preclinical findings , ketamine has been examined clinically as an adjuvant to opioids for the treatment of acute and chronic pain and in settings of opioid - induced hyperalgesia.6,10 however , among clinical studies , there has been a great variability in ketamine doses and duration of administration . additionally , the beneficial effects of ketamine have not been consistently reported , and some groups have been unable to show opioid - sparing effects of ketamine in clinical settings associated with opioid - induced hyperalgesia.11,12 similarly , in chronic pain , there are no definitive data supporting the use of ketamine for the treatment of chronic regional pain syndrome ( crps ) or other types of chronic pain.13 further , in critical reviews of available trials , researchers can only conclude that there might be a beneficial role for ketamine as an adjuvant to opioids to treat pain in the postoperative period , cancer pain , and chronic pain.9,12,14,15 consequently , as outlined in clinical guidelines , the evidence to support the use of ketamine for the treatment of acute and chronic pain is at best moderate.16,17 however , despite lack of conclusive clinical evidence to support its use , clinicians from various disciplines in many countries continue to use ketamine as an adjuvant to opioids for acute and chronic pain management.1821 some argue that in the setting of limited effective pharmacologic options , one might be left with no alternative other than less studied strategies such as ketamine infusions . our institution has used subanesthetic ketamine ( defined as not > 1 mg / kg / h ) to treat acute and chronic pain in children , adolescents , and young adults since 2006 . we have previously reported our experience using ketamine for the treatment of cancer pain , postoperative , and chronic pain.5,11 in this study , we sought to examine the use of subanesthetic ketamine in a large number of patients admitted to a tertiary pediatric hospital whose pain proved unresponsive to routine pharmacologic interventions . specifically , we sought to determine the overall effect of ketamine on pain scores and opioid intake , to identify factors associated with larger or smaller effects and to examine the frequency with which ketamine was associated with clinically meaningful reductions in pain scores and opioid intake . the children s national health system institutional review board approved this longitudinal cohort study and a waiver for written informed consent in 2014 ( pro00004569 ) . requirement for patient written consent to review their records was waived as this was a minimal risk study and the data collected were devoid of patient identifiers . patients who had received subanesthetic doses of ketamine for pain management were eligible for inclusion in this study . we reviewed a list of all patients ( obtained from the hospital pharmacy records ) who were admitted between january 2006 and april 2014 and had received an order for continuous infusion of intravenous ketamine . patients who lacked a pain medicine consult , underwent a spinal fusion procedure ( those patients were part of a previously reported investigational study11 ) , had incomplete record of pain scores and/or opioid intake , or received ketamine only for the purpose of sedation were excluded . data collected included sex , age , race , ketamine infusion duration , pain duration ( acute vs chronic ) , pain diagnoses ( vaso - occlusive episodes [ voe ] , visceral , crps , headache , or other [ mucositis , diabetic neuropathy , neurofibromatosis , and postural orthostatic tachycardia syndrome ] ) , primary clinical diagnosis ( accidental trauma , surgical trauma , and postoperative pain [ patients who had a planned surgical procedure during hospitalization ] , functional gastrointestinal disorder [ chronic and/or recurrent gastrointestinal symptoms with no identified etiology ] , inflammatory disease [ pancreatitis or crohn s disease ] , sickle cell disease , malignancy , or other ) , and primary pain location . patients with pain in more than two locations , we retrieved documentation of hemodynamic changes that warranted treatment with vasoactive agents , as well as reports of psychotropic effects , nausea , vomiting , and changes in sleep pattern . we then calculated average daily pain scores before ( day prior to ketamine administration ) and after ( 24 h , 48 h , and on the day after discontinuation ) ketamine infusion . pain scores were measured using the numeric rating scale ( nrs ) , the wong - baker faces , or the face , legs , activity , cry , and consolability ( flacc ) , as appropriate . at the times indicated earlier , we also collected total daily opioid intake and then calculated the daily oral morphine - equivalent ( per body weight in kilograms ) intake using the opioid conversion tool available at www.globalrph.com/narcoticonv.htm.22 the primary outcome was change in average pain scores from baseline to postketamine infusion . secondary outcomes included changes in oral morphine - equivalent intake from baseline measurements to postketamine infusion and proportion of ketamine infusions that resulted in clinically meaningful reductions in pain scores and opioid intake . we defined clinically meaningful change in pain scores or opioid intake as a 20% reduction in pain scores and in morphine - equivalent intake from baseline to the day after ketamine discontinuation ( day after ketamine - baseline / baseline 100 ) . such a decline in pain score ( 20% ) is larger than the 12.5% decrease in pain score suggested by others as being minimally clinically significant for adolescents with chronic pain.23 at our institution , ketamine prescribed by the pain medicine division can be administered on regular inpatient care units where oxygen delivery set - up , suction , and resuscitation equipment are available . trained nurses administer ketamine infusions using a locked infusion pump and patients are maintained on a cardiorespiratory monitor ( heart and respiratory rate , blood pressure , and pulse oximeter ) . criteria to receive subanesthetic doses of ketamine infusions include inadequate analgesia with standard pain therapy , opioid tolerance associated with undesirable side effects , opioid - induced hyperalgesia , neuropathic pain , postoperative pain in patients in whom opioid use was undesirable , or an opioid - sparing effect was needed for clinical reasons . exclusion criteria for ketamine administration include history of psychosis , intracranial hypertension , severe labile hypertension , or poorly controlled cardiac arrhythmia , hypersensitivity to ketamine , and age <3 months . in our institution , ketamine infusions order sets are available , and the suggested starting doses are as follows : 1 ) for opioid naive patients , ketamine is started at 0.050.4 mg / kg / h ; 2 ) for opioid tolerant patients ( as documented by the pain service ) , 0.051 mg / kg / h ; and 3 ) for patients with documented opioid - induced hyperalgesia , 1 mg / kg / h . during infusions , ketamine doses could be titrated according to its indication and pain scores up to 1 mg / kg / h as allowed by the board of nursing . the distributions of raw pain scores , daily opioid intake , and changes over time were tested for normality . because the data were skewed , the wilcoxon signed - rank test was used to test for statistical significance of within - individuals changes between baseline and posttreatment outcome measurements . box plots were used to examine how the individual - level changes ( between baseline and the day after ketamine discontinuation ) in raw pain scores and daily opioid intake were distributed across the subgroups of the eight patient characteristics ( age , sex , race , duration of pain , clinical diagnosis , type of pain , location of pain , and duration of ketamine infusion ) , and the distributions were tested for normality . because the data were skewed for certain characteristics , nonparametric tests were used for all statistical analyses . the kruskal wallis test was used to compare the distribution of values across the subgroups of each patient characteristic separately . when an overall difference was detected , dunn s test ( with a bonferroni adjustment for multiple comparisons ) was then used to compare all possible pairs of subgroups and identify any statistically significant detectable differences . the proportion of patients ( 95% confidence interval [ ci ] ) who achieved clinically meaningful reductions in pain and daily opioid intake were graphed in order to visually compare how the proportions varied across the subgroups of all eight patient characteristics . fisher s exact test was used to examine whether the proportions varied across the subgroups of sex , race , duration of pain , clinical diagnosis , type of pain , or location of pain . a nonparametric test for trend was used to examine whether the proportion of patients with a clinically meaningful reduction in pain scores and opioid intake systematically declined across ordered age groups or by the duration of ketamine infusion . stata 14.1/ic ( statacorp llc , college station , tx , usa ) was used to conduct all analyses . p - values < 0.05 were considered statistically significant except when bonferroni s correction for multiple comparisons was applied . in those instances , the children s national health system institutional review board approved this longitudinal cohort study and a waiver for written informed consent in 2014 ( pro00004569 ) . requirement for patient written consent to review their records was waived as this was a minimal risk study and the data collected were devoid of patient identifiers . patients who had received subanesthetic doses of ketamine for pain management were eligible for inclusion in this study . we reviewed a list of all patients ( obtained from the hospital pharmacy records ) who were admitted between january 2006 and april 2014 and had received an order for continuous infusion of intravenous ketamine . patients who lacked a pain medicine consult , underwent a spinal fusion procedure ( those patients were part of a previously reported investigational study11 ) , had incomplete record of pain scores and/or opioid intake , or received ketamine only for the purpose of sedation were excluded . data collected included sex , age , race , ketamine infusion duration , pain duration ( acute vs chronic ) , pain diagnoses ( vaso - occlusive episodes [ voe ] , visceral , crps , headache , or other [ mucositis , diabetic neuropathy , neurofibromatosis , and postural orthostatic tachycardia syndrome ] ) , primary clinical diagnosis ( accidental trauma , surgical trauma , and postoperative pain [ patients who had a planned surgical procedure during hospitalization ] , functional gastrointestinal disorder [ chronic and/or recurrent gastrointestinal symptoms with no identified etiology ] , inflammatory disease [ pancreatitis or crohn s disease ] , sickle cell disease , malignancy , or other ) , and primary pain location . patients with pain in more than two locations , we retrieved documentation of hemodynamic changes that warranted treatment with vasoactive agents , as well as reports of psychotropic effects , nausea , vomiting , and changes in sleep pattern . we then calculated average daily pain scores before ( day prior to ketamine administration ) and after ( 24 h , 48 h , and on the day after discontinuation ) ketamine infusion . pain scores were measured using the numeric rating scale ( nrs ) , the wong - baker faces , or the face , legs , activity , cry , and consolability ( flacc ) , as appropriate . at the times indicated earlier , we also collected total daily opioid intake and then calculated the daily oral morphine - equivalent ( per body weight in kilograms ) intake using the opioid conversion tool available at www.globalrph.com/narcoticonv.htm.22 secondary outcomes included changes in oral morphine - equivalent intake from baseline measurements to postketamine infusion and proportion of ketamine infusions that resulted in clinically meaningful reductions in pain scores and opioid intake . we defined clinically meaningful change in pain scores or opioid intake as a 20% reduction in pain scores and in morphine - equivalent intake from baseline to the day after ketamine discontinuation ( day after ketamine - baseline / baseline 100 ) . such a decline in pain score ( 20% ) is larger than the 12.5% decrease in pain score suggested by others as being minimally clinically significant for adolescents with chronic pain.23 at our institution , ketamine prescribed by the pain medicine division can be administered on regular inpatient care units where oxygen delivery set - up , suction , and resuscitation equipment are available . trained nurses administer ketamine infusions using a locked infusion pump and patients are maintained on a cardiorespiratory monitor ( heart and respiratory rate , blood pressure , and pulse oximeter ) . criteria to receive subanesthetic doses of ketamine infusions include inadequate analgesia with standard pain therapy , opioid tolerance associated with undesirable side effects , opioid - induced hyperalgesia , neuropathic pain , postoperative pain in patients in whom opioid use was undesirable , or an opioid - sparing effect was needed for clinical reasons . exclusion criteria for ketamine administration include history of psychosis , intracranial hypertension , severe labile hypertension , or poorly controlled cardiac arrhythmia , hypersensitivity to ketamine , and age <3 months . in our institution , ketamine infusions order sets are available , and the suggested starting doses are as follows : 1 ) for opioid naive patients , ketamine is started at 0.050.4 mg / kg / h ; 2 ) for opioid tolerant patients ( as documented by the pain service ) , 0.051 mg / kg / h ; and 3 ) for patients with documented opioid - induced hyperalgesia , 1 mg / kg / h . during infusions , ketamine doses could be titrated according to its indication and pain scores up to 1 mg / kg / h as allowed by the board of nursing . the distributions of raw pain scores , daily opioid intake , and changes over time were tested for normality . because the data were skewed , the wilcoxon signed - rank test was used to test for statistical significance of within - individuals changes between baseline and posttreatment outcome measurements . box plots were used to examine how the individual - level changes ( between baseline and the day after ketamine discontinuation ) in raw pain scores and daily opioid intake were distributed across the subgroups of the eight patient characteristics ( age , sex , race , duration of pain , clinical diagnosis , type of pain , location of pain , and duration of ketamine infusion ) , and the distributions were tested for normality . because the data were skewed for certain characteristics , nonparametric tests were used for all statistical analyses . the kruskal wallis test was used to compare the distribution of values across the subgroups of each patient characteristic separately . when an overall difference was detected , dunn s test ( with a bonferroni adjustment for multiple comparisons ) was then used to compare all possible pairs of subgroups and identify any statistically significant detectable differences . the proportion of patients ( 95% confidence interval [ ci ] ) who achieved clinically meaningful reductions in pain and daily opioid intake were graphed in order to visually compare how the proportions varied across the subgroups of all eight patient characteristics . fisher s exact test was used to examine whether the proportions varied across the subgroups of sex , race , duration of pain , clinical diagnosis , type of pain , or location of pain . a nonparametric test for trend was used to examine whether the proportion of patients with a clinically meaningful reduction in pain scores and opioid intake systematically declined across ordered age groups or by the duration of ketamine infusion . stata 14.1/ic ( statacorp llc , college station , tx , usa ) was used to conduct all analyses . p - values < 0.05 were considered statistically significant except when bonferroni s correction for multiple comparisons was applied . in those instances , we reviewed 1542 records , containing orders for subanesthetic ketamine administration ( figure 1 ) . records containing ketamine orders not written by the pain medicine division were incomplete or were from patients who had undergone spinal fusion ( ketamine was administered as part of a postoperative pain management and previously reported11 ) were excluded . two hundred and fifty - one patients and 441 infusions , which occurred between january 2006 and april 2014 , met the study eligibility criteria . of the 441 remaining records , 57 were excluded as pain scores , and/or opioid intake data were missing during the first or second day of ketamine administration or for the 24 h after discontinuation of ketamine . records from 24 ketamine infusions were eliminated because they consisted of a second ketamine infusion during a single hospital admission . the final study cohort included data from 230 different patients who , during 360 separate hospital admissions , received subanesthetic ketamine infusions for pain therapy . of those , 180 patients were admitted once , while the other 50 were each admitted between two and ten times . for the purpose of this study , over the 360 ketamine infusions , most patients were from 12 to 18 years of age ( 58% ) , 6 to 11 years ( 17% ) , 19 to 21 years ( 14% ) , 3 to 5 years ( 5% ) , > 22 years ( 4% ) , and 1.1 to 2.9 years ( 3% ) . seventy - eight percent of infusions ( 280 ) were administered to patients with acute pain and 22% to patients with chronic pain . additionally , most ketamine infusions ( 50% ) were administered to patients with sickle cell disease who had been admitted for voe , whereas 17% who had postoperative pain after surgical trauma and 13% had malignancy - associated pain ( table 2 ) . most patients had generalized pain ( 25% ) , followed by abdominal ( 22% ) , lower extremity ( 21% ) , and chest ( 15% ) pain . we collected average pain scores on the day prior to initiation of ketamine and on the first day of ketamine infusion . in the hospital admissions ( n=331 of 441 ) where both values were originally available in the electronic medical record , the average pain score on the day prior to ketamine initiation ( 6.85 [ 95% ci : 6.587.11 ] ) and on the first day of ketamine infusion were similar ( 6.84 [ 95% ci : 6.597.08 ] ) , p=0.499 . given that in all infusions , the average pain score values were available on the first day of infusion , we considered that as the baseline pain score for the final study cohort of 360 hospital admissions . overall , ketamine infusions were associated with significant reductions in average pain scores from the first day of infusion ( 6.65 [ 95% ci : 6.396.91 ] ) to the day after discontinuation of ketamine ( 4.38 [ 95% ci : 4.064.69 ] ) , p<0.001 . the effect of ketamine infusions on raw pain scores ( within - individual change ) was similar comparing all age groups ( p=0.842 ) , males and females ( p=0.065 ) , all races ( p=0.721 ) , acute and chronic pain ( p=0.460 ) , and pain types ( p=0.894 ) , data not shown . in contrast , the effect of ketamine infusions on pain scores varied according to clinical diagnosis ( p=0.01 ) , infusion duration ( p=0.004 ) , and pain location ( p=0.004 , figure 2 ) . specifically , greater median reductions in pain scores were observed in patients with malignancy - associated pain and patients with inflammatory disease ( pancreatitis and crohn s disease ) , whereas the lowest were in patients with functional gastrointestinal disorder and other ( cystic fibrosis , diabetes mellitus , neurofribromatosis 1 , postural orthostatic tachycardia syndrome , and two cases simply coded as other , p=0.011 for overall difference , figure 2a ) . regarding infusion durations , longer ketamine infusions yielded greater median reductions in pain scores ( p=0.004 , figure 2b ) . further , patients with back ( 2.57 [ 4.75 , 1.56 ] , median [ 25 , 75 percentiles respectively ] ) , abdominal ( 1.91 [ 3.88 , 0.6 ] ) , and generalized ( 1.87 [ 3.43 , 0.75 ] ) pain had the greatest reductions , whereas patients with chest pain ( 0.5 [ 2.37 , 0.33 ] ) had the lowest reductions in pain scores ( p=0.004 for overall difference ) . regarding opioid intake , ketamine infusions were associated with significant decreases in overall average oral morphine - equivalent intake from the first day of infusion ( 2.74 mg / kg / d [ 2.473.00 ] , mean [ 95% ci ] ) to the day after discontinuation of ketamine ( 2.06 mg / kg / d [ 1.812.31 ] ) , p<0.001 . there was no impact of age , sex , race , pain duration ( acute vs chronic ) , or pain location or type on the effect of ketamine on oral morphine - equivalent intake ( all p0.322 ) . however , the ketamine effect on opioid intake varied according to clinical diagnosis ( p=0.030 , figure 2c ) and duration of infusion ( p=0.022 , figure 2d ) . specifically , greater median reductions in opioid intake were observed in patients with malignancy - associated pain and patients with sickle cell disease , whereas lesser reductions were observed in patients with accidental trauma and postoperative pain after surgical trauma ( p=0.030 for overall difference , figure 2c ) . regarding ketamine infusion duration , similar to the observation of reduction in pain scores , longer ketamine infusions yielded greater reductions in oral morphine - equivalent intake ( p=0.022 , figure 2d ) . during ketamine administration , there were no records of hallucinations , changes in sleep pattern , or hemodynamic changes or arrhythmias requiring therapy in any patient . we then examined the proportion of ketamine infusions that yielded clinically meaningful changes in pain scores and opioid intake , here defined as a 20% reduction in pain scores and a 20% reduction in morphine - equivalent intake after discontinuation of the infusion compared to baseline levels , respectively . overall , there was a 20% reduction in pain scores in 58% of all ketamine infusions . the proportion of infusions that yielded a 20% or greater reduction in pain scores varied according to clinical diagnosis ( p=0.003 ) , pain location ( p=0.012 ) , age ( p=0.039 ) , and duration of infusion ( p=0.017 , figure 3 ) . for example , the highest proportion of infusions associated with clinically meaningful reduction in pain scores were observed among infusions administered to cancer patients and patients with inflammatory diseases and the lowest among patients with functional gastrointestinal disorders ( figure 3a ) . there was a trend for higher frequency of meaningful reduction in pains scores in males ( 63% ) than in females ( 54% ) , p=0.056 . conversely , the frequency of meaningful reduction in pain scores did not vary with the type ( p=0.391 ) and duration of pain ( p=0.224 ) or race ( p=0.268 ) . regarding the frequency of a meaningful reduction in opioid intake , in 52% of ketamine infusions the frequency of infusions associated with 20% reduction in opioid intake varied according to duration of ketamine infusion ( p=0.030 ) , with longer durations infusions being more likely to result in a meaningful reduction in opioid intake ( data not shown ) . conversely , the frequency of a meaningful reduction in opioid intake did not vary with the clinical diagnosis , type of pain , duration of pain , location of pain , age , sex , or race . we reviewed 1542 records , containing orders for subanesthetic ketamine administration ( figure 1 ) . records containing ketamine orders not written by the pain medicine division were incomplete or were from patients who had undergone spinal fusion ( ketamine was administered as part of a postoperative pain management and previously reported11 ) were excluded . two hundred and fifty - one patients and 441 infusions , which occurred between january 2006 and april 2014 , met the study eligibility criteria . of the 441 remaining records , 57 were excluded as pain scores , and/or opioid intake data were missing during the first or second day of ketamine administration or for the 24 h after discontinuation of ketamine . records from 24 ketamine infusions were eliminated because they consisted of a second ketamine infusion during a single hospital admission . the final study cohort included data from 230 different patients who , during 360 separate hospital admissions , received subanesthetic ketamine infusions for pain therapy . of those , 180 patients were admitted once , while the other 50 were each admitted between two and ten times . for the purpose of this study , the median age was 14 years ( interquartile range : 1017 years ) . over the 360 ketamine infusions , most patients were from 12 to 18 years of age ( 58% ) , 6 to 11 years ( 17% ) , 19 to 21 years ( 14% ) , 3 to 5 years ( 5% ) , > 22 years ( 4% ) , and 1.1 to 2.9 years ( 3% ) . seventy - eight percent of infusions ( 280 ) were administered to patients with acute pain and 22% to patients with chronic pain . additionally , most ketamine infusions ( 50% ) were administered to patients with sickle cell disease who had been admitted for voe , whereas 17% who had postoperative pain after surgical trauma and 13% had malignancy - associated pain ( table 2 ) . most patients had generalized pain ( 25% ) , followed by abdominal ( 22% ) , lower extremity ( 21% ) , and chest ( 15% ) pain . we collected average pain scores on the day prior to initiation of ketamine and on the first day of ketamine infusion . in the hospital admissions ( n=331 of 441 ) where both values were originally available in the electronic medical record , the average pain score on the day prior to ketamine initiation ( 6.85 [ 95% ci : 6.587.11 ] ) and on the first day of ketamine infusion were similar ( 6.84 [ 95% ci : 6.597.08 ] ) , p=0.499 . given that in all infusions , the average pain score values were available on the first day of infusion , we considered that as the baseline pain score for the final study cohort of 360 hospital admissions . overall , ketamine infusions were associated with significant reductions in average pain scores from the first day of infusion ( 6.65 [ 95% ci : 6.396.91 ] ) to the day after discontinuation of ketamine ( 4.38 [ 95% ci : 4.064.69 ] ) , p<0.001 . the effect of ketamine infusions on raw pain scores ( within - individual change ) was similar comparing all age groups ( p=0.842 ) , males and females ( p=0.065 ) , all races ( p=0.721 ) , acute and chronic pain ( p=0.460 ) , and pain types ( p=0.894 ) , data not shown . in contrast , the effect of ketamine infusions on pain scores varied according to clinical diagnosis ( p=0.01 ) , infusion duration ( p=0.004 ) , and pain location ( p=0.004 , figure 2 ) . specifically , greater median reductions in pain scores were observed in patients with malignancy - associated pain and patients with inflammatory disease ( pancreatitis and crohn s disease ) , whereas the lowest were in patients with functional gastrointestinal disorder and other ( cystic fibrosis , diabetes mellitus , neurofribromatosis 1 , postural orthostatic tachycardia syndrome , and two cases simply coded as other , p=0.011 for overall difference , figure 2a ) . regarding infusion durations , longer ketamine infusions yielded greater median reductions in pain scores ( p=0.004 , figure 2b ) . further , patients with back ( 2.57 [ 4.75 , 1.56 ] , median [ 25 , 75 percentiles respectively ] ) , abdominal ( 1.91 [ 3.88 , 0.6 ] ) , and generalized ( 1.87 [ 3.43 , 0.75 ] ) pain had the greatest reductions , whereas patients with chest pain ( 0.5 [ 2.37 , 0.33 ] ) had the lowest reductions in pain scores ( p=0.004 for overall difference ) . regarding opioid intake , ketamine infusions were associated with significant decreases in overall average oral morphine - equivalent intake from the first day of infusion ( 2.74 mg / kg / d [ 2.473.00 ] , mean [ 95% ci ] ) to the day after discontinuation of ketamine ( 2.06 mg / kg / d [ 1.812.31 ] ) , p<0.001 . there was no impact of age , sex , race , pain duration ( acute vs chronic ) , or pain location or type on the effect of ketamine on oral morphine - equivalent intake ( all p0.322 ) . however , the ketamine effect on opioid intake varied according to clinical diagnosis ( p=0.030 , figure 2c ) and duration of infusion ( p=0.022 , figure 2d ) . specifically , greater median reductions in opioid intake were observed in patients with malignancy - associated pain and patients with sickle cell disease , whereas lesser reductions were observed in patients with accidental trauma and postoperative pain after surgical trauma ( p=0.030 for overall difference , figure 2c ) . regarding ketamine infusion duration , similar to the observation of reduction in pain scores , longer ketamine infusions yielded greater reductions in oral morphine - equivalent intake ( p=0.022 , figure 2d ) . during ketamine administration , there were no records of hallucinations , changes in sleep pattern , or hemodynamic changes or arrhythmias requiring therapy in any patient . we then examined the proportion of ketamine infusions that yielded clinically meaningful changes in pain scores and opioid intake , here defined as a 20% reduction in pain scores and a 20% reduction in morphine - equivalent intake after discontinuation of the infusion compared to baseline levels , respectively . overall , there was a 20% reduction in pain scores in 58% of all ketamine infusions . the proportion of infusions that yielded a 20% or greater reduction in pain scores varied according to clinical diagnosis ( p=0.003 ) , pain location ( p=0.012 ) , age ( p=0.039 ) , and duration of infusion ( p=0.017 , figure 3 ) . for example , the highest proportion of infusions associated with clinically meaningful reduction in pain scores were observed among infusions administered to cancer patients and patients with inflammatory diseases and the lowest among patients with functional gastrointestinal disorders ( figure 3a ) . there was a trend for higher frequency of meaningful reduction in pains scores in males ( 63% ) than in females ( 54% ) , p=0.056 . conversely , the frequency of meaningful reduction in pain scores did not vary with the type ( p=0.391 ) and duration of pain ( p=0.224 ) or race ( p=0.268 ) . regarding the frequency of a meaningful reduction in opioid intake , in 52% of ketamine infusions the frequency of infusions associated with 20% reduction in opioid intake varied according to duration of ketamine infusion ( p=0.030 ) , with longer durations infusions being more likely to result in a meaningful reduction in opioid intake ( data not shown ) . conversely , the frequency of a meaningful reduction in opioid intake did not vary with the clinical diagnosis , type of pain , duration of pain , location of pain , age , sex , or race . in this cohort study involving a large number of children , adolescents , and young adults , we examined the effects of subanesthetic ketamine infusions on pain intensity and opioid intake . we found that continuous subanesthetic ketamine infusions administered to children , adolescents , and young adults with inadequate pain control in a regular inpatient care unit setting were safe and not associated with significant psychotropic effects or hemodynamic side effects that warranted treatment . we do acknowledge that increases in blood pressure could occur during administration of ketamine ; however , we recorded only instances where changes in blood pressure warranted treatment with vasoactive drugs . further , while there were no records of hallucinations , it is possible that the presence of hallucinations in young children could have manifested as dysphoria and those were not recorded . nevertheless , ketamine was associated with significant decreases both in pain scores and opioid intake and these effects varied according to clinical diagnosis , infusion duration , and pain location . the greatest reductions in pain scores were observed in patients with cancer pain and patients with inflammatory processes including pancreatitis and crohn s disease , whereas patients with functional gastrointestinal disorder , diabetes mellitus , neurofibromatosis 1 , and postural orthostatic tachycardia syndrome had less reduction in pain scores . we also found that ketamine infusions in patients with cancer pain , inflammatory diseases , and postoperative pain were associated with a greater ( over 75% of infusions ) frequency of meaningful reduction in pain scores ( here defined as a 20% reduction ) . therefore , this study is informative in that its findings can be used to help design much - needed randomized trials to evaluate the effect of ketamine in the treatment of pain in children , adolescents , and young adults with poorly controlled pain . over the past few years , the use of ketamine for the treatment of cancer pain has been the focus of significant debate.15,24,25 the discussion ensued after publication of a randomized placebo - controlled clinical trial enrolling 185 adult participants with cancer pain , who received ketamine or placebo delivered subcutaneously.26 that trial showed that ketamine , administered over 5 days in a dose - escalating regimen , had no clinical benefit compared to placebo and was associated with increased adverse events.26 however , researchers argue that the trial used a subcutaneous regimen not commonly utilized , the ketamine doses were escalated rapidly , and the study population was very heterogeneous , all of which are potential limitations of that trial.25 therefore , researchers argue that those issues should tailor the interpretation of the findings26 and those negative results should not discard the potential role of ketamine in patients with cancer pain.25 after publication of those negative results , a survey of clinicians demonstrated that the practice of using ketamine was continued by 30% of the respondents24 and the use of ketamine to treat cancer pain and other types of pain by many specialists has continued.19,24 on balance , one must recognize that the existing data on the use of ketamine in children with cancer pain are very limited , most reports consist of small case series , and randomized clinical trials of ketamine to treat cancer pain in children are lacking . nevertheless , we have sided with those who argue that , while clinical trials are needed , the practice of using ketamine in children with cancer pain might be one to be adopted when other options are unavailable . concerning acute postoperative pain , we found in the present study that the opioid - sparing effect of ketamine was minimal . this finding is consistent with our results from a previous randomized placebo - controlled trial , where ketamine administered in the intraoperative and postoperative periods did not yield an opioid - sparing effect in adolescents with scoliosis undergoing spinal fusion.11 interestingly , a more recent trial and a new meta - analysis12 also showed lack of beneficial effect of ketamine in postoperative pain.27 it is important to note that one limitation of the most recent study in adolescents undergoing spinal fusion27 as well as ours11 is that neither study examined the effect of ketamine on chronic postsurgery pain . nevertheless , our findings from the present study are discrepant to those showing that low - dose ketamine infusion during the perioperative period yields opioid - sparing effects in adults undergoing major surgeries.28 while we acknowledge that , in adults , there are data to support the use of ketamine in the perioperative period , in our institution , we no longer routinely use ketamine to treat postoperative pain in our pediatric patients . one must acknowledge the limitations of this study , which call for caution in interpreting its results . first , we report on the use of ketamine in a heterogeneous population of children , adolescents , and young adults and lack a control group . further , while there was consistency regarding the ketamine administration regimen and indications for its use , timing of administration , and titration of opioids and ketamine doses were left to the discretion of the pain medicine attending . additionally , for the purpose of this study , we regarded ketamine infusions administered to the same patients as independent infusions and we did not account for the potential confounding effect of repeated measures by repeatedly administering ketamine to some patients . in conclusion , despite its limitations , this study suggests that in an inpatient setting , the treatment of pain in children , adolescents , and young adults with subanesthetic doses of ketamine is feasible and safe in regular patient care units . the study is also informative in that it suggests that ketamine might have a differential beneficial effect in patients with certain pain syndromes . additionally , this study raises a number of hypotheses to be tested in future clinical trials to more definitively determine which patients would benefit from this therapeutic modality . finally , our findings can be used to determine sample and effect sizes for future randomized trials to evaluate the role of ketamine for the treatment of pain in children , adolescents , and young adults .

backgroundsubanesthetic doses of ketamine , an n - methyl - d - aspartate receptor antagonist used as an adjuvant to opioid for the treatment of pain in adults with acute and chronic pain , have been shown , in some instances , to improve pain intensity and to decrease opioid intake . however , less is known about the role of ketamine in pain management in children , adolescents , and young adults.purposewe examined the effects of subanesthetic ketamine on pain intensity and opioid intake in children , adolescents , and young adults with acute and chronic pain syndromes treated in an inpatient setting.methodsthis is a longitudinal cohort study of patients treated with subanesthetic ketamine infusions in regular patient care units in a tertiary pediatric hospital . primary outcomes included changes in pain scores and morphine - equivalent intake.resultsthe study cohort included 230 different patients who during 360 separate hospital admissions received subanesthetic ketamine infusions for pain management . overall , ketamine infusions were associated with significant reductions in mean pain scores from baseline ( mean pain scores 6.64 [ 95% ci : 6.386.90 ] ) to those recorded on the day after discontinuation of ketamine ( mean pain scores 4.38 [ 95% ci : 4.064.69 ] ) , p<0.001 . importantly , the effect of ketamine on pain scores varied according to clinical diagnosis ( p=0.011 ) , infusion duration ( p=0.004 ) , and pain location ( p=0.004 ) . interestingly , greater reductions in pain scores were observed in patients with cancer pain and patients with pain associated with pancreatitis and crohn s disease . there were no records of psychotomimetic side effects requiring therapy.conclusionthese data suggest that administration of subanesthetic ketamine for pain management is feasible and safe in regular inpatient care units and may benefit children , adolescents , and young adults with acute and chronic pain . this study is informative and can be helpful in determining sample and effect sizes when planning clinical trials to determine the role of subanesthetic ketamine infusions for pain management in pediatric patients .

trigeminal neuralgia ( tn ) is a disorder of cranial nerve ( cn ) v that results in severe episodes of shock - like or lancinating pain in one or more of its three divisions ( v1v3 ) . idiopathic tn and cases due to vascular compression of cn v are categorized as classical tn . patients diagnosed with symptomatic tn experience trigeminal - related facial pain secondary to a brain tumor , skull deformity , or multiple sclerosis ( ms ) . evidence suggests that the majority of cases of tn are the consequence of focal compression of the entry zone of the root of the trigeminal nerve , while only 2% of cases are observed in patients diagnosed with ms . other than excruciating facial pain , there are no other direct medical symptoms associated with tn , and the condition does not decrease life expectancy . however , many patients with tn struggle with accomplishing tasks that affect quality of life , which is how this disorder elicits a negative impact on the social and mental wellness of the patients who suffer from this illness . following the diagnosis of tn however , many patients experience only limited relief from medication or are unable to endure the side effects of the prescribed drugs , and in turn seek neurosurgical intervention . currently , surgical approaches include microvascular decompression ( mvd ) or a number of techniques that target the trigeminal ganglion or root which involve the destruction or blockage of portions of those anatomical structures [ 1 , 2 ] . although the neurosurgical modalities are preferred in many clinical situations and have proven to be effective in achieving initial pain control , they are known to come with a variety of complications , and facial pain recurrence is likely . stereotactic radiosurgery ( srs ) has proven to be an effective management approach for patients with medically and surgically refractory tn as a primary and repeat treatment modality . the use of radiosurgery in the treatment of tn dates back to sweden in the 1950 's , where professor lars leksell performed radiogangliotomies directed at the gasserian ganglion . since the time of leksell , advancements in radiosurgery and imaging technologies has led to the increasing popularity of srs as a treatment option for patients with tn . one form of srs that can be delivered to a patient is through a machine called the gamma knife ( gk ) . the gk device is a cobalt-60-based machine , with 201 separate 4 to 18 mm collimator openings , that emits multiple gamma rays that converge on a target specified by computer planning . for specific medication intolerable patient subsets , gamma knife radiosurgery ( gkrs ) can be used as an initial management approach , or as a secondary management approach following radiosurgery or one or more of the various surgical modalities . as the evidence examining the role of gkrs in the management of patients with tn is increasing , it is of utmost importance for physicians to understand the criteria associated with gkrs , so that the optimal course of treatment for their patients can be prescribed . an evidence - based review on the evaluation and treatment of tn by gronseth et al . found level c evidence indicating that gasserian ganglion percutaneous techniques , gkrs , and mvd may be considered for facial pain management for medically refractory patients . however , questions remain regarding optimal treatment modalities in specific patient subsets . for this reason , the goal of this paper is to provide a modern review of the literature thoroughly analyzing the efficacy of gkrs in the treatment of patients with tn , as well as evaluating the treatment planning and methods associated with this evolving modality . to identify contemporary studies assessing the clinical outcomes of patients treated with gkrs for tn , a pubmed search from 2006 to april 2011 was performed . keywords for search included gamma knife or gamma knife radiosurgery or stereotactic radiosurgery trigeminal neuralgia or tic douloureux . studies analyzed in this review included retrospective cohort studies and prospective cohort studies with 5 evaluated patients . studies published only in abstract form and studies published in a language other than english were excluded from our analysis . due to our broad search strategy and the vast amount of world literature , we reviewed a total of 19 studies [ 4 , 5 , 925 ] analyzing the efficacy of patients with tn who were treated once with gkrs ( table 1 ) . thirteen of the 19 evaluated studies [ 4 , 5 , 919 ] utilized the barrow neurological institute ( bni ) pain intensity scale as a measurement of response to treatment ( see section 3 ) . of these 13 studies , only two [ 9 , 13 ] analyzed patients treated with gkrs as an initial management approach . with a median followup of 31 months , sheehan et al . classified 87% of patients in bni class i - iiib , while chen et al . classified 91% of patients in bni class i - iiib ( median followup = 15 months ) . chen et al . also reported that five of the 44 patients ( 11% ) treated with gkrs developed hypoesthesia following the procedure . the other 11 bni pain intensity scale studies we reviewed included patients where previous surgical procedures were performed in a fraction of patients [ 4 , 5 , 1012 , 1519 ] or all patients . of the 10 studies where previous surgical procedures were performed in a fraction of patients , nine reported outcomes in terms of categorizing patients in bni class i - iiib [ 4 , 5 , 1012 , 1518 ] . classified 58.6% of patients in bni class i - iiib ( median followup = 48 months ) , kondziolka et al . classified 71% of patients in bni class i - iiib at three years , dhople et al . classified 72% of patients in bni class i - iiib ( median followup = 29 months ) , han et al . classified 76.7% of patients in bni class i - iiib ( mean followup = 58 months ) , dhople et al . classified 81% of patients in bni class i - iiib ( median followup = 5.6 years ) , matsuda et al . classified 82% of patients in bni class i - iiib ( median followup = 37 months ) , little et al . classified 83% of patients in bni class i - iiib ( median followup = 6.3 years ) , dellaretti et al . classified 89.5% of patients in bni class i - iiib ( mean followup = 20.3 months ) , and park and hwang classified 94% of patients in bni class i - iiib with a minimum followup of 3 years . reported clinical outcomes with respect to bni class i , which contained only 5.7% of patients . the study that evaluated gkrs where previous surgical procedures were performed in 100% of patients classified 85% of patients in bni class i - iiib , with a median followup of 36 months . we also reviewed two studies that used the excellent - good - fair - poor ( egfp ) categorical scale to assess patient outcomes [ 20 , 21 ] ( see section 3 ) . treated 30 patients with tn with gkrs at iran gamma knife center between 2006 and 2007 . the authors reported that 40% of patients had an excellent outcome , 10% of patients had a good outcome , 33% of patients had a fair outcome , and 17% of patients had a poor outcome following the procedure . after surgery , 15 patients ( 54% ) reported an excellent outcome , one patient ( 4% ) reported a good outcome , two patients ( 7% ) reported a fair outcome , and 10 patients ( 36% ) reported a poor outcome . the complications from mvd included facial numbness in six patients ( 21% ) , dysesthesias in three patients ( 11% ) , and delayed facial palsy in one patient ( 4% ) . four of the 19 studies we evaluated [ 2225 ] used other methodologies in determining the effectiveness of gkrs . in a prospective controlled trial , rgis et al . analyzed 100 patients with tn treated with gkrs and reported that 83 patients ( 83% ) were completely pain free , 58 of which ( 58% ) discontinued all medication following the procedure ( minimum followup = 12 months ) . ten patients ( 10% ) experienced radiation - induced complications , which included facial paresthesia or hypesthesia . performed a study investigating the short - term efficacy of gkrs in 67 patients with medically refractory tn . overall , 77.6% of patients witnessed some degree of pain relief , with 32.6% of those patients becoming completely pain free . of the 67 patients , 10 ( 14.9% ) experienced complications from the procedure , which included hypoesthesia and paresthesia . sixty eight patients ( 42.5% ) underwent prior invasive treatments . in clinical analysis , it was found that 61% of patients were pain free without medication , 29% of patients were pain free with medication , and 10% of patients did not respond to gkrs . thirty eight patients ( 49.4% ) exhibited some level of pain improvement following gk treatment , with 23 of those patients ( 29.9% ) reporting a pain - free outcome . twelve patients ( 15.6% ) experienced complications , which were reported to be mild facial sensory changes and mild facial nerve dysfunction . as gkrs has proven to be an effective initial treatment for tn , numerous reports have been published analyzing patients treated on multiple occasions ( > 1 ) with gkrs . we reviewed six studies evaluating patients treated more than once with gkrs [ 2732 ] ( table 2 ) . of these six articles , two [ 29 , 32 ] utilized the bni pain intensity scale . sixteen patients ( 76.2% ) exhibited compelling improvements and were placed in bni class i - ii . huang et al . analyzed 65 medically refractory patients with tn who were treated with gkrs as a second treatment modality . of these 65 patients , 30 ( 46% ) had undergone gkrs as an initial management approach . the authors placed 22 patients ( 34% ) in bni class i , 11 patients ( 17% ) in bni class ii , four patients ( 6% ) in bni class iiia , and five patients ( 8% ) in bni class iiib . overall , with a median followup of 64 months , 65% of patients reported successful results in terms of pain control rates . a total of three of the six reviewed studies evaluated patients using the egfp categorical scale [ 28 , 30 , 31 ] . analyzed 37 patients treated a second time with gkrs for recurrent tn and reported that 17 patients ( 46% ) achieved excellent pain relief , nine patients ( 24% ) achieved good pain relief , five patients ( 14% ) achieved fair pain relief , and six patients ( 16% ) achieved poor pain relief . however , the authors concluded that 57% of patients experienced some form of trigeminal dysfunction following repeat radiosurgery . , huang et al . treated 28 patients with repeat gkrs and reported that 12 patients ( 43% ) exhibited excellent pain relief , five patients ( 18% ) exhibited good pain relief , and two patients ( 7% ) exhibited fair pain relief . in addition , the authors found a statistically significant ( p = 0.047 ) correlation between cumulative radiation doses > 115 gy and facial numbness . in a separate study , huang et al . specifically , a total of eight patients underwent mvd a mean of 7.6 months following repeat gkrs . of the eight patients , seven ( 87.5% ) were completely pain free at a mean of 21 months following neurosurgery . this data supports the use of mvd if multiple gk procedures are deemed ineffective . kimball et al . treated 53 patients with repeat gkrs and analyzed the patients not lost during followup using the marseille scale , which categorizes patients into one of five classes , with a higher class statistically indicating a worse prognosis for the patient . with a mean followup of 42 months , 20 patients ( 43.5% ) were categorized in marseille class i - ii , six patients ( 13% ) were categorized in marseille class iii - iv , and 20 patients ( 43.5% ) were categorized in marseille class v. the authors also reported a statistically significant ( p = 0.047 ) correlation between facial numbness and superior long - term pain relief . a total of 22 patients ( 48% ) experienced trigeminal dysfunction of any kind , while 21 patients ( 46% ) experienced numbness in the face . since gkrs can be performed as both initial and salvage treatment options for patients who suffer from tn , its efficacy has been compared in patients who undergo one versus multiple radiosurgery procedures . we reviewed eight studies to further examine this matter [ 3 , 3339 ] ( table 3 ) . four of the eight studies utilized the bni pain intensity scale to evaluate patient outcomes [ 3 , 3335 ] . , it was reported that 75% , 60% , and 58% of patients with idiopathic tn had bni scores of i iiib at 1 , 3 , and 5 years , respectively . the 1- , 3- , and 5-year - bni scores of i iiib in patients with ms - related tn were 56% , 30% , and 20% , respectively . the authors concluded that repeat gkrs exhibited similar success rates when compared to the initial procedure . similar to verheul et al . , park et al . did not find differences in terms of time to initial response , time to pain recurrence , and overall pain relief when comparing patients who underwent one versus two gk treatments . however , it was observed that patients who received two gk treatments were more likely to have facial sensory changes when compared to patients treated a single time with radiosurgery . little et al . performed a study where 79 patients with typical tn were treated with gkrs as a salvage procedure . approximately five years following salvage gkrs , the authors reported that 50% of patients experienced pain relief and 20% of those patients were completely pain free . in addition , a statistically significant ( p = 0.029 ) correlation between gkrs failure and prior mvd was found . treated 37 patients ( 78% had failed prior surgery ) with ms - related tn with gkrs . nine patients ( 24% ) underwent gkrs as their first procedure . the reported 1 , 3 , and 5 year bni scores of i iiib were 82.6% , 73.9% , and 54% , respectively . the other four studies we reviewed utilized the egfp categorical scale as a measurement of response to treatment [ 3639 ] . two of the evaluated studies [ 36 , 37 ] were conducted by fountas et al . and analyzed patients treated with gkrs for idiopathic tn based on whether or not they had undergone previous surgical or radiosurgical procedures for facial pain control . one of the studies evaluated 106 patients ( 19 previous radiosurgery procedures ) and concluded that the treatment group without a previous history of surgical or radiosurgical procedures exhibited superior clinical outcomes , with 1-year and 2-year complete pain relief rates of 82.5% and 78% , respectively . the 1-year and 2-year complete pain relief rate in the patient group with a history of surgical or radiosurgical procedures was 69.4% and 63.5% , respectively . as expected , similar results were found in the other study by fountas et al . ; however , no prior radiosurgical procedures were performed in the patient group with a history of prior procedures . huang et al . conducted a study where 89 patients with idiopathic tn were treated with gkrs as an initial management approach , 20 of which underwent a subsequent gkrs procedure for facial pain recurrence . following the initial radiosurgical procedure , 50 patients ( 56% ) had an excellent response , 12 patients ( 13.5% ) had a good response , and 7 patients ( 7.9% ) had a fair response . following the second radiosurgical procedure , 11 patients ( 55% ) had an excellent response and one patient ( 5% ) had a good response . in a separate study , huang et al . assessed 21 patients with benign tumor - related tn who were treated with gkrs as an initial or repeat procedure . following the initial gk procedure to the tumor , 12 patients ( 57% ) had an excellent response and 1 patient ( 5% ) had a good response . a total of eight patients were treated with a subsequent gkrs procedure targeted at the ipsilateral trigeminal root or ganglion due to facial pain recurrence . following the second radiosurgical procedure , the authors reported four patients ( 50% ) with an excellent response . we identified six studies comparing patients treated with gkrs with patients treated with one of the various surgical modalities [ 2 , 4044 ] ( table 4 ) . the authors of this review acknowledge the importance of percutaneous techniques in the management of tn ; however , our modern literature search predominantly yielded comparison studies analyzing the efficacy of mvd when compared to gkrs . specifically , four of the six studies [ 2 , 4042 ] analyzed patients treated with gkrs against patients treated with mvd . specifically , 36 were treated with mvd ( 45% ) , while 44 were treated with gkrs ( 55% ) . the mvd treatment arm statistically differed from the gkrs treatment arm with respect to age ( median of 54 versus 74 years ) , preoperative symptom duration ( median of 2.6 versus 7.5 years ) , and the presence of major comorbidities ( 2.8 versus 58.3% ) . the authors reported that patients treated with mvd exhibited superior levels of initial ( 100% ) , 2 year ( 88% ) , and 5 year ( 80% ) actuarial pain - free rates when compared to the patients treated with gkrs ( 78 , 50 , and 33% , resp . ) , with a p value of 0.0002 . in addition to increased levels of patient satisfaction , as reported by required patient surveys , the mvd treatment group also had a decreased level of permanent mild ( 5.6% ) and severe sensory loss ( 0% ) when compared to the gkrs treatment group ( 6.8% and 2.3% , resp . ) . two patients ( 5.6% ) in the mvd group experienced permanent mild paresthesias or numbness , one ( 2.8% ) patient experienced a cerebrospinal fluid leak from the wound , and one patient ( 2.8% ) experienced hearing loss and diplopia . three patients ( 6.8% ) in the gkrs group experienced permanent mild paresthesias or numbness , one patient ( 2.3% ) experienced a more permanent sensory numbness , and one patient ( 2.3% ) experienced a transient headache and nausea following the gk procedure . all patients were diagnosed with typical tn and did not undergo previous gk or mvd procedures . it was reported that patients treated with mvd exhibited superior levels of complete pain relief at 12 ( 68% ) and 18 months ( 68% ) when compared to the gkrs group , who 's complete pain relief rate was 58% at 12 months and 24% at 18 months ( p = 0.089 ) . the treatment arms did not statistically differ in terms of 90% pain relief at 12 and 18 months . this study could be criticized due to the large difference in the number of patients constituting the two treatment arms . oh et al . evaluated a total of 45 elderly patients ( > 65 years of age ) diagnosed with idiopathic tn who were treated with either mvd ( 27 patients ) or gkrs ( 18 patients ) . it was reported that three mvd patients ( 11% ) and three gk patients ( 17% ) underwent previous percutaneous procedures . the mean followup period was reported to be 35.9 months for the mvd group and 33.1 months for the gkrs group . according to the bni pain intensity scale , the mvd group had a superior prognosis , with 17 patients ( 63% ) classified in bni class i - ii compared with the 10 patients ( 56% ) in the gkrs group classified in bni class i - ii . the observed complications following mvd included constant headache in 11 patients ( 40.7% ) , facial paresthesia in five patients ( 18.5% ) , paresthesia of the tongue in two patients ( 7.4% ) , infection at the site of incision in one patient ( 3.7% ) , an acute subdural hemorrhage in one patient ( 3.7% ) , temporary hearing loss in one patient ( 3.7% ) , and otitis media with cerebrospinal leakage in one patient ( 3.7% ) . two patients ( 11% ) in the gkrs group experienced paresthesia . compared the clinical outcomes of 19 patients treated with mvd with 15 patients treated with gkrs . nine gk patients ( 60% ) and four mvd patients ( 21% ) underwent previous surgical procedures . the treatment arms statistically differed ( p = 0.0005 ) with respect to mean patient age , with the mean age of the gkrs group exceeding the mvd group by 13 years ( 74 versus 61 years ) . in addition , patient satisfaction was graded on a scale of 1 ( unsatisfied ) to 10 ( completely satisfied ) . it was reported that the mean tn complexity grade was statistically different ( p < 0.001 ) between the treatment arms ( gk = 5.8 ; mvd = 3 ) . the average response following the procedure was determined to be 3.4 for the mvd group and 2.4 for the gkrs group ( p = 0.017 ) . also , it was found that the satisfaction score for the mvd group was superior to the gkrs group ( 8.7 versus 6.4 ) , with a p value of 0.02 . the authors reported a statistically significant correlation between tn complexity grade and clinical response ( p < 0.001 ) , as well as tn complexity grade and patient satisfaction ( p < 0.001 ) . to date , no randomized trials have been conducted analyzing the outcomes of patients with tn who are treated with mvd compared to gkrs . in a large review on tn management , zakrzewska and linskey found evidence that mvd is an effective treatment for long - term facial pain relief but comes with an increased risk of ipsilateral hearing loss . in addition , the authors concluded that single - dose srs is an effective treatment for long - term facial pain relief but puts patients at risk for facial numbness or facial paresthesias . investigation into this matter in the form of a randomized controlled trial would provide the best evidence in terms of facial pain relief and procedure - related complications . in addition , we reviewed two studies comparing patients treated with gkrs with patients treated with posterior fossa exploration ( pfe ) [ 43 , 44 ] , both of which were conducted by pollock and colleagues at the mayo clinic college of medicine . one of the studies was a specific prospective comparison of 91 patients treated with pfe and 49 patients treated with gkrs for idiopathic tn as an initial management approach . the treatment arms statistically differed in terms of age ( gkrs = 67.1 years ; pfe = 58.2 years ) , with a p value < 0.001 . it was reported that patients treated with pfe were more likely to be pain free and off medications at 1 year ( 84% ) and 4 years ( 77% ) when compared to the gkrs group ( 66 and 56% , resp . ) retreatment for recurrent facial pain was performed in 15% of the patients in the pfe treatment arm and 35% of patients in the gkrs treatment arm ( p = 0.009 ) . also , it was found that nonbothersome facial numbness occurred more frequently in the gkrs group ( p = 0.04 ) . an additional study from the mayo clinic evaluated patients with recurrent tn who underwent 3 or more surgical procedures . the authors reported that patients treated with pfe exhibited superior levels of complete pain relief at 3 years of followup when compared to patients treated with gkrs , balloon compression , and glycerol rhizotomy ( p < 0.01 ) and underwent additional surgery for recurrent facial pain less often when compared to patients treated with the other modalities ( p = 0.02 ) . clinical 006futcomes did not differ between patients treated with gkrs and patients treated with the percutaneous techniques . srs can be performed by a variety of tools , which include gkrs , cyberknife technology , and linear accelerator ( linac)-based treatment . our analysis yielded one study whose primary endpoint was to devise a method using cyberknife treatment planning that would mimic the dosimetric characteristics of the gk treatment plan in five patients undergoing radiosurgery for tn . both the isodose lines and critical structures were identified using the gkrs treatment plan and were transferred to the cyberknife treatment planning system . it was reported that the average length of the trigeminal nerve receiving a dose of 60 gy was 4.5 mm for the gk , 4.5 mm for the nonisocentric cyberknife , and 4.4 mm for the isocentric cyberknife . the authors found it more difficult to minimize the dose to critical structures when using cyberknife technology . also , the dose falloff of gkrs was found to be steeper when compared to cyberknife technology due to , what the authors hypothesized , the large number of gamma rays produced which converge on the focal point with precision . as previously mentioned , the gk machine 's primary functional unit is cobalt-60 , which is used to emit photon energy through 201 separate 4 to 18 mm collimator openings that converge on a target specified by a treatment planning system . balamucki et al . performed a study examining if the half life of cobalt ( 5.26 years ) relates to the outcomes for patients being treated for tn with gkrs . the authors collected data on 239 gkrs procedures performed at their institution between 1999 and 2004 . patient surveys were used to measure responses to radiosurgery . with the followup time ranging from one to six months , it was reported that 80% of patients experienced some degree of pain relief and that 56% of those patients were pain free . the authors concluded that clinical outcomes remained consistent during the first half life of cobalt-60 . an area of controversy in the treatment of patients with tn is defining the optimal maximum radiosurgery dose that can be delivered to specific patient subsets . we analyzed five studies whose primary endpoint was to assess gkrs - dosing efficacy [ 4852 ] . kim et al . utilized the bni pain intensity scale to assess 66 tn patients treated with a gk maximum dose of 80 gy and 44 tn patients treated with a gk dose of 85 gy . although the two groups did not statistically differ in terms of facial rain relief and procedure - related complications , the authors did report that patients treated with a gk dose of 85 gy experienced a more rapid response to treatment when compared to the patients treated with a gk dose of 80 gy . arai et al . analyzed 165 patients with tn treated with a gkrs dose of 80 gy . specifically , the authors divided the patients into two groups , which differed in the radiation dose rate received ( low - dose rate = 1.212.05 gy / min ; high - dose rate = 2.063.74 gy / min ) . using the bni pain intensity scale as a clinical evaluation method , it was reported that the low - dose - rate group and the high - dose - rate group did not statistically differ in terms of initial pain relief , maintenance of pain relief , and clinical complications . patients in group one were treated with a gk dose < 90 gy with no beam channel plugging , patients in group two were treated with a gk dose equal to 90 gy with no beam channel plugging , and patients in group three were treated with a gk dose equal to 90 gy with beam channel plugging . although the trend did not reach full statistical significance ( p = 0.054 ) , patients in group three exhibited the highest level of pain relief , while patients in group one exhibited the lowest level of pain relief . the authors also observed that the three groups statistically differed ( p < 0.0001 ) in terms of trigeminal nerve dysfunction , with patients in group three experiencing the highest rate of mild and bothersome complications and patients in group one experiencing the lowest rate of mild and bothersome complications . similar to the results of massager et al . , morbidini - gaffney et al . reported positive outcomes in patients treated with gk doses > 85 gy . the authors also found that patients treated with two isocenters were more likely to have superior bni pain intensity scale scores during their course of followup when compared to patients treated with a single isocenter . the median initial dose was 80 gy , and the median retreatment dose was 45 gy . although the authors did not report any predictors in terms of facial pain control and patient morbidity , they did compare the results of their study with seven published retreatment articles and found that successful levels of pain control ( > 50% ) were significantly correlated with cumulative gkrs doses > 130 gy , as well as new trigeminal nerve dysfunction ( > 20% ) . in addition to dose selection efficacy in select patient cohorts , the radiosurgical target of cn v is another subject matter that requires further clinical investigation . we reviewed three studies [ 5355 ] analyzing specific gkrs targeting methods in the treatment of tn and one study that examined the accuracy of gkrs to its image - guided target . compared patients treated with gkrs targeted at the dorsal root entry zone ( 59 patients ) with patients whose radiosurgical target was the retrogasserian zone of the trigeminal nerve ( 41 patients ) . with a median followup of 30 months , the dorsal root entry target group exhibited statistically superior levels of initial complete pain remission ( p = 0.003 ) and experienced less complications than the retrogasserian zone group ( p = 0.009 ) . chen et al . also reported positive results with the dorsal root entry zone - targeting approach , with a success rate of 82.8% and a complication rate of 15% . park et al . compared the dorsal root entry zone and retrogasserian zone - targeting methods in the treatment of 39 patients with medically refractory tn . the authors reported that the two treatment arms did not statistically differ in treatment success ( bni class i - iiib ) with respect to the bni pain intensity scale . however , patients treated with the retrogasserian zone - targeting method experienced a substantially shorter time of response to gkrs than patients treated with the dorsal root entry zone - targeting method ( p = 0.044 ) . although the two groups did not statistically differ with regard to treatment - related morbidities , it was found that the patients whose targeting approach was the dorsal root entry zone experienced a greater amount of bothersome complications than the retrogasserian zone group . massager et al . analyzed the targeting accuracy of gkrs in 65 patients treated for tn whose six month followup mri showed focal contrast enhancement of the trigeminal nerve . the authors found that the median deviation of the coordinates between the intended radiosurgical target and the center of contrast enhancement was 0.91 mm in euclidean space . the median radiation dose fitting into the contrast enhancement region was determined to be 77 8.7 gy . this small deviation from the gkrs target explains the high accuracy and precise nature of the machine . the two most common methods of measuring patient outcomes from gkrs in the management of tn are the barrow neurological institute pain intensity scale ( table 5 ) and the excellent - good - fair - poor ( egfp ) categorical scale ( table 6 ) . the bni pain intensity scale divides patients into one of five classes , with a higher class indicating a worse prognosis for the patient . patients in bni class iiia do not experience trigeminal pain but require the use of medication . patients in bni class iiib experience some trigeminal pain that can be satisfactorily managed with medication . patients in bni class iv experience some trigeminal pain that is not satisfactorily managed with medication . patients in bni class excellent outcomes are defined as complete pain relief without the need of medication . good outcomes are defined as complete pain relief with the need of medication . poor outcomes are defined as a < 50% pain relief rate or treatment failure . for patients with medically refractory forms of tn , gkrs has proven to be an effective initial and repeat treatment option . cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs . however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs . although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive . further evidence in the form of a phase iii - randomized trial is needed to confirm the clinical outcomes of patients treated with either modality . questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter .

since its introduction by leksell , gamma knife radiosurgery ( gkrs ) has become increasingly popular as a management approach for patients diagnosed with trigeminal neuralgia ( tn ) . for this reason , we performed a modern review of the literature analyzing the efficacy of gkrs in the treatment of patients who suffer from tn . for patients with medically refractory forms of the condition , gkrs has proven to be an effective initial and repeat treatment option . cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs . however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs . although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive . questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter .

this intervention has potential complications such as surgical site infection ( ssi ) , one of the most frequent nosocomial infections , with a reported prevalence of 2 - 38.7% . prevention of ssi can be achieved by surgical hand disinfection , a standard and obligatory procedure used in all hospitals . the surgical hand antisepsis , performed before donning sterile glove and gown , is defined as a process to remove transient microorganisms and reduce the resident skin flora . due to potential risk of surgical glove perforations occurring during surgery , it is necessary to reduce skin flora as high as possible , and use antibacterial solutions before gloves are worn . since joseph lister had shown the importance of hand washing in the control of postoperative ssi , researchers have been trying to introduce preparations that have maximum efficacy on the density of resident flora . hence , a variety of formulations have been produced and presented to the pharmaceutical market . up to now , based on the formulations produced , two main groups of surgical hand disinfectants have been presented : ( a ) antibacterial soaps , used in traditional hand disinfection procedure , and ( b ) alcohol - based hand solutions , which are used in surgical hand rub procedure , as a waterless and brushless method . alcohol - based hand rubs are known to be the most effective surgical hand antiseptics and are often preferred to antimicrobial soaps because they are broad - spectrum agents and have high antibacterial effect , act faster and in the shortest time , can be applied easily , and are better tolerated by skin . as marchetti et al . reported , although antiseptic - based soap ( betadine ) significantly reduced the skin flora , just sterillium and softaman ( two alcohol - based hand rubs ) , and hibiscrub ( chlorhexidine ) could meet the requirement of pren 12791 ( an european standard for evaluating the antibacterial efficacy of surgical hand antiseptics ) . hsieh , in a systemic literature review , showed hand rubbing for 3 min with an alcoholic disinfectant was more effective than 6 min of hand scrubbing using chlorhexidine gluconate ( chg ) 4% . recently , hand rubs have also been increasingly used in the islamic republic of iran . with the widespread use of alcoholic disinfectants , health service managers are always trying to choose more efficient and cost - effective antiseptics . in addition , researchers around the world are constantly trying to compare the efficacy of these formulations to determine the effectiveness of them . for example , in a research conducted on 3 and 5 min hand disinfection with two hand rub products ( ethanol- and isopropanol - based hand rubs ) , isopropanol hand rub caused better result in decreasing the skin flora . kampf and ostermeyer , in a controlled trial , compared the efficacy of two alcoholic disinfectants ( sterillium rub and avagard ) ; their results showed that based on pren 12791 , sterillium rub was more effective than avagard . iranian pharmaceutical market is mainly composed of a variety of imported and domestic surgical hand disinfectants of different prices . today in iran , economic sanctions have been led to sharp increases the price of imported products , therefore the ministry of health emphasizes the use of similar local products . hence this study was conducted to compare the efficacy of two imported alcoholic hand rubs , sterilium and decosept , with that of domestic hand rub antiseptic , septicidine . if the domestic product 's effectiveness is similar to that of foreign products , its use is suggested as it is less expensive . this quasi - experimental study was a clinical trial consisting of one group , with before and after design . this study was approved by the research ethics committee of hamadan university of medical sciences . the study was conducted on 20 healthy volunteers who were students or personnel of the university . inclusion criteria were : ( a ) age more than 18 years , ( b ) having clean and short nails , and ( c ) not having used any substance with antibacterial activity 1 week before the study . exclusion criteria were : ( a ) pregnancy , ( b ) afflicted with any skin diseases , and ( c ) presence of any cuts or abrasion on hands . the sample size was estimated to be 20 subjects for each treatment ( paired sample ) , considering the kamp fetal article data and using the following equation for = 0.05 and = 0.2 : the following surgical hand rubs were used : sterllium ( 45% 2-propanol , 30% 1-propanol , 0.2% mecetronium etilsulfate ; bode chemie , hamburg , germany ) , septicidine pc ( 50% ethanol , 25% isopropanol , 0.5% chg ; behban shimi , tehran , iran ) , decosept ha ( 44.7% 2-propanol , 21.9% 1-propanol , 0.1% benzalkonium chloride ; borer chemie , zuchwil , switzerland ) . for recovering of skin flora , at least 1 week interval had been elapsed before every interven tion . before treatment , subjects washed their hands with soft soap ( sapokalinus2 ) . after rinsing and drying of hands with non - sterile paper towel , bacterial culture samples of finger tips of both hands were taken ( pre - value count ) . then both hands were rubbed with one of the products under test . after disinfecting , to assess the immediate antibacterial effect , a sample as well as pre - value was taken from one randomly selected hand and the unsampled hand was gloved for 3 h. to evaluate the 3 h antibacterial effect , after removing the glove , sampling was performed similar to that of the immediate sample . washing hands up to wrist was achieved according to the method recommended by the word health organization ( who ) and en12791 with 10 ml of sapokalinus for 1 min . distal phalanges of each hand were rubbed in 9-cm diameter petri dish containing 10 ml of tryptic soy broth ( tsb ) . for pre - value , dilutions of 10 and 10 of sampling fluid were prepared in tsb . for each dilution , 0.1 ml was spread over the surface of a tryptic soy agar ( tsa ) plate . for post - value sampling , 1 and 0.1 ml of undiluted sampling fluid and 0.1 ml from its 10 dilution all plates were incubated aerobically at 37c 1c for 2448 h. the interval between sampling and incubating was less than 30 min . the hand rubbing was practiced in accordance with the instruction of manufacturer as follows : for all hand antiseptics , the procedure was the same as that for standard hand rub , up to wrist . during the procedure , if hands nearly dried , additional volume of the product was applied to hands so they were being kept moist during disinfection phase . but the duration of application and , consequently , the volume of product were different for each product : for decosept , they were 3 min and 812 ml , for septicidine 6 min and 1014ml , and for sterillium 1.5 min and 47 ml , respectively . the three hand rubs were dispensed sterilely into the hollow of subjects palm . in order to normalize the data , pre and post treatment colony counts per milliliter of sampling fluid ( cfu / ml ) were inverted to 10 logarithmic values . the number of colony forming units ( cfu ) per plate for each dilution was recorded , and then the number of cfu per milliliter was calculated . the plate counts between 15 and 300 were chosen in order to calculate the cfu / ml . if the cfu of post - value plates were less than 15 , these values were counted . if values in the range that could be entered into calculations were obtained from more than one dilution , their mean was used as the final logarithmic value . the following surgical hand rubs were used : sterllium ( 45% 2-propanol , 30% 1-propanol , 0.2% mecetronium etilsulfate ; bode chemie , hamburg , germany ) , septicidine pc ( 50% ethanol , 25% isopropanol , 0.5% chg ; behban shimi , tehran , iran ) , decosept ha ( 44.7% 2-propanol , 21.9% 1-propanol , 0.1% benzalkonium chloride ; borer chemie , zuchwil , switzerland ) . each subject applied all the hand rubs under investigation . for recovering of skin flora , at least 1 week interval had been elapsed before every interven tion . before treatment , subjects washed their hands with soft soap ( sapokalinus2 ) . after rinsing and drying of hands with non - sterile paper towel , bacterial culture samples of finger tips of both hands were taken ( pre - value count ) . then both hands were rubbed with one of the products under test . after disinfecting , to assess the immediate antibacterial effect , a sample as well as pre - value was taken from one randomly selected hand and the unsampled hand was gloved for 3 h. to evaluate the 3 h antibacterial effect , after removing the glove , sampling was performed similar to that of the immediate sample washing hands up to wrist was achieved according to the method recommended by the word health organization ( who ) and en12791 with 10 ml of sapokalinus for 1 min . distal phalanges of each hand were rubbed in 9-cm diameter petri dish containing 10 ml of tryptic soy broth ( tsb ) . for pre - value , dilutions of 10 and 10 of sampling fluid were prepared in tsb . for each dilution , 0.1 ml was spread over the surface of a tryptic soy agar ( tsa ) plate . for post - value sampling , 1 and 0.1 ml of undiluted sampling fluid and 0.1 ml from its 10 dilution all plates were incubated aerobically at 37c 1c for 2448 h. the interval between sampling and incubating was less than 30 min . the hand rubbing was practiced in accordance with the instruction of manufacturer as follows : for all hand antiseptics , the procedure was the same as that for standard hand rub , up to wrist . during the procedure , if hands nearly dried , additional volume of the product was applied to hands so they were being kept moist during disinfection phase . but the duration of application and , consequently , the volume of product were different for each product : for decosept , they were 3 min and 812 ml , for septicidine 6 min and 1014ml , and for sterillium 1.5 min and 47 ml , respectively . the three hand rubs were dispensed sterilely into the hollow of subjects palm . in order to normalize the data , pre and post treatment colony counts per milliliter of sampling fluid ( cfu / ml ) were inverted to 10 logarithmic values . the number of colony forming units ( cfu ) per plate for each dilution was recorded , and then the number of cfu per milliliter was calculated . the plate counts between 15 and 300 were chosen in order to calculate the cfu / ml . if the cfu of post - value plates were less than 15 , these values were counted . if values in the range that could be entered into calculations were obtained from more than one dilution , their mean was used as the final logarithmic value . the pre - value and immediate post - value log10 of the three products were : sterillium : 4.3 0.44 and 0.32 0.57 , decosept : 4.07 0.53 and 0.81 0.84 , and septicidine : 3.95 0.94 and 1.28 1.14 , respectively . t - test showed the differences were significant , that is , the three hand rubs had immediate effect . using anova test for comparison of immediate effect revealed significant difference among them , that is , the effects of products were not similar . sterillium had the most and septicidine had the least immediate effect [ table 1 ] . comparison of the bacterial effect ( immediate and 3 h ) of the three surgical alcohol - based hand rubs after 3 h , the bacterial density of hands increased , but these counts ( 3 h post - value ) were significantly lower than the pre - value : sterillium : 3.92 0.63 and 1.80 1.14 , decosept : 3.86 1.44 and 1.94 1.10 , and septicidine : 3.98 1.02 and 1.58 1.23 ( t - test ; p < 0.0001 ) . this means the 3 h effect of the antiseptics was the same [ table 1 ] . septicidine did not cause fast and immediate effect as the other products , but comparison of log10 immediate value and log10 after 3 h of t he products using t - test showed that only septicidine could retain the antibacterial effect after 3 h under the glove [ table 2 ] . comparison of immediate effect and 3 h effect of the three surgical alcohol - based hand rubs the density and composition of the skin normal flora may be influenced by various factors , including anatomical locale , age , sex , moisture , ph , and immune system . it has been estimated that about 1010 cfu / cm inhabit on the skin of a human adult . the skin microbes found in the most superficial layers of the epidermis are gram - positive cocci ( staphylococcus epidermidis and micrococcus sp . ) and corynebacteria such as propionibacterium sp . these are generally nonpathogenic or commensal ; even some of them are mutualistic ( offer a benefit ) . sometimes potentially pathogenic staphylococcus aureus is found on the face and hands in individuals who are nasal carriers . nevertheless , these flora do not cause infection , if the skin is intact . in patients undergoing surgery s. aureus and s. epidermidis were reported to be the two most common organisms causing ssi . one predisposing factor of ssi is the microorganisms carried by the hands of surgical team in case of using perforated surgical gloves ; hence , surgical hand antisepsis is carried out to eliminate the transient flora and diminish the resident skin flora as much as possible . in this study , we found that all three alcoholic hand rubs were able to significantly reduce the skin colony count immediately and 3 h after disinfection . this result is similar to previously reported results . despite the fact that these clinical trials used different methods , their results showed significant reduction in skin flora after the interventions . the efficacy of alcohol - based hand formulation is influenced by ( a ) the type , concentration , and volume of alcohol used , ( b ) duration of application , and ( e ) other disinfectant or auxiliary agent . in our study , sterillium , containing 1-propanol 30% , the best effective alcohol , and 2-propranol 45% ( total 75% ) , led to the immediate decrease of resident hand flora . similar to this , in some studies , it was found to be the most effective alcoholic hand rub among others with different alcohol types a nd longer application time . among the short chain alcohols , comparison of an 80% ethanol - based hand rub , irrespective of the application time ( 1.5 , 3 , and 5 min ) , with propan-1-ol 60% ( 3 min ) showed less significant effect than that of propane - based rub , and comparison of 1.5 and 3 min disinfection with propan-2-ol 75% and propan-1-ol 60% ( 3 min ) demonstrated less significant effect of isopropanol - based hand rub . the 6 min hand rub with septicidine ( contains ethanol 50% , 2-propanol 25% , and chg 0.5% ) caused the least immediate effect ; but after 3 h disinfection , any significant increase in resident flora did not reduce . it was concluded that septicidine could retain the antibacterial effect during 3 h ; in other words , its effect persisted better , which may be related to the ingredient in septicidine ( chg ) . although reduction in resident flora after 3 h in comparison to the pre - value was significant for the two other formulations , skin flora increased after 3 h in comparison to the immediate value . to the best of our knowledge , there is no study concerning the effectiveness of septicidine . one minute application of a hand rub ( hibistat containing chg 0.5% , isopropanol 70% ; its formulation is nearly similar to septicidine ) , in comparison to 3 min disinfection with propan-1-ol 60% , caused significantly less immediate and 3 h effect . effectiveness of each alcohol - based solution must be considered with time span . in this study , decosept , sterillium , and septicidine , respectively , within 3 , 1.5 , and 6 min , could achieve effective results . very short application time may fail to show antibacterial efficacy or long application may not result in any further effect . hence , it is an important factor to determine how long should be the contact time . reported that hand rubbing within 1.5 and 3 min with sterillium and 1.5 min with sensiva ( containing propan-1-ol 45% , propan-2-ol 28% , and lactic acid 3% ) showed similar results , whereas 1.5 min disinfection with desderman ( ethanol 78.2% , 2-biphenylol 0.1% ) did not show acceptable results . in another study , two who formulations , ethanol 80% and isopropanol 75% , with 5 min application time were as effective as applying them for 1.5 and 3 min . even when the most powerful alcohol , propan-1-ol 70% , plus chg 0.5% and ethanol 78% plus biphenyl-2-ol 0.1% were used for 1 min , they could not produce suitable effect . for each hand rub , the best antibacterial efficacy can be achieved by a certain application time that is recommended by the manufacturer . in clinic , this can not be practiced . some study reported surgical team disinfected their hand for shorter application time , which certainly resulted in less efficacy ; therefore , it is necessary that surveillances are frequently undertaken and the importance of this influencing factor must be emphasized to surgeons and surgical technologists . short application time of hand disinfectants is certainly accompanied with less volume of antiseptic products used . because of saving time and applied volume , short application of disinfectant without any decrease in the quality of antiseptic effect is a known favorable clinical condition , which makes healthcare facilities use such products . antiseptic dose is also a factor influencing their efficacy . in our study , the applied volume for total disinfection time was : 47 , 812 , and 1014 ml , respectively , for sterillium , decosept , and septicidine . kampf and ostermyer studied the efficacy of 3 min disinfection with different doses of propan-1-ol . based on the applied volume of the product under test , which was necessary keep the hand moist , they designated volunteers to three groups ( 6 , 9 , and 12 ml ) . as no significant differences were found among interventions , they concluded that the required volume of alcoholic hand rubs is the volume necessary to keep the hand wet , which varied according to the size , temperature , and activity of hands . to conclude , in this experiment , the alcohol concentrations of products were somewhat equal . the alcohol concentrations of sterillium , septicidine and decosept are 75 , 75 and 66.6 percent respectively , that their concentrations were in range of effective alcohol concentration . in a study , sterillium rub containing ethanol 80% at 3 min disinfection led to better significantly effect on skin flora than avagard ( ethanol 61% plus chg 1% ) . the authors concluded that the difference between the two disinfectants was related to the high concentration of ethanol in sterillium rub , whereas chg 1% could not enhance the antibacterial efficacy of avagard . based on the results of the study , although decosept and septicidine had significant effect on skin flora , considering the factors that influence the antibacterial efficacy of alcohol - based hand rub ( type , concentration , applied volume , and duration of contact with antiseptic ) , sterillium was the best product .

background : it is proved that surgical hand disinfectant contains alcohol , and has favorable properties such as strong and rapid antibacterial effect , ease of application , and suitable effect on skin . therefore , nowadays use of them has been gradually replacing traditional surgical hand scrub with antibacterial soap . hence , several domestic and imported products are available to the healthcare facilities in iran . this study was done in order to determine the antibacterial effect of decosept , sterillium , and septicidine on skin flora.materials and methods : this clinical trial was carried out on 20 volunteers . subjects disinfected their hands with three test products . at first , subjects washed their hands with soap . then pre - value sample was taken from the finger tips in tryptic soy broth ( tsb).1 after that , the hands were disinfected with one of the surgical hand rubs with as much volume as necessary to keep the hands wet , at the recommended application time . immediate post - value sample was taken from one hand and the other hand was gloved for 3 h. after removing the surgical glove , 3 h post - value sample ( sustained effect ) was taken from the hand.results:all products remarkably decreased the colony forming units ( cfu ) immediately ( p < 0.0001 ) and 3 h ( p < 0.0001 ) after disinfection . analysis of variance ( anova ) revealed significant differences among immediate post - values ( p < 0.005 ) . septicidine was significantly the least effective than the others , whereas 3 h effect of the three products was similar ( p = 0.630 , anova).conclusions : our results confirm the effectiveness of the three alcohol - based hand rubs . considering the short application time , less volume used , and more antibacterial effect , however , sterillium seems to be a better choice .

in the early 2000s , the grading of recommendations assessment , development and evaluation ( grade ) working group developed a framework in which the certainty in evidence was based on numerous factors and not solely on study design which challenges the pyramid concept.8 study design alone appears to be insufficient on its own as a surrogate for risk of bias . certain methodological limitations of a study , imprecision , inconsistency and indirectness , were factors independent from study design and can affect the quality of evidence derived from any study design . for example , a meta - analysis of rcts evaluating intensive glycaemic control in non - critically ill hospitalised patients showed a non - significant reduction in mortality ( relative risk of 0.95 ( 95% ci 0.72 to 1.25)9 ) . the quality of this evidence is rated down due to the methodological imitations of the trials and imprecision ( wide ci that includes substantial benefit and harm ) . hence , despite the fact of having five rcts , such evidence should not be rated high in any pyramid . the quality of evidence can also be rated up . for example , we are quite certain about the benefits of hip replacement in a patient with disabling hip osteoarthritis . although not tested in rcts , the quality of this evidence is rated up despite the study design ( non - randomised observational studies).10 therefore , the first modification to the pyramid is to change the straight lines separating study designs in the pyramid to wavy lines ( going up and down to reflect the grade approach of rating up and down based on the various domains of the quality of evidence ) . another challenge to the notion of having systematic reviews on the top of the evidence pyramid relates to the framework presented in the journal of the american medical association user 's guide on systematic reviews and meta - analysis . the guide presented a two - step approach in which the credibility of the process of a systematic review is evaluated first ( comprehensive literature search , rigorous study selection process , etc ) . if the systematic review was deemed sufficiently credible , then a second step takes place in which we evaluate the certainty in evidence based on the grade approach.11 in other words , a meta - analysis of well - conducted rcts at low risk of bias can not be equated with a meta - analysis of observational studies at higher risk of bias . for example , a meta - analysis of 112 surgical case series showed that in patients with thoracic aortic transection , the mortality rate was significantly lower in patients who underwent endovascular repair , followed by open repair and non - operative management ( 9% , 19% and 46% , respectively , p<0.01 ) . clearly , this meta - analysis should not be on top of the pyramid similar to a meta - analysis of rcts . after all , the evidence remains consistent of non - randomised studies and likely subject to numerous confounders . therefore , the second modification to the pyramid is to remove systematic reviews from the top of the pyramid and use them as a lens through which other types of studies should be seen ( ie , appraised and applied ) . the systematic review ( the process of selecting the studies ) and meta - analysis ( the statistical aggregation that produces a single effect size ) are tools to consume and apply the evidence by stakeholders . changing how systematic reviews and meta - analyses are perceived by stakeholders ( patients , clinicians and stakeholders ) has important implications . for example , the american heart association considers evidence derived from meta - analyses to have a level a ( ie , warrants the most confidence ) . a evidence could have been high , moderate , low or of very low quality.12 the quality of evidence drives the strength of recommendation , which is one of the last translational steps of research , most proximal to patient care . one of the limitations of all pyramids and depictions of evidence hierarchy relates to the underpinning of such schemas . the construct of internal validity may have varying definitions , or be understood differently among evidence consumers . a limitation of considering systematic review and meta - analyses as tools to consume evidence may undermine their role in new discovery ( eg , identifying a new side effect that was not demonstrated in individual studies13 ) . ebm teachers can compare it to the existing pyramids to explain how certainty in the evidence ( also called quality of evidence ) is evaluated . it can be used to teach how evidence - based practitioners can appraise and apply systematic reviews in practice , and to demonstrate the evolution in ebm thinking and the modern understanding of certainty in evidence .

a pyramid has expressed the idea of hierarchy of medical evidence for so long , that not all evidence is the same . systematic reviews and meta - analyses have been placed at the top of this pyramid for several good reasons . however , there are several counterarguments to this placement . we suggest another way of looking at the evidence - based medicine pyramid and explain how systematic reviews and meta - analyses are tools for consuming evidence that is , appraising , synthesising and applying evidence .

hypertension is a common chronic medical condition affecting people in pakistan and the rest of the world . it is an important modifiable risk factor for cardiovascular morbidity and mortality , particularly for stroke ( accounting for 51% of all stroke deaths worldwide ) , ischemic heart disease ( 45% of all deaths ) , chronic kidney disease , congestive heart failure , aortic aneurysm , and peripheral arterial disease . prevalence of hypertension ( systolic blood pressure > 140 mm hg or diastolic blood pressure > 90 mm hg , or on antihypertensive medications ) in pakistan has increased from 17% in 1980 to 35% in 2008 in adults aged 18 years and older . the increasing prevalence of hypertension together with a deficient control makes this one of the frequent conditions that require urgent medical attention . the prevalence of uncontrolled hypertension varies around the world , with the lowest prevalence in rural india ( 3.4% in men and 6.8% in women ) and the highest prevalence in poland ( 68.9% in men and 72.5% in women ) . however , the control of hypertension was 23% from a community based data in urban population from karachi , pakistan . uncontrolled hypertension can progress to hypertensive crisis defined as a systolic blood pressure > 180 mm hg or a diastolic blood pressure > 120 mm hg . hypertensive crisis can be further classified as a hypertensive urgency or hypertensive emergency depending on end - organ involvement including cardiac , renal , and neurologic injury . hypertensive urgency refers to severe hypertension without evidence of new or worsening end - organ injury while hypertensive emergency refers to a severe hypertension that is associated with new or progressive end - organ damage . hypertensive crises ( 76% urgencies , 24% emergencies ) represented more than one - fourth of all medical urgencies / emergencies . hypertensive urgencies frequently present with headache ( 22% ) , epistaxis ( 17% ) , and psychomotor agitation ( 10% ) and hypertensive emergencies frequently present with chest pain ( 27% ) , dyspnea ( 22% ) , and neurological deficit ( 21% ) . the reason for uncontrolled hypertension in pakistan is high due to lack of awareness , knowledge , adherence , and attitudes of pakistani patients with hypertension . however there is no data on patients with hypertensive crisis from tertiary care hospitals in pakistan . additionally the number of patients who complicate towards stroke , myocardial infarction , and chronic kidney disease is also not known . hence , it is essential to have figures on prevalence and clinical presentation from our setup . therefore , we conducted this study to determine the prevalence of hypertensive crisis , its management , and its outcome in patients presenting to a tertiary care center in karachi . this was a retrospective study conducted at the aga khan university , karachi , pakistan . the aga khan university hospital ( akuh ) has 563 beds in operation and provides services to over 50,000 hospitalized patients and to over 600,000 outpatients annually . ethical approval from the ethics review committee of the aga khan university ( 1985-med - erc-11 ) was taken for conduct of the study . adult inpatients ( > 18 yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included . controlled blood pressure was defined as systolic blood pressure ( sbp ) < 140 mm hg or diastolic blood pressure ( dbp ) < 90 mm hg . uncontrolled hypertension was defined as sbp > 140 mm hg and dbp > 90 mm hg in both diabetic and nondiabetic patients who were either aware of their problem or under pharmacological treatment . the sample was drawn using computerized medical record system international classification of diseases-9-coordination and maintenance ( icd-9-cm ) at health information management system in the hospital . patients admitted with primary diagnosis of hypertension crisis , uncontrolled hypertension , hypertensive emergency , and hypertensive urgency were selected through the icd-9-cm ( i-10 : essential ( primary ) hypertension , i-11 : hypertensive heart disease , i-12 : hypertensive renal disease , i-13 : hypertensive heart and renal disease , and i-15 : secondary hypertension ) . patients whose medical records did not contain minimal clinical information to allow case classification ( hypertensive urgency or emergency ) were excluded from the study . data over a period of 5 years , from year 2005 till year 2010 , was used . a sample of 1336 consecutive patients fulfilling the inclusion criterion was selected . all patients gave a general consent on admission ; however informed consent was not taken as data was extracted later through icd-9-cm . data on demographics , comorbid conditions , clinical symptoms , blood pressure readings at subsequent time intervals , length of stay , and antihypertensive drug therapy was recorded by trained data collectors . a history of physician - diagnosed diabetes mellitus ( dm ) , chronic kidney disease ( ckd ) , ischemic heart disease ( ihd ) , and stroke was noted from the patient 's medical record file . dm was defined as fasting plasma glucose 126 mg / dl at a prior visit . ckd was defined as rise in serum creatinine of > 1.2 mg / dl for 3 months . blood pressure readings , at different time intervals , were recorded from vital sheets for nursing services . hypertensive crisis was defined as a systolic blood pressure > 180 mm hg or a diastolic blood pressure > 120 mm hg . management of patient was assessed by recording the list of medication from the computer generated pharmacy sheet attached inside the medical record file . antihypertensive treatment was divided into two types : medication given per oral and medications given intravenously . i values where available were recorded using the patient profile viewer , an online hospital database software . mean length of stay was recorded by calculating the time interval between the patient 's admission date / time and discharge date / time from er / ward . various complications like myocardial infarction , stroke , aortic dissection , acute renal failure , and pulmonary edema developed during the hospital stay were recorded using the discharge summary notes filled by the primary consultant . myocardial infarction was diagnosed when blood levels of sensitive and specific biomarkers such as cardiac troponin or ckmb are increased in the clinical setting of acute myocardial ischemia with electrocardiographic changes . according to who definition , stroke was defined as a rapidly developing clinical sign of focal ( at times global ) disturbance of cerebral function , lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin . aortic dissection was referred as the condition when a separation has occurred in aortic wall intima as diagnosed on ct scan , causing blood flow into a new false channel composed of the inner and outer layers of the media . acute renal failure was diagnosed when the plasma urea nitrogen ( pun ) or serum creatinine did not stabilize within 72 hours . acute pulmonary edema was defined as alveolar or interstitial edema verified by chest x - ray and/or with o2 saturation < 90% on room air prior to treatment accompanied by severe respiratory distress , with crackles over the lungs and orthopnea . a minimum sample size of 237 patients was required to estimate a proportion of 19% of patients with hypertensive crises presenting to er , with bound on error of 5% and alpha of 5% . mean and standard deviation were used for qualitative variables and frequency and percentage for qualitative variables . chi - square test was used to compare categorical variables and student 's t - test was used to compare quantitative variables . a total of 73,063 hypertensive patients presented to the er between years 2005 and 2010 out of which a sample of 1336 ( 1.8% ) patients was taken for this study . the prevalence ( % ) of uncontrolled hypertension was 28.9% ( 387 ) . prevalence of hypertensive crisis overall was 16.3% ( 218 ) and among those with uncontrolled hypertension is 56.3% ( 218 ) . mean age ( sd ) of patients presenting to the er was 56.7 ( 14.7 ) and 175% ( 45.2 ) of patients were male . overall , dyslipidemia was the most common comorbidity in patients presenting with uncontrolled hypertension to the er with the prevalence of 43.2% ( 167 ) followed by diabetes mellitus , 36.9% ( 143 ) , and ischemic heart disease , 21.4% ( 83 ) , and 13.9% ( 54 ) of them were smokers . the baseline characteristics of patients overall , with hypertensive crisis and without hypertensive crisis , are given in table 1 . headache was the most common presenting symptom , 35.7% ( 138 ) , followed by dyspnea , 32.6% ( 126 ) , chest pain , 21.4% ( 83 ) , dizziness , 21.2% ( 82 ) , vomiting , 17.3% ( 67 ) , epistaxis , 5.2% ( 20 ) , and neurologic deficit , 3.6% ( 14 ) . on comparison of patients with hypertensive crisis with those with no hypertensive crisis the clinical symptoms that were statistically significant were as follows : headache was present in 42.2% ( 92 ) versus 27.2% ( 46 ) p value = 0.002 and chest pain was present in 17.4% ( 38 ) versus 26.6% ( 45 ) p value = 0.02 . the mean ( sd ) systolic blood pressure ( sbp ) recorded in patients with hypertensive crisis versus no hypertensive crisis in er was 202 ( 17.971 ) and 158 ( 13.387 ) ( p value 0.001 ) . the mean ( sd ) diastolic blood pressure in patients with hypertensive crisis versus no hypertensive crisis in er was 108 ( 17.429 ) mm hg and 87 ( 14.984 ) mm hg , respectively , ( p value 0.001 ) . the trend of blood pressure recorded in the er is shown in figures 1 and 2 . antihypertensive medications used in management of patients with hypertensive crisis . calcium channel blocker was the most widely used oral antihypertensive medication in the er , 35.4% ( 137 ) . intravenous ( iv ) nitrate was the most commonly administered iv medication in er , 22.7% ( 88 ) . the mean ( sd ) drop in sbp in patients with hypertensive crisis who received intravenous medications versus oral medications was 53.1 ( 29 ) mm hg and 43 ( 27 ) , respectively ( p value = 0.01 ) . the mean drop in dbp in patients with hypertensive crisis who received intravenous versus perioral medications was 25.8 ( 19 ) and mm hg and 17.8 ( 22 ) mm hg , respectively ( p value = 0.006 ) . comparison of blood pressure trends in patients with and without hypertensive crisis on intravenous or per oral medication is shown in table 2 . the maximum drop in systolic blood pressure and diastolic blood pressure was achieved by sodium nitroprusside : 80 ( 15 ) mm hg and 37.5 ( 7.77 ) mm hg . types of intravenous medications and drop in sbp and dbp are shown in table 3 . the total length of stay ( in days ) for patients with hypertensive crisis was 2.46 ( 0.164 ) whereas the total length of stay ( in days ) for patients without hypertensive crisis was 2.20 ( 0.158 ) . overall prevalence ( % ) of complications was 47.7% ( 104 ) in patients with hypertensive crisis . acute renal failure was the most common complication with the prevalence ( % ) of 41.3% ( 43 ) followed by myocardial infarction 28.8% ( 30 ) and pulmonary edema 18.26% ( 19 ) . stroke accounted for 6.5% ( 12 ) of complications . on comparison of patients with hypertensive crisis and patients without it , myocardial infarction occurred in 7.2% ( 15 ) versus 9% ( 15 ) , stroke occurred in 2.4% ( 5 ) versus 4.2% ( 7 ) , and acute kidney injury occurred in 11.5% ( 24 ) versus 11.4% ( 19 ) patients , respectively . on further categorization of patients with hypertensive crisis who received intravenous antihypertensive in er versus not receiving intravenous medication , there was no significant difference in the complications in both groups . we have shown in this study on patients presenting to er with hypertensive crisis that the prevalence of hypertensive crisis is high ( 56.3% ) . mean length of stay was longer in patients with hypertensive crisis and acute renal failure was the most common complication in these patients . the incidence of hypertensive crisis was 47.22% in a study conducted in bosnia and 16% in a study conducted in brazil [ 22 , 23 ] . the reasons for high prevalence of patients with hypertensive crisis in our setup are multiple . lack of knowledge about control of hypertension and poor compliance to antihypertensive medications is a major issue in pakistan . also , lack of proper health infrastructure in public sector leading to inability of the poor population to access healthcare adds onto the severity of hypertension in these patients . in previous studies [ 25 , 26 ] , the most frequent clinical sign of patients presenting to the er was headache ( 22% and 42% ) and dizziness was reported among 30% of emergency department patients . this study showed ( 36% ) headache as the most common clinical sign followed by dizziness ( 21% ) which supports the previous findings about the characteristics of hypertensive crisis patients . complications of hypertensive crisis reported in previous studies conducted in bahrain and italy were acute coronary syndrome ( 32% ) , left ventricular heart failure ( 38% ) , and stroke ( 29.3% ) and cerebral infarction ( 24% ) , pulmonary edema ( 23% ) , and hypertensive encephalopathy ( 16% ) . however acute renal failure , myocardial infarction , and pulmonary edema are the most common complications in our setup . the reason for high prevalence of acute renal failure in our setup may be the higher number of patients with hypertensive emergency . the most common intravenous medication used to control hypertensive crisis in this study was nitrates . recommendation for use of intravenous nicardipine was reported by a study conducted in 2005 whereas sodium nitroprusside , because of its direct vasodilator effect and immediate onset of action , was recommended for hypertensive emergency in a subsequent study in 2006 . we observed in this study that sodium nitroprusside was the most potent intravenous antihypertensive in dropping blood pressure . however it was not the most commonly used drug in this study . in a similar study , intravenous labetalol was reported as the most frequently used antihypertensive medication for emergency department patients presenting with hypertensive urgency . this indicates that the use of intravenous nitrate as the most common antihypertensive for management of hypertensive crisis ( apart from use in patients with cardiac cause ) is not completely in accordance with the recommendations . however in some studies , oral labetalol , beta blockers , diuretics , ace inhibitors , and calcium channel blockers have been recommended . calcium channel blockers have also been found to be the most common antihypertensives used for management of hypertensive in this population and have been found to have good control rates . calcium channel is now recommended as a first line antihypertensive in the recent nice guidelines . the reduction in systolic and diastolic blood pressure reported in our study shows a smooth decline in order to avoid risks of potential side effects of a much rapid decline . it is recommended that the initial reduction in mean arterial pressure ( map ) in case of hypertensive emergency should not be more than 20%25% below the pretreatment blood pressure or that map be reduced within the first 3060 minutes to 110115 mm hg . this can be comparable to our study which reported an overall decrease of blood pressure of about 52 mm hg in sbp from intravenous medication from the time of admission until 2 hours after admission in er . a rapid decline in blood pressure is associated with acute deterioration in renal function , ischemic , cardiac , or cerebral events , and occasional retinal artery occlusion and acute blindness . the goal is to reduce bp by 1015% over a period of 3060 minutes , with the exception of the patient that presents with aortic dissection or acute intracranial bleed . however treatment should be individualized to each patient based on the type and extent of end - organ damage , degree of bp elevation , and the specific side effects that each medication could have on a patient 's preexisting comorbidities . the strength of this study is that it is the first study from this region to report figures on prevalence of hypertensive crisis , as well as prevalence of complications as a result of hypertensive crisis among patients . moreover , this study also reports management of these patients in terms of the treatment received in the er and into the ward and their mean length of stay . however there are limitations in this study ; firstly it has limited external validity as the sample is not representative for an entire population . it represents population visiting a single - tertiary care hospital and , hence , it is not representative of the entire population of pakistan . secondly , compliance issues with the medication regimen have not been taken into consideration ; hence , the medications administered to the patients may be misjudged . thirdly , some patients may also have gotten discharged against medical advice ; hence , the optimal length of stay for these patients may not have been achieved . fourth , this retrospective study can not strongly give a cause and effect association and we have not reported the hypertensive urgencies and emergencies separately . we did not report data separately on hypertensive emergency and urgency and retinopathy could not be examined and reported in all patients . the prevalence of hypertensive crisis is high in our subjects . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in the er and ward .

objectives . hypertension , if uncontrolled , can lead to hypertensive crisis . we aim to determine the prevalence of hypertensive crisis , its management , and outcome in patients presenting to a tertiary care center in karachi . methods . this was a cross - sectional study conducted at the aga khan university , karachi , pakistan . adult inpatients ( > 18 yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included . results . out of 1336 patients , 28.6% ( 387 ) had uncontrolled hypertension . the prevalence of hypertensive crisis among uncontrolled hypertensive was 56.3% ( 218 ) . per oral calcium channel blocker ; 35.4% ( 137 ) and intravenous nitrate ; 22.7% ( 88 ) were the most commonly administered medication in the er . the mean ( sd ) drop in sbp in patients with hypertensive crisis on intravenous treatment was 53.1 ( 29 ) mm hg and on per oral treatment was 43 ( 27 ) mm hg . the maximum mean ( sd ) drop in blood pressure was seen by intravenous sodium nitroprusside ; 80 ( 51 ) mm hg in sbp . acute renal failure was the most common complication with a prevalence of 11.5% ( 24 ) . conclusion . the prevalence of hypertensive crisis is high . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in our setup .

the last few years have seen a rapid expansion in the use and availability of ablation techniques with hundreds of papers published . radiofrequency ( rf ) remains the frontrunner in terms of cost , ease of set - up , versatility and flexibility but other techniques are catching up . ablation with cryotherapy and microwave , which were previously only available at open laparotomy due to the large size of the probes , are now readily performed percutaneously , with a predictable reduction in morbidity . cryotherapy offers the opportunity to monitor the ice - ball with ultrasound ( us ) , computed tomography ( ct ) or magnetic resonance imaging ( mri ) . cell death occurs around 40c and this isotherm lies approximately 5 mm inside the ice - ball . more powerful microwave generators have been developed and active research into better impedance matching with specific tissues should result in improved performance . microwave has several theoretical advantages ; it can be designed to share a spherical geometry with most tumours and has a more predictable dose response curve . simultaneous use of multiple energy sources ( probes / electrodes ) is now available in all the different technologies . improvements in guidance include the development of real - time co - registration techniques , most commonly ct and us . five percent dextrose is instilled into the area adjacent to the ablation to displace vulnerable structures , usually bowel , away from the ablation zone . a displacement of as little as 1 cm is enough to protect the bowel . ablation is now accepted as the first line of treatment in patients with limited volume hepatocellular carcinoma ( hcc ) who are not candidates for transplantation . there is continuing debate in most other areas but the evidence is increasing for an important role in liver metastases , renal carcinoma , inoperable lung tumours and some bone tumours . nephron - sparing surgery presents a challenge to the surgeon . yet , there is increasing evidence that even moderate degrees of renal failure can significantly affect survival following cardiovascular events . this will increase the focus of all physicians on the need to preserve renal function wherever and whenever possible . one study found that 95.2% of patients had a glomerular filtration rate ( gfr ) > 60 ml / min per 1.73 m at 3 years post rfa compared with 70.7% post partial nephrectomy and only 39.9% post radical nephrectomy . therefore patients with a solitary kidney and others with borderline renal function will increasingly be treated with ablation . renal tumours up to 3.5 cm in diameter can be destroyed in situ by laser , rf or cryotherapy with virtually no damage to the surrounding normal renal tissue . multiple renal tumours are not rare and can be difficult to resect without complications but complications are rare after ablation , particularly if a percutaneous approach is used . bowel injury can be prevented by dextrose isolation but it remains necessary to maintain a distance of > 1 cm from the proximal ureter or cool the ureter via a stent as ablation can cause stricturing . several series have now been published including one retrospective comparison with partial nephrectomy which showed comparable oncologic efficacy albeit with a shorter mean follow - up in the radiofrequency group ( 30 months versus 47 months ) . an apparently promising meta - analysis was heavily skewed away from recent innovations by the inclusion of early technology , failure to analyse by tumour size and location , and the stipulation that success after a single procedure was the main endpoint . percutaneous ablative techniques are relatively easy to repeat compared with either surgery or laparoscopic cryotherapy . the question as to whether cryotherapy or radiofrequency is better is yet to be resolved and would be better addressed with mature technology and in a specific tumour cohort , e.g. tumours < 3.5 cm . this is predicted to be the single largest growth area in ablation over the next few years . good results can be achieved in small , peripheral tumours . both inoperable primary and limited numbers of metastatic tumours computed tomography fluoroscopy facilitates electrode placement as small , scirrhous lung lesions can be difficult to penetrate with a larger calibre needle . pneumothorax occurs in about 40% , a similar incidence to that seen with trucut biopsy , but only a small percentage ( 1015% ) require drainage . the likelihood of a pneumothorax increases with the length of aerated lung that is traversed by the electrode and is more common when treating multiple tumours . infection is an unusual complication and in our series only occurred in patients with underlying asthma or chronic obstructive airways disease . cavitation does not usually indicate infection but cavitating lesions are more likely to become infected . during treatment a penumbra of ground glass opacification ( ggo ) develops around the tumour representing the ablation zone and a surrounding inflammatory reaction . histological studies have shown that the zone of cell death lies 24 mm inside the outer margin of the ground glass shadowing . this has been corroborated in a clinical study , with > 6 months follow - up , where the width of ggo at treatment was correlated with the development and location of recurrence . a minimum ggo of 4.5 mm is recommended to ensure complete ablation . over time the ablation zone becomes increasingly dense and then reduces in size . at 12 months , up to 33% of successfully treated small lesions will have shrunk to a linear scar . recurrence is identified by enlargement of the ablation zone , or a change in the shape of the zone indicating enlargement in one area or the development of focal nodular enhancement . tumours < 3.0 cm can usually be ablated at a single session , larger tumours , 3.05.0 cm , may require more than one ablation or other additional therapy . multivariate analysis has shown size to be the dominant feature determining complete ablation , but contact with > 3 mm blood vessels or bronchi also increases the chance of recurrence . current indications include patients with small volume , but inoperable metastases and early primary lung cancer in medically inoperable patients . early clinical studies report 3- and 5-year survival of between 46% and 57% in patients with colorectal metastases , . combinations of radiotherapy and rfa have been used to good effect in primary lung cancer in inoperable patients . the rapture study showed that lung function recovered quickly after an initial immediate post - treatment reduction and was maintained at pre - treatment values 12 months later . one of the first accepted indications for ablation was the minimally invasive treatment of benign osteoid osteomas . malignant primary bone tumours are usually treated by chemotherapy , radiotherapy and surgery . however , if aggressive therapy is delivered at an early stage , recurrence can be very difficult to treat . treatment by rfa may be curative , but is more likely to form part of a palliative treatment regimen . ct or mr radiofrequency ablation and cryoablation have been advocated in the symptomatic palliation of bone metastases following radiotherapy . initial results suggest that ablation can produce significant reductions in pain levels and analgesic requirements . it is important to select patients with a clearly defined and understood dominant site of bone pain . some authors promote the combination of ablation and cementoplasty , others argue that cementoplasty alone would be adequate . nephron - sparing surgery presents a challenge to the surgeon . yet , there is increasing evidence that even moderate degrees of renal failure can significantly affect survival following cardiovascular events . this will increase the focus of all physicians on the need to preserve renal function wherever and whenever possible . one study found that 95.2% of patients had a glomerular filtration rate ( gfr ) > 60 ml / min per 1.73 m at 3 years post rfa compared with 70.7% post partial nephrectomy and only 39.9% post radical nephrectomy . therefore patients with a solitary kidney and others with borderline renal function will increasingly be treated with ablation . renal tumours up to 3.5 cm in diameter can be destroyed in situ by laser , rf or cryotherapy with virtually no damage to the surrounding normal renal tissue . multiple renal tumours are not rare and can be difficult to resect without complications but complications are rare after ablation , particularly if a percutaneous approach is used . bowel injury can be prevented by dextrose isolation but it remains necessary to maintain a distance of > 1 cm from the proximal ureter or cool the ureter via a stent as ablation can cause stricturing . several series have now been published including one retrospective comparison with partial nephrectomy which showed comparable oncologic efficacy albeit with a shorter mean follow - up in the radiofrequency group ( 30 months versus 47 months ) . an apparently promising meta - analysis was heavily skewed away from recent innovations by the inclusion of early technology , failure to analyse by tumour size and location , and the stipulation that success after a single procedure was the main endpoint . percutaneous ablative techniques are relatively easy to repeat compared with either surgery or laparoscopic cryotherapy . the question as to whether cryotherapy or radiofrequency is better is yet to be resolved and would be better addressed with mature technology and in a specific tumour cohort , e.g. tumours < 3.5 cm . this is predicted to be the single largest growth area in ablation over the next few years . good results can be achieved in small , peripheral tumours . both inoperable primary and limited numbers of metastatic tumours computed tomography fluoroscopy facilitates electrode placement as small , scirrhous lung lesions can be difficult to penetrate with a larger calibre needle . pneumothorax occurs in about 40% , a similar incidence to that seen with trucut biopsy , but only a small percentage ( 1015% ) require drainage . the likelihood of a pneumothorax increases with the length of aerated lung that is traversed by the electrode and is more common when treating multiple tumours . infection is an unusual complication and in our series only occurred in patients with underlying asthma or chronic obstructive airways disease . cavitation does not usually indicate infection but cavitating lesions are more likely to become infected . during treatment a penumbra of ground glass opacification ( ggo ) develops around the tumour representing the ablation zone and a surrounding inflammatory reaction . histological studies have shown that the zone of cell death lies 24 mm inside the outer margin of the ground glass shadowing . this has been corroborated in a clinical study , with > 6 months follow - up , where the width of ggo at treatment was correlated with the development and location of recurrence . a minimum ggo of 4.5 mm is recommended to ensure complete ablation . over time the ablation zone becomes increasingly dense and then reduces in size . at 12 months , up to 33% of recurrence is identified by enlargement of the ablation zone , or a change in the shape of the zone indicating enlargement in one area or the development of focal nodular enhancement . tumours < 3.0 cm can usually be ablated at a single session , larger tumours , 3.05.0 cm , may require more than one ablation or other additional therapy . multivariate analysis has shown size to be the dominant feature determining complete ablation , but contact with > 3 mm blood vessels or bronchi also increases the chance of recurrence . current indications include patients with small volume , but inoperable metastases and early primary lung cancer in medically inoperable patients . early clinical studies report 3- and 5-year survival of between 46% and 57% in patients with colorectal metastases , . combinations of radiotherapy and rfa have been used to good effect in primary lung cancer in inoperable patients . the rapture study showed that lung function recovered quickly after an initial immediate post - treatment reduction and was maintained at pre - treatment values 12 months later . one of the first accepted indications for ablation was the minimally invasive treatment of benign osteoid osteomas . malignant primary bone tumours are usually treated by chemotherapy , radiotherapy and surgery . however , if aggressive therapy is delivered at an early stage , recurrence can be very difficult to treat . treatment by rfa may be curative , but is more likely to form part of a palliative treatment regimen . ct or mr radiofrequency ablation and cryoablation have been advocated in the symptomatic palliation of bone metastases following radiotherapy . it is important to select patients with a clearly defined and understood dominant site of bone pain . some authors promote the combination of ablation and cementoplasty , others argue that cementoplasty alone would be adequate . a trial to establish the relative merits of the two techniques the last few years have seen a sharp increase in our understanding of ablation , maturation of the technology , an improvement in the safety profile , ablation efficacy and monitoring techniques and the publication of results in larger patient cohorts . ablation will soon be sufficiently established to allow trials comparing ablation with conventional therapies in specific patient groups .

abstractthe last few years have seen a rapid expansion in the use and availability of ablation techniques with hundreds of papers published . radiofrequency remains the front - runner in terms of cost , ease of set - up , versatility and flexibility but other techniques are catching up . ablation with cryotherapy and microwave , which were previously only available at open laparotomy due to the large size of the probes , are now readily performed percutaneously , with a predictable reduction in morbidity . ablation is now accepted as the first line of treatment in patients with limited volume hepatocellular carcinoma who are not candidates for transplantation . there is continuing debate in most other areas but the evidence is increasing for an important role in liver metastases , renal carcinoma , inoperable lung tumours and some bone tumours .

non - small cell lung carcinoma ( nsclc ) constitutes approximately 80% of lung cancer , with a 5-year survival of only 15% . further study on the pathogenetic mechanism of lung cancer is needed to establish novel diagnostic or treatment strategies for this lethal disease . ca is a ubiquitous cellular signal mediating various cellular activities such as proliferation , differentiation , and gene transcription . store - operated ca channels ( soccs ) and receptor - operated ca channels ( roccs ) are two important pathways regulating the basal intracellular ca concentration . both channels are thought to be formed of transient receptor potential channel ( trpcs ) family protein members ( trpc17 ) and/or ca - release - actived ca channel ( crac / orai ) family members ( orai13 ) , and are activated by stromal interacting molecules ( stim1 and stim2 ) ( lu et al . , 2008 ) . recently , emerging evidence has uncovered that abnormal expression of trpcs were related to the development of various kinds of tumors , such as renal cell carcinoma , hepatoma , prostatic carcinoma , neuroblastoma imr-32 cells , and breast cancer ( nasman et al . , 2006 , thebault et al . , 2006 , veliceasa et al . , 2007 , , 2008 , guilbert et al . , 2008 , aydar et al . , 2009 , saito et al . , 2011 ) . recently , a study identified that trpc expression correlates to lung cancer differentiation ( jiang et al . , especially , another study showed that higher levels of trpc3 expression in tumor cells are an independent predictor of a better prognosis in patients with adenocarcinoma of the lung ( ouadid - ahidouch et al . our previous study has also demonstrated that trpcs played a role in the progresses of nsclc ( zhang et al . , 2010 ) . since trpcs play an important role in the cell function including enzyme activity , emiocytosis , and cell proliferation and apoptosis ( liao et al . , 2007 , peel et al . , 2008 , we hypothesized that the polymorphisms in soccs and roccs component and regulatory genes might contribute to genetic susceptibility to lung cancer . to test this hypothesis , we conducted a two - stage case - control study with a total of 2433 lung cancer cases and 2433 cancer - free controls to evaluate the effects of gene polymorphisms in the 9 selected genes related to soccs and roccs ( trpc1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 ) . to our knowledge , this is the first study to explore the associations between a comprehensive panel of polymorphisms in genes related to soccs and roccs and lung cancer risk , and to identify subgroups that would be more likely to have higher lung cancer risk . the study design and subject recruitment have been described previously ( zhang et al . , 2013 ) . the first - stage discovery study included 1422 lung cancer cases and 1422 controls , which were genetically unrelated ethnic han chinese and were from the first affiliated hospital of guangzhou medical university ( guangzhou , guangdong , china ) as described in a previous study ( zhang et al . , 2013 ) . study was conducted on participants ( 1011 cases and 1011 controls ) derived from xianyang , central hospital ( xiangyang , hubei , china ) to verify the results from the first - stage analysis . all the total 2433 patients with histopathologically confirmed incident lung cancer were consecutively recruited from september 2009 to september 2013 . the 2433 cancer - free controls , frequency matched to patients by sex and age ( 5 years ) were randomly selected from the health examination center of the same hospital during the same time period when patients were recruited . generally , all subjects met the following criteria : ( 1 ) both cases and controls were genetically unrelated han chinese ; ( 2 ) all the lung cancer patients in the study were newly diagnosed and histopathologically confirmed ; ( 3 ) all the control subjects were no self - reported cancer history and frequency matched to the cases by sex and age ( 5 years ) . the characteristics of the cases and controls selected for this two - stage study with a total of 2433 cases and 2433 controls are summarized in table 1 . information on demographic data and environmental exposure history such as tobacco smoking were also obtained by the professional interviewers . individuals who had smoked < 100 cigarettes in the past lifetime were identified as never smokers ; otherwise , they were defined as smokers ( those smokers who stopped smoking for > 1 year were identified as former smokers ) . pack - years were calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked . this study was approved by each participating center 's institutional ethical committee and was conducted according to the principles of the declaration of helsinki . written informed consent was obtained from all subjects . after informed written consent was obtained , a ~ 5 ml venous blood sample with egta - na2 as anticoagulant was collected for each participant . the genomic dna was extracted with qiagen blood dna kit ( qiagen , valencia , ca ) . nine genes related to soccs and roccs were selected : trpc 1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 . for each of them , we selected the tagsnps by haploview 4.2 software within 10 kb upstream of the transcriptional start site or 10 kb downstream of the transcriptional stop site . the genotypes of 236 selected tagsnps and their associations with lung cancer were described in table s1 ( p < 0.05 ) and fig . s1 snp frequency and ld data were based on the international hapmap project database , release 24 , human genome build 36 . genotypes were analyzed and exported using the illumina beadstudio software . to ensure quality control , genotyping was done without knowledge of case / control status of the subjects , and the polymorphism analysis was made by two persons independently . > 15% of the samples were randomly performed for confirmation , and the results were 100% concordant . the study design and subject recruitment have been described previously ( zhang et al . , 2013 ) . the first - stage discovery study included 1422 lung cancer cases and 1422 controls , which were genetically unrelated ethnic han chinese and were from the first affiliated hospital of guangzhou medical university ( guangzhou , guangdong , china ) as described in a previous study ( zhang et al . , 2013 ) . study was conducted on participants ( 1011 cases and 1011 controls ) derived from xianyang , central hospital ( xiangyang , hubei , china ) to verify the results from the first - stage analysis . all the total 2433 patients with histopathologically confirmed incident lung cancer were consecutively recruited from september 2009 to september 2013 . the 2433 cancer - free controls , frequency matched to patients by sex and age ( 5 years ) were randomly selected from the health examination center of the same hospital during the same time period when patients were recruited . generally , all subjects met the following criteria : ( 1 ) both cases and controls were genetically unrelated han chinese ; ( 2 ) all the lung cancer patients in the study were newly diagnosed and histopathologically confirmed ; ( 3 ) all the control subjects were no self - reported cancer history and frequency matched to the cases by sex and age ( 5 years ) . the characteristics of the cases and controls selected for this two - stage study with a total of 2433 cases and 2433 controls are summarized in table 1 . information on demographic data and environmental exposure history such as tobacco smoking were also obtained by the professional interviewers . individuals who had smoked < 100 cigarettes in the past lifetime were identified as never smokers ; otherwise , they were defined as smokers ( those smokers who stopped smoking for > 1 year were identified as former smokers ) . pack - years were calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked . this study was approved by each participating center 's institutional ethical committee and was conducted according to the principles of the declaration of helsinki . written informed consent was obtained from all subjects . after informed written consent was obtained , a ~ 5 ml venous blood sample with egta - na2 as anticoagulant was collected for each participant . the genomic dna was extracted with qiagen blood dna kit ( qiagen , valencia , ca ) . nine genes related to soccs and roccs were selected : trpc 1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 . for each of them , we selected the tagsnps by haploview 4.2 software within 10 kb upstream of the transcriptional start site or 10 kb downstream of the transcriptional stop site . the genotypes of 236 selected tagsnps and their associations with lung cancer were described in table s1 ( p < 0.05 ) and fig . s1 snp frequency and ld data were based on the international hapmap project database , release 24 , human genome build 36 . genotypes were analyzed and exported using the illumina beadstudio software . to ensure quality control , genotyping was done without knowledge of case / control status of the subjects , and the polymorphism analysis was made by two persons independently . > 15% of the samples were randomly performed for confirmation , and the results were 100% concordant . test was used to evaluate differences in the distributions of demographic characteristics , selected variables , and genotypes of the variants between the cases and controls . goodness of fit to the hardy - weinberg equilibrium ( hwe ) expectation in control was also evaluated by the -test for each snp . akaike 's information criteria ( aic ) ( akaike , h. , ieee trans . automat ac-19 , 716 - 723 ( 1974 ) ) were applied to select the most parsimonious genetic model for each snp . odds ratios ( ors ) and its corresponding 95% confidence intervals ( cis ) were measured by an unconditional logistic regression model with adjustment for age , sex , and pack - year of smoking . stratification analyses were also done by variables of interest , such as age , sex , smoking status , pack year , and sex - smoking . the pairwise ld among the snps was calculated using lewontin 's standardized coefficient d and ld coefficient r ( lewontin , 1988 ) and haplotype blocks were defined by the method of gabriel et al . ( gabriel et al . , 2002 ) in the publicly available haploview software with default settings ( http://www.broad.mit.edu/personal/jcbarret/haplo/ ) . in addition , phase 2.1 bayesian algorithm was used to validate the haplotype frequencies estimated by haplo.stats ( stephens and donnelly , 2003 ) ( http://mayoresearch.mayo.edu/mayo/research/schaid_lab/index.cfm ) . statistic power ( gauderman , 2002 ) was done by quanto 1.2 ( http://hydra.usc.edu/gxe ) . p = 0.05 was the criterion of statistical significance and all statistical tests were two - sided . as shown in table 1 , concordant results were observed in both discovery and replication populations with significant differences identified in the smoking status , pack years , and sex - smoking ( p < 0.001 for all ) and no significant deviation in distributions of age and sex between case and control groups ( p > 0.05 for all ) . the frequency distributions of smoking status were not homogeneous ( breslow - day test p = 5.8 10 ) , reflecting different lifestyle between the discovery and replication populations . we further combined the two populations into stratification analysis and cumulative effect analysis in order to increase the study power . in addition , these variables were further adjusted by age , sex and pack - year of smoking in the multivariate logistic regression model to control possible confounding on the main effects of the studied polymorphisms . we selected 236 tagsnps from nine genes related to soccs and roccs : trpc1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 . p < 0.05 in any of the discovery , replication , and combined populations . among these tagsnps , we found consistently significant associations of trpc4 rs9547991 and rs978156 and trpc7 rs11748198 with lung cancer risk among the above three kinds of groups ( p < 0.05 for all ) . all observed genotype distributions among these groups agreed with the hwe ( p 0.05 for all ) . when combined discovery and replication populations , the significances were more significant than any of them in additive model . in table s2 , the results of the first - stage study revealed that trpc4 rs9547991 and rs978156 variant genotypes significantly increased the lung cancer risk in additive model ( adjusted or = 1.29 , 95%ci = 1.031.62 ; adjusted or = 1.21 , 95%ci = 1.051.40 , respectively ) . trpc7 rs11748198 significantly increased the lung cancer risk in additive model ( rs11748198 : adjusted or = 1.26 , 95%ci = 1.041.53 ) . in the second - stage study , the associations of trpc4 rs9547991 and rs978156 , and trpc7 rs11748198 with lung cancer risk were validated with ors of 1.38 and 1.21 , and 1.29 , respectively . of course , the associations remained significant after all subjects were combined ( rs9547991 : adjusted or = 1.33 , 95%ci = 1.111.59 ; rs978156 : adjusted or = 1.21 , 95%ci = 1.081.35 ; rs11748198 : adjusted or = 1.28 , 95%ci = 1.101.47 ) . then , further detailed analysis was taken about the relationship between these three snps and the three groups by pooling all of the discovery and validation stage in additive models . we achieved significant associations for trpc4 rs9547991 and rs978156 ( pcombined = 1.6 10 , or = 1.33 at 5q31.1 ; pcombined = 6.6 10 , or = 1.21 13q13.3 , respectively ) , and trpc7 rs11748198 ( pcombined = 1.3 10 , or = 1.28 13q13.3 ) ( table 2 ) . furthermore , we evaluated combined effects of the three snps variants ( trpc4 rs9547991 a > g and rs978156 c > t , trpc7 rs11748198 g > t ) on lung cancer risks . as shown in table 3 , lung cancer risk was significantly increased with the increasing number of variant alleles of the three snps in a dose - dependent manner ( p for trend = 7.2 10 ) . compared with those carrying 0 variant allele , subjects carrying 1 variant alleles had a 1.29-fold increased risk of lung cancer ( 95%ci = 1.151.46 ) . for further study , the relationships between combined variant alleles and environmental characteristics in combined population were also taken . 16 variant alleles had a more significantly increased lung risks than 0 variant alleles in younger people ( age 60 , p = 1.9 10 ) ; in both sex ( p = 1.5 10 for male ; p = 5.0 10 for female ) ; in current ( p = 1.1 10 ) and never smokers ( p = 12.0 10 ) ; in both 25 pack year and 0 pack year ( p = 2.9 10 and p = 12.0 10 , respectively ) ; in both male smokers and female non - smoker ( both p > 0.7 10 ) as well as in discovery population . we also found that there were significant multiplicative interactions between pack year , sex - smoking , source of population and allele genes with pmax 0.0328 ( table 4 ) . we further assessed the associations of snps rs9547991 , rs978156 and rs11748198 variant genotypes with lung cancer risk stratified by selected variables . s2 and table s3 , compared with the common wild - type homozygous genotype , the adverse effects of rs9547991 and rs978156 were more evident in male ( rs9547991 : adjusted or = 1.53 and 95% ci = 1.231.92 ; rs978156 : adjusted or = 1.21 and 95% ci = 1.041.41 ) ; in former smokers ( rs9547991 : adjusted or = 2.77 and 95% ci = 1.335.57 ; rs978156 : adjusted or = 1.63 and 95% ci = 1.072.49 ) ; in male smokers ( rs9547991 : adjusted or = 1.68 and 95% ci = 1.292.17 ; rs978156 : adjusted or = 1.24 and 95% ci = 1.041.498 ) than the rest of all . similar association strengths were observed among younger subjects ( age 60 ) compared with that in older subjects ( age > 60 ) between all subgroups for the three snps ( rs9547991 : adjusted or = 1.61 and 95% ci = 1.232.09 ; rs978156 : adjusted or = 1.38 and 95% ci = 1.551.66 ; rs11748198 : adjusted or = 1.38 and 95% ci = 1.111.73 ) . interestingly , stronger effects of rs11748198 were shown among female ( adjusted or = 1.67 , 95% ci = 1.252.24 ) and never smokers ( adjusted or = 1.40 , 95% ci = 1.111.76 ) than the rest of all . we also performed haplotype analysis in the combined population to assess the effect of the haplotype containing rs9547991 and rs978156 variant alleles on lung cancer risks ( table s4 ) . when compared with the most frequent ac haplotype , the haplotype carrying any of the variant allele all showed significant risk effects ( adjusted or > 1 ) , which were consistent with that in the analysis of single snp . then the combined genotypes risk was also evaluated . we found that there was a significantly increased risk of lung cancer as the risk genotype number increased compared with those with 0 variants , implying that these variants might have a joint effect on the risk of lung cancer ( p for trend = 9.6 10 ) . ld information of these two snps ( trpc4 rs9547991 and rs978156 ) , calculated from genotyping data of 2433 controls of the combined study , was d = 0.91 and r = 0.04 . as shown in table 1 , concordant results were observed in both discovery and replication populations with significant differences identified in the smoking status , pack years , and sex - smoking ( p < 0.001 for all ) and no significant deviation in distributions of age and sex between case and control groups ( p > 0.05 for all ) . the frequency distributions of smoking status were not homogeneous ( breslow - day test p = 5.8 10 ) , reflecting different lifestyle between the discovery and replication populations . we further combined the two populations into stratification analysis and cumulative effect analysis in order to increase the study power . in addition , these variables were further adjusted by age , sex and pack - year of smoking in the multivariate logistic regression model to control possible confounding on the main effects of the studied polymorphisms . we selected 236 tagsnps from nine genes related to soccs and roccs : trpc1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 . p < 0.05 in any of the discovery , replication , and combined populations . among these tagsnps , we found consistently significant associations of trpc4 rs9547991 and rs978156 and trpc7 rs11748198 with lung cancer risk among the above three kinds of groups ( p < 0.05 for all ) . all observed genotype distributions among these groups agreed with the hwe ( p 0.05 for all ) . when combined discovery and replication populations , the significances were more significant than any of them in additive model . in table s2 , the results of the first - stage study revealed that trpc4 rs9547991 and rs978156 variant genotypes significantly increased the lung cancer risk in additive model ( adjusted or = 1.29 , 95%ci = 1.031.62 ; adjusted or = 1.21 , 95%ci = 1.051.40 , respectively ) . trpc7 rs11748198 significantly increased the lung cancer risk in additive model ( rs11748198 : adjusted or = 1.26 , 95%ci = 1.041.53 ) . in the second - stage study , the associations of trpc4 rs9547991 and rs978156 , and trpc7 rs11748198 with lung cancer risk were validated with ors of 1.38 and 1.21 , and 1.29 , respectively . of course , the associations remained significant after all subjects were combined ( rs9547991 : adjusted or = 1.33 , 95%ci = 1.111.59 ; rs978156 : adjusted or = 1.21 , 95%ci = 1.081.35 ; rs11748198 : adjusted or = 1.28 , 95%ci = 1.101.47 ) . then , further detailed analysis was taken about the relationship between these three snps and the three groups by pooling all of the discovery and validation stage in additive models . we achieved significant associations for trpc4 rs9547991 and rs978156 ( pcombined = 1.6 10 , or = 1.33 at 5q31.1 ; pcombined = 6.6 10 , or = 1.21 13q13.3 , respectively ) , and trpc7 rs11748198 ( pcombined = 1.3 10 , or = 1.28 13q13.3 ) ( table 2 ) . the genotype distributions of the three significant snps were also in table 2 . furthermore , we evaluated combined effects of the three snps variants ( trpc4 rs9547991 a > g and rs978156 c > t , trpc7 rs11748198 g > t ) on lung cancer risks . as shown in table 3 , lung cancer risk was significantly increased with the increasing number of variant alleles of the three snps in a dose - dependent manner ( p for trend = 7.2 10 ) . compared with those carrying 0 variant allele , subjects carrying 1 variant alleles had a 1.29-fold increased risk of lung cancer ( 95%ci = 1.151.46 ) . for further study , the relationships between combined variant alleles and environmental characteristics in combined population were also taken . 16 variant alleles had a more significantly increased lung risks than 0 variant alleles in younger people ( age 60 , p = 1.9 10 ) ; in both sex ( p = 1.5 10 for male ; p = 5.0 10 for female ) ; in current ( p = 1.1 10 ) and never smokers ( p = 12.0 10 ) ; in both 25 pack year and 0 pack year ( p = 2.9 10 and p = 12.0 10 , respectively ) ; in both male smokers and female non - smoker ( both p > 0.7 10 ) as well as in discovery population . we also found that there were significant multiplicative interactions between pack year , sex - smoking , source of population and allele genes with pmax 0.0328 ( table 4 ) . we further assessed the associations of snps rs9547991 , rs978156 and rs11748198 variant genotypes with lung cancer risk stratified by selected variables . s2 and table s3 , compared with the common wild - type homozygous genotype , the adverse effects of rs9547991 and rs978156 were more evident in male ( rs9547991 : adjusted or = 1.53 and 95% ci = 1.231.92 ; rs978156 : adjusted or = 1.21 and 95% ci = 1.041.41 ) ; in former smokers ( rs9547991 : adjusted or = 2.77 and 95% ci = 1.335.57 ; rs978156 : adjusted or = 1.63 and 95% ci = 1.072.49 ) ; in male smokers ( rs9547991 : adjusted or = 1.68 and 95% ci = 1.292.17 ; rs978156 : adjusted or = 1.24 and 95% ci = 1.041.498 ) than the rest of all . similar association strengths were observed among younger subjects ( age 60 ) compared with that in older subjects ( age > 60 ) between all subgroups for the three snps ( rs9547991 : adjusted or = 1.61 and 95% ci = 1.232.09 ; rs978156 : adjusted or = 1.38 and 95% ci = 1.551.66 ; rs11748198 : adjusted or = 1.38 and 95% ci = 1.111.73 ) . interestingly , stronger effects of rs11748198 were shown among female ( adjusted or = 1.67 , 95% ci = 1.252.24 ) and never smokers ( adjusted or = 1.40 , 95% ci = 1.111.76 ) than the rest of all . we also performed haplotype analysis in the combined population to assess the effect of the haplotype containing rs9547991 and rs978156 variant alleles on lung cancer risks ( table s4 ) . when compared with the most frequent ac haplotype , the haplotype carrying any of the variant allele all showed significant risk effects ( adjusted or > 1 ) , which were consistent with that in the analysis of single snp . then the combined genotypes risk was also evaluated . we found that there was a significantly increased risk of lung cancer as the risk genotype number increased compared with those with 0 variants , implying that these variants might have a joint effect on the risk of lung cancer ( p for trend = 9.6 10 ) . ld information of these two snps ( trpc4 rs9547991 and rs978156 ) , calculated from genotyping data of 2433 controls of the combined study , was d = 0.91 and r = 0.04 . lung cancer remains to be a challenging disease due to a 5-year survival of only 15% and the accompanied high medical costs . using genetic markers for determining risk may help to identify high risk population for early screening , diagnosis , and therapy , which may also improve clinical outcome . this is the first study to explore the associations between a comprehensive panel of polymorphisms of trpcs genes and lung cancer risks and to explore subgroups that would more likely have higher cancer risks . we evaluated 236 tagsnps in the 9 selected genes from the socc and the rocc pathways for their associations with lung cancer risks . in this two - stage case - control study with a total of 2433 lung cancer cases and 2433 controls , we found that rs9547991 and rs978156 in trpc4 and rs11748198 in trpc7 were potentially susceptibility markers of lung cancer in chinese population . especially , our study provides the epidemiological evidence supporting a connection between comprehensive trpcs snps and lung cancer risks . the mammalian trpcs channels are encoded by at least 28 genes ( bodding , 2007 ) . most of these proteins have a putative topology of six transmembrane domains with a pore loop between the fifth and sixth segments . evidences of genetic linkage to diseases and studies from numerous independent laboratories have strongly suggested that trpcs channels have substantial importance in mammalian biology and might be valuable therapeutic drug targets . members of trpcs have been found to be implicated in abnormal proliferation , differentiation , and cancer formation ( bodding , 2007 ) . for example , trpc6 mutations cause familial focal segmental glomerulosclerosis ( winn et al . , 2005 ) , and another study has been linked a snp in trpc6 to idiopathic pulmonary hypertension ( yu et al . , 2009 ) . gain - of - function mutation in trpc4 protects against myocardial infarction ( mi ) in diabetes ( jung et al . , 2011 ) . in the present study , two of the three significant snps were in trpc4 , which is known to have important functions contributing to lung cancer risk . trpc4 is widely expressed in the vasculature ( yip et al . , 2004 ) , where it participates in the generation of intracellular ca signals that regulate functions such as endothelial permeability ( tiruppathi et al . , 2002 ) and , the best - characterized physiological role for trpc4 is in the regulation of endothelial cell function . trpc4 has been demonstrated that is a required component of socc channels in vascular endothelial cells and that trpc4 is part of the ca entry signal transduction channel regulating vascular tone ( abramowitz and birnbaumer , 2009 ) . similarly , vascular endothelial cells derived from trpc4 knock - out ( trpc4 / ) mice showed impaired store - operated ca entry channels ( soce ) ( freichel et al . , 2001 ) . therefore , studies showed that trpc4-dependent ca entry is a key determinant of increased permeability in the mouse pulmonary vasculature ( tiruppathi et al . , 2002 ) . in a study of the properties of lung endothelial cells derived from the same trpc4 mice , tiruppathi et al . ( tiruppathi et al . , 2002 ) expanded the observations of freichel et al . ( freichel et al . , 2001 ) , and identified that absence of trpc4 was associated with a loss of endothelial cell responses to thrombin , suggesting a critical involvement of trpc4 in microvascular permeability . trpc4 antisense oligonucleotides were shown to partially inhibit soce in mouse mesangial cells , implying that trpc4 might also form part of endogenous soccs in that kind cells ( wang et al . furthermore , trpc4 appears to be involved in mediating some aspects of hypoxia - induced gene expression and cell proliferation . culture of human pulmonary artery endothelial cells under hypoxic conditions results in increased trpc4 expression of mrna and protein , enhanced soce ( fantozzi et al . , 2003 ) . in addition , haplotype analysis was also evaluated to further explore effects of haplotypes and combined genotypes of trpc4 on lung cancer risks , because haplotype - based analysis might be more informative than single snp analysis and resequencing dna samples carrying the high - risk haplotypes might be able to improve risk assessment . especially , the two most significant trpc4 snps we identified are all located in the intron region , which may contribute to alterations in gene expression or splicing . alternatively , it is also possible that these snps are linked to other causal variants in trpc4 . in our previous study ( zhang et al . , 2010 ) , although studies have demonstrated that it is expressed in the heart , lung , and eye in mice ( okada et al . , 1999 ) . the main reason was that the relatively small sample size we chose might be difficult to detect the probably low expression of trpc7 . in the present study , one of the three most significant snps was in trpc7 . unlike trpc4 , trpc7 have been detected less frequently and also have not been studied extensively . trpc7 , the final member of trpc family , demonstrates properties very similar to trpc3 and trpc6 with regard to its voltage - current relationship ( trpc7 is most closely related to trpc3 with 81% identity , and demonstrates 75% identity with trpc6 in mice ) , and activated by diacylglycerol ( dag ) ( beck et al . , 2006 ) . the differences between the three channel types may lie in their ion selectivity , in which trpc6 is reported to be somewhat ca - selective , while trpc3 and trpc7 do not appear to be . trpc7 has demonstrable sensitivity to skf96365 ( a novel inhibitor of receptor - mediated ca entry ) , and is relatively insensitive to lanthanides . up to now , the component(s ) required for coupling of the trpc7 to store depletion is unknown . it is unlikely that trpc7 alone is responsible for specific biological function among trpcs , since trpc7 is co - expressed with other trpcs in most of the tissues . it is possible that trpc7 interacts with inositol triphosphate ( ip3 ) receptors to suppress their activity . alternatively , trpc7 may be also localized in the endoplasmic reticulum ( er ) membrane and contribute to passive ca release from stores . it has been suggested that trpc7 plays key roles in the ca signaling pathway because of its unique activation properties such as constitutive activity ( okada et al . , 1999 ) . a study revealed that trpc7 mediated angiotensin ii - induced myocardial apoptosis ( satoh et al . , 2007 ) . however , another report showed that trpc3 and trpc6 , but not trpc7 , were essential for angiotensin ii - induced cardiac hypertrophy . but trpc7 can display some functions via association with other proteins , such as trpc6 , which positively regulates calcineurin - nfat ( nuclear factor of activated t cells ) signaling , and was related with cardiac hypertrophy ( nishida et al . , 2010 ) . trpc7 may also form heteromeric channels with trpc6 , and be involved in cardiac failure . in this study , it is possible that the variant allele of trpc7 rs11748198 may affect gene transcription thus altering protein level . alternatively overall , our study suggested the association of trpc4 and trpc7 polymorphisms with lung cancer risks . we applied a gene sets - based approach to comprehensively evaluate the effect of the three significant snps on the risk of lung cancer . when combined the effects of the three significant snps , subjects carrying 1 variant alleles had a higher increased risk of lung cancer compared with those carrying 0 variant allele . lung cancer risks significantly increased with the increasing number of variant alleles of the three snps in a dose - dependent manner . those with 2 risk genotypes had the highest risk of lung cancer , suggesting combined variations were detrimental and had a larger effect than any single variant . this emphasizes the importance of including multiple snps within a shared pathway for examining joint effects in the risk assessment . despite the strengths and biologic plausibility of the associations observed in the present study , inherited biases in our study may have led to spurious findings . firstly , further fine mapping and functional assays are necessary to reveal potential molecular mechanisms . however , subjects in the two - stage study covered the population of southeastern and northern chinese , which made the population representativeness more stable and reasonable . in addition , it would be interesting to exam these snps in minority populations . finally , although our data are largely internally validated , future replication studies in independent populations are needed to validate some of the results . our study provided evidence indicating that rs9547991 and rs978156 in trpc4 and rs11748198 in trpc7 were potentially susceptibility markers of lung cancer in chinese population . s2 supplementary tables . locations and ld structure of the 236 selected tagsnps in trpcs pathway . this work was supported by guangdong natural science foundation team grant ( 1035101200300000 ) , the national natural science foundation of china ( 81170052 , 81071917 , 81173112 , 81520108001 , and 81220108001 ) , the guangdong province universities and colleges pearl river scholar funded scheme to w. lu ( 2014 ) and the key project of department of education of guangdong province ( cxzd1142 ) , a guangdong department of education research grant ( cxzd1025 ) , guangzhou department of education yangcheng scholarships ( 12a001s ) , guangzhou department of education team grant for innovation ( 13c08 ) , guangdong natural science foundation ( 1614050002587 ) and guangzhou municipal university research projects ( 1201430298 ) . the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript .

objectivestore operated calcium channels ( soccs ) and receptor - operated calcium channels ( roccs ) are important pathways participating in regulation of intracellular ca2 + concentration in various cell types . the purpose of our study is to determine whether genetic variations in key components of soccs and roccs are associated with lung cancer risk.methodswe identified 236 tagsnps in 9 key genes related to soccs and roccs ( trpc1 , trpc3 , trpc4 , trpc6 , trpc7 , orai1 , orai2 , stim1 , and stim2 ) and evaluated their association with lung cancer risk in a two - stage case - control study with a total of 2433 lung cancer cases and 2433 cancer - free controls using illumina high throughput genotyping platform.resultswe found consistently significant associations of trpc4 rs9547991 and rs978156 , and trpc7 rs11748198 with increased risk of lung cancer among the three kinds of sources of populations ( additive model in combined population : adjusted or = 1.33 , 95% ci = 1.111.59 for rs9547991 ; adjusted or = 1.21 , 95% ci = 1.081.35 for rs978156 ; and adjusted or = 1.28 , 95% ci = 1.101.47 for rs11748198 ) . when combining the effects of trpc7 rs11748198 , and trpc4 rs9547991 and rs978156 , subjects carrying 1 variant alleles had a 1.29-fold increased risk of lung cancer ( 95% ci = 1.151.46 ) , compared with those carrying 0 variant allele . lung cancer risk significantly increased with the increasing number of variant alleles of the three snps in a dose - dependent manner ( p for trend = 7.2 10 7).conclusionthese findings suggested that trpc4 rs9547991 and rs978156 , and trpc7 rs11748198 were candidate susceptibility markers for lung cancer in chinese population . our study provides the epidemiological evidence supporting a connection between trpc members and lung cancer risks .

samples of stem , root , and leafy branches preserved in 70% ethanol were collected from a population near saipipi , savaii , samoa . a healer preparation from falealupo prostratin ( 1 ) was detected in each sample , and these materials were used for method development and validation . another collecting expedition was made to the island of savaii , samoa , in january 2005 . thirty - six samples , consisting of vouchers and ethanol - preserved pharmaceutical grade collections , were taken from three natural populations near the villages of saipipi , tafua , and falealupo . six trees were sampled from each population , and gps coordinates , elevation , dbh , height , petiole color , and surrounding vegetation were recorded for each tree sampled . from three plants at each site three different morphological samples were collected : roots ( bark + wood ) , stem ( bark + wood ) , and leafy branches , with fruit and/or flowers if present . only stem samples were collected from the other three trees at each site for a total of 12 samples per site . nine stem samples were collected on additional expeditions to the island of tutuila in american samoa in may and november of 2005 for a total of 45 samples including 27 stemwood samples . samples were shipped in vacuum - sealed aluminum vessels in 70% ethanol ( except for the tutuila island collections , which were shipped in 70% 2-propanol ) . the alcohol fractions were separated and the plant material air - dried in a fume hood . vouchers were deposited at the herbarium of the california state university fullerton s department of biological science . plant material was finely chopped then pulverized with a coffee grinder , and approximately 1 g of dry sample tissue was suspended in 25 ml of acetone in a 55 c bath for 10 min . 4 disk , dried in a savant aes1000 speedvac , suspended in 500 l of 80% ethanol in hplc grade water , and then filtered with a 0.22 m millipore ultrafree mc centrifugal filter device . nontarget organics were removed from the alcohol fraction with a waters sep - pak c18 cartridge using reversed - phase elution ( conditioning : sequential washes of 5 ml of 52% acetonitrile , 5 ml of 26% acetonitrile , and 2.5 ml of 100% hplc water ; loading : 100 l of sample ; separation : 1 ml of hplc water , 2.5 ml of 26% acetonitrile , and 2.5 ml of 40% acetonitrile ) ; the last fraction was filtered with a 0.22 m ultrafree mc centrifugal filter , dried , and suspended in 40 l of 80% ethanol for injection . the ethanol or 2-propanol fractions and the acetone extract for each of 45 samples were analyzed by hplc in triplicate to measure prostratin ( 1 ) concentration in g per g. prostratin was separated by reversed - phase elution using a waters nova - pak c18 column , 4 m bead , 300 3.9 mm , on a gradient hplc system ( waters 717 automated injector , waters 1525 binary solvent delivery system , and empower data analysis system ) at 30 c . identification was using a waters 2487 dual - wavelength uv absorbance detector using an authenticated standard ( icn mp biomedicals ) at 254 nm . aliquots of 10 l of each sample were injected , eluted over 15 min with a linear gradient mixing from 32% to 40% acetonitrile using filtered and degassed hplc grade water ( fisher ) and hplc grade acetonitrile ( sigma chromasolv ) , with a flow of 1.0 ml / min . method validation was completed in compliance with the specifications in the united states pharmacopoeia ( usp ) , general chapter 1225,(33 ) and meyer.(34 ) ruggedness was evaluated by calculating the precision of biweekly triplicate injections at one concentration during the entire range of the study ( rsd 5.21% ) . linearity was evaluated by plotting peak area as a function of analyte concentration , and regression analysis was performed : slope = 207.78 ; intercept = 1468.70 ; correlation coefficient = 0.9997 ; residual sum of squares = 716 109 646.17 . the limit of detection ( lod ) and limit of quantification ( loq ) were determined as 2.5 and 25 pmol , respectively , with a range to 30 nmol . accuracy ( recovery = 96% ) was calculated by spiking blank matrix with known amounts of 1 in triplicate at five concentrations . repeatability was assessed with triplicate injections at five concentrations on two consecutive days ( rsd 1.81% ) . intermediate precision was calculated biweekly over the range of the study with triplicate injections at three concentrations ( rsd 5.87% ) . the observed concentrations of 1 were ranked , and 95% confidence limits for the distribution of these concentrations around the median were constructed using eq 8.2.2 in snedecor and cochran.(35 ) to determine if the median prostratin ( 1 ) concentrations of all populations were equal , statistical hypotheses were tested using a kruksalwallis h test , the nonparametric analogue of anova . the resultant h statistic was tested for statistical significance at the p < 0.05 level using standard tables . a chi - square test for independence was employed to ascertain if the occurrence of exceptionally high concentration plants was equal between populations and also to determine if the falealupo population had a greater than expected number of exceptionally high - yielding prostratin plants . we used a similar chi - square procedure to test the tafua and tutuila populations to see if they had a higher than expected number of exceptionally low - yielding 1 plants . spearman s rank correlation coefficient was calculated to assess the correlation between prostratin concentration and diameter at breast height ( dbh ) . a kruskalwallis h test was employed to establish if 1 was equally distributed throughout plant parts ( leafy branches , stem , and root ) ; to determine if plants with high stem concentrations also have high concentrations in the leaf or roots , we calculated a spearman s rank correlation coefficient and tested for significant correlation at the p < 0.05 level .

homalanthus nutans , used by samoan healers to treat hepatitis , produces the antiviral compound 12-deoxyphorbol 13-acetate , prostratin ( 1 ) . prostratin is being developed as an adjuvant therapy to clear latent viral reservoirs , the major obstacle to eradication of hiv - aids within the human body . a validated reversed - phase hplc method was developed to assay concentrations of 1 in h. nutans . a survey of four distinct populations on two different samoan islands revealed significant variability in content . the stem tissue ( range 0.252.6 g / g 1 ) , used by healers in indigenous therapies , gave a higher median concentration of prostratin ( 3.5 g / g ) than root or leaf tissues ( 2.9 and 2.5 g / g , respectively ) . the high variability and skewness of these data indicate that cultivar selection for drug production will be important for this species . the reversed - phase hplc assay will allow plants to be selected for agricultural development and genetic analysis by identifying those individuals above and below a 95% confidence interval for the median concentration .

breast cancer , the most common cancer in women worldwide , accounted for 1.7 million new cases in 2012 , comprising a quarter of all new cancer cases . while traditional risk factors for breast cancer include age , family history of cancer , and reproductive and menstrual history , the national cancer institute also recognizes overweight , lack of physical activity , and consumption of alcohol as risk factors . metabolic syndrome ( ms ) is a cluster of pathophysiological disorders comprising central obesity , insulin resistance , high blood pressure , and dyslipidemia . reaven 's definition of ms in 1988 was followed by definitions from the world health organization , national cholesterol education program 's adult treatment panel iii ( ncep atp iii ) , american heart association / national heart , lung , and blood institute , and the international diabetes federation . these criteria include the presence of three or more of the following : abdominal obesity ( waist circumference 35 inches in women ) , triglycerides 150 mg / dl , high density lipoprotein cholesterol ( hdl - c ) < 50 mg / dl , blood pressure ( bp ) 130/85 mmhg , and fasting glucose 110 mg / dl . ms is estimated to be prevalent in at least a quarter of the adults in the americas , in europe , and in india . ms has been identified as a risk factor for several cancers , particularly breast , pancreatic , colorectal , and prostate cancers [ 1015 ] . individual components of ms , for example , abdominal obesity , high blood glucose , high bp , high triglycerides , and low hdl , are positively associated with the development of certain cancers , most notably breast cancer [ 1627 ] . while studies show a positive association of breast cancer with diabetes [ 19 , 2833 ] and obesity [ 16 , 34 , 35 ] , others show a negative association with obesity in premenopausal women [ 3638 ] . mixed results also characterize hypertension [ 22 , 23 , 39 , 40 ] and dyslipidemia [ 22 , 41 , 42 ] as risk factors for breast cancer . in addition , although individual components of ms may not be strongly associated with the development of breast cancer , their combination may elevate the risk [ 13 , 14 , 4356 ] . for example , ms may activate different molecular pathways through endocrine , metabolic , and immune cell changes , which in turn influence breast tumorigenesis . such pathways that enhance breast cancer cell proliferation and inhibit apoptosis include ( 1 ) increased levels of circulating estrogen , for example , estradiol [ 52 , 54 , 57 ] , ( 2 ) higher levels of insulin [ 58 , 59 ] , ( 3 ) decreased level of circulating adiponectin , ( 4 ) increased plasma leptin concentration , and ( 5 ) increased production of proinflammatory cytokines , such as interleukin-6 and tumor necrosis factor alpha . previous epidemiologic studies on ms and breast cancer risk show contrary results . for example , only four [ 13 , 14 , 43 , 51 ] of eight studies [ 13 , 14 , 43 , 48 , 51 , 6264 ] reported a statistically significant association between ms and risk of breast cancer . this might invite a conclusion that the association between ms and breast cancer risk is unknown . however , such an inference would be based on the vote - counting approach , an approach that ignores the magnitude of the association . a recent systematic review and meta - analysis of ms and postmenopausal breast cancer found that ms was moderately associated with the risk of postmenopausal breast cancer . however , to the best of our knowledge , no meta - analytic research has addressed the conflicting results from individual studies of ms and breast cancer risk in all adult women . therefore , the purpose of this study was to use the aggregate data meta - analytic approach to examine the association between ms and breast cancer risk in women . the a priori inclusion criteria for this study were as follows : ( 1 ) observational studies using cohort ( both prospective and retrospective ) , case - control , or nested case - control study designs ; ( 2 ) studies examining the association between ms ( presence of a cluster of three or more metabolic abnormalities ) and breast cancer incidence , as defined by the authors ; ( 3 ) studies with adult females 18 years of age as participants ; ( 4 ) english - language studies published as journal articles , doctoral dissertations , or masters ' theses ; ( 5 ) published and indexed studies up to june 30 , 2012 ; and ( 6 ) studies reporting sufficient data ( e.g. , rate ratios , risk ratios , odds ratios , standardized incidence ratios , hazard ratios , or frequencies ) for calculating a common effect size . neither lobular carcinoma in situ nor ductal carcinoma in situ breast cancer cases were excluded from the study . studies not meeting all inclusion criteria were excluded from this review . excluded studies were those that ( 1 ) were not published as full reports , such as conference abstracts and letters to the editors ; ( 2 ) only examined individual components of ms ; ( 3 ) measured the ms variables at time of cancer diagnosis ; ( 4 ) used cancer mortality , rather than incidence , as the outcome ; and ( 5 ) were published in a language other than english . a comprehensive and systematic search was conducted using four electronic databases : pubmed , cumulative index to nursing and allied health literature ( cinahl ) , web of science , and proquest ( from their commencement to june 30 , 2012 ) . since the term ms dates back to the late 1950s , with variations in use as early as the 1920s , the start dates of each of the databases were used as the commencement date for study search : web of science ( 1900 ) , cinahl ( 1952 ) , pubmed ( 1966 ) , and proquest ( 1861 ) . major keywords used in the search for potentially eligible studies included metabolic syndrome ( insulin resistance syndrome , syndrome x ) and breast cancer ( neoplasm and breast ) . using the most recent publication , trials published as duplicate reports ( parallel publications ) an initial cut - off point for the inclusion of studies was not used given the difficulty in establishing such a point , as well as our concern about the potential loss of studies that met our eligibility criteria . at the first screening , one author ( rb ) screened all abstracts and selected articles for full - text examination . at the second level of the study selection process , two of the authors ( rb and th ) examined the full - text articles and then selected the included studies following mutual discussion and consensus . two of the authors ( rb and th ) reviewed every study selected and independently extracted data from studies onto electronic coding forms . attempts were made to contact authors of three of the original studies for missing information [ 13 , 62 , 64 ] , but only one provided the requested information . after initial coding , the two coders ( rb and th ) reviewed each item for agreement . using cohen 's kappa ( k ) statistic , the overall interrater agreement rate prior to correcting discrepant items was 0.96 for all included studies . risk of bias was assessed using a modified version of strengthening the reporting of observational studies in epidemiology ( strobe ) checklist . the items assessed included ( 1 ) study design , ( 2 ) adjustments for confounders , ( 3 ) selection of participants and their eligibility criteria , ( 4 ) measurement of predictor variables , ( 5 ) breast cancer diagnosis , ( 6 ) study size , ( 7 ) handling of missing data , and ( 8) reasons for nonparticipation of individuals at each stage of the study . a description of the criteria for risk of bias assessment is shown in table 1 . two of the authors ( rb and th ) conducted all assessments , independently of each other . risk estimates were used to examine the association between ms and risk of breast cancer . these were derived from reported relative risks , odds ratios , hazard ratios , incident rate ratios , or standardized incidence ratios , together with corresponding 95% confidence intervals ( cis ) , from the original studies . where necessary and possible adjusted risk estimates were pooled for analysis from multivariable models in the original studies . however , for two case - control studies that were included [ 14 , 51 ] , adjusted odds ratios were used because of the lack of the requisite data to convert odds ratios to rrs . all rr results were pooled using a random - effects model , an approach that incorporates between - study heterogeneity into the model . a z - score two - tailed alpha value 0.05 was considered to be statistically significant . in addition , 95% cis were calculated for each result from each study as well as for pooled estimates . an alpha level 0.10 for the q statistic was considered to be evidence of statistically significant heterogeneity . while somewhat arbitrary , i values of 25% , 50% , and 75% were considered to represent low , moderate , and high amounts of heterogeneity . publication bias was assessed using the trim and fill approach of duval and tweedie . in addition , rosenthal 's fail - safe n test was used to compute the number of missing null studies that would be needed to nullify the overall pooled rr as being statistically significant . influence analysis was conducted with each study result deleted from the model once , in order to examine the effects of each on the overall pooled results . cumulative meta - analysis , ranked by year , was also conducted in order to examine the accumulation of results over time . a separate pooled analysis , limited to postmenopausal women , was conducted because studies show that ms in postmenopausal women increases the risk of breast cancer [ 13 , 14 , 43 , 48 , 51 , 62 ] . in addition , pooled analyses were conducted with the following caveats post hoc : ( 1 ) deletion of results from two case - control studies because odds ratios were used instead of rr [ 14 , 51 ] , ( 2 ) deletion of results from studies that were not prospective cohort designs [ 13 , 14 , 51 ] , and ( 3 ) limiting the results to studies that controlled for four or more of the important confounders ( as listed in table 1 ) [ 14 , 43 , 48 , 51 ] . given the potential for diabetes and diabetes medications to affect breast cancer risk , post hoc data analysis was also conducted with studies that included participants with diabetes and/or taking medications for diabetes , deleted from the model [ 14 , 43 , 64 ] . figure 1 presents a flow diagram of the selection of studies for the meta - analysis . of the 291 studies screened , 47 ( 16.2% ) were selected for full - text review : 25 from pubmed [ 14 , 4355 , 64 , 7483 ] , 17 from the web of science [ 13 , 39 , 63 , 8497 ] , one from cinahl , and four from proquest [ 62 , 99101 ] . eight ( 17.0% ) of the 47 studies that underwent a full - text review met the eligibility criteria [ 13 , 14 , 43 , 48 , 51 , 6264 ] . one article presented results for two independent cohorts ; thus each cohort was treated independently . the study designs included four prospective cohorts [ 48 , 6264 ] , one retrospective cohort , one prospective nested case - control study , and two case - control studies [ 14 , 51 ] . the baseline year for cohort inception ranged from 1983 to 2004 , with average follow - up ranging between 2.7 and 13.5 years . six studies conducted analyses on postmenopausal women [ 13 , 14 , 43 , 48 , 51 , 62 ] . the results of each cohort or case - control study were initially reported as a hazard ratio [ 13 , 48 , 63 ] , incidence rate ratio [ 43 , 62 ] , standardized incidence ratio , or odds ratio [ 14 , 51 ] . methods for exposure assessment , cancer identification , and control of confounders varied across the eight included studies ( table 3 ) . seven studies identified the outcome ( breast cancer ) through histological reports or medical reports or from a cancer registry [ 13 , 14 , 43 , 48 , 51 , 62 , 64 ] , while one used self - report . only three studies examined invasive breast cancer cases [ 43 , 48 , 64 ] . one study also reported on the in situ breast cancer cases but there were only seven such cases in that study . another study analyzed all breast cancer cases ( in situ and invasive ) as well as invasive cancers separately , and results remained unchanged . all of the studies were considered to be at low risk for selection of participants and meeting eligibility criteria in addition to providing adequately powered sample sizes . out of eight studies , a majority were also considered low risk with respect to study design ( six studies ) and measurement of the outcome variable ( seven studies ) . in terms of handling potential confounders , half the studies were low risk , three were high risk , and one was unclear risk . missing confounding variables included education , smoking status , alcohol use , family history of cancer , contraceptive use , or hormonal history . similarly , half the studies had objective measurements of predictor variables , while the remainder relied on self - report , and were consequently considered high risk . four studies deleted the participants with missing variables in their analyses ( high risk ) , while two did not report how they handled missing data . lastly , six studies were considered high risk because they did not report the reasons for nonparticipation of subjects at each stage of follow - up . overall , a statistically significant increase of 47% in the risk for incident breast cancer was observed for adult females with ms ( rr : 1.47 , 95% ci , 1.151.87 ; z = 3.13 ; p < 0.002 ; q = 26.28 , p < 0.001 ; i = 69.55% ) ( figure 2 ) . with the exception of one study , all other studies had rr in the direction of increased risk [ 13 , 14 , 43 , 48 , 51 , 62 , 64 ] . funnel plot results for potential publication bias are shown in figure 3 . using the trim and fill approach that resulted in two imputations , the risk decreased by 16% but remained significant ( rr : 1.31 , 95% ci , 1.011.70 ) . null studies would be needed to nullify the statistically significant association between ms and breast cancer risk in adult females . no statistically significant outliers were identified ( p = 0.060.82 ) . with each study deleted from the model once , results remained positive and statistically significant ( figure 4 ) . the pooled rr fell within a range of 20% ( rr = 1.361.56 ) and none of the cis for the point estimates was less than 1.0 . cumulative meta - analysis , ranked by year , revealed that results have been statistically significant since 2011 ( figure 5 ) . deleting the two case - control studies from the model , the rr for incident breast cancer for women with ms decreased by 18% but was still statistically significant with moderate heterogeneity ( rr : 1.29 , 95% ci , 1.0031.67 ; z = 1.98 ; p = 0.05 ; q = 14.13 , p = 0.01 ; i = 64.61% ) . when limited to studies with only prospective designs , the rr decreased by 30% but remained statistically significant with very low heterogeneity ( rr : 1.17 , 95% ci , 1.011.36 ; z = 2.04 ; p = 0.04 ; q = 4.30 , p = 0.37 ; i = 7.04% ) . when limited to postmenopausal women , breast cancer risk increased by 34% and was still statistically significant with high heterogeneity ( rr : 1.81 , 95% ci , 1.282.56 ; z = 3.37 ; p = 0.001 ; q = 23.36 , p = 0.001 ; i = 74.32% ) . when limiting the results to studies that controlled for four or more of the important confounders ( as listed in table 1 ) [ 14 , 43 , 48 , 51 ] , breast cancer risk increased by 17% and was statistically significant with moderate heterogeneity ( rr : 1.64 , 95% ci , 1.232.20 ; z = 3.34 ; p = 0.001 ; q = 8.55 , p = 0.07 ; i = 53.21% ) . lastly , when data were analyzed after deleting from the model those studies that had participants with diabetes or taking medications for diabetes [ 14 , 43 , 64 ] , the rr was slightly larger than the overall finding but the 95% ci included 1.0 ( rr : 1.48 , 95% ci , 0.922.4 ; z = 1.61 ; p = 0.11 ; q = 17.4 , p = 0.02 ; i = 76.96% ) . the purpose of this aggregate data meta - analysis was to examine the association between ms and the risk for breast cancer in adult females . overall , the results suggest that there was a modest positive association between ms and risk of breast cancer . this finding is strengthened by the robustness of results from other analyses . these include ( 1 ) examination for publication bias , ( 2 ) influence analysis with each study being deleted from the model once , ( 3 ) deletion of the two case - control studies with odds ratios from the overall model , ( 4 ) limiting the analysis to prospective designs , ( 5 ) including only postmenopausal women in the analysis , and ( 6 ) limiting the results to studies that controlled for four or more of the important confounders . in addition , the results from cumulative meta - analysis , ranked by year , indicate an increasingly statistically significant association since 2011 . in contrast , despite a slightly increased mean rr , overlapping cis were observed when studies that included participants with diabetes or taking medications for diabetes were deleted from the model [ 14 , 43 , 64 ] . however , whether this reduced precision is the result of these specific characteristics or some other factors , for example , loss of power with a reduced number of studies , is not known . assessment for risk of bias indicated that a majority of studies were at low risk regarding study design , cancer assessment , and sample size . however , a majority were at high risk or unclear risk in terms of handling of missing data and nonparticipation of subjects at each stage of follow - up . it is suggested that future studies provide complete information on the handling of missing data and on the nonparticipation of subjects at each stage of follow - up . when limited to postmenopausal women , a stronger association between ms and breast cancer was observed . this association was stronger in case - control and retrospective cohort study designs compared to prospective cohort study designs . these findings concur with those from a recent meta - analysis on ms and breast cancer risk in postmenopausal women . several studies have shown that ms in this group increases the risk of breast cancer [ 43 , 46 , 102 ] , suggesting that the etiology of breast cancer may differ among pre- and postmenopausal women . first , obese postmenopausal women produce higher levels of estrogens , which in turn increase the biologically available fraction of circulating estradiol by reducing plasma concentration of sex hormone binding globulin ( shbg ) . low plasma shbg levels are associated with insulin resistance [ 104 , 105 ] and other components of ms [ 106 , 107 ] . second , adipose tissue produces two adipokines ( cytokine - like factors ) , leptin and adiponectin , that affect breast cancer biology . higher plasma leptin levels are associated with obesity [ 54 , 57 , 109 ] , insulin resistance [ 110 , 111 ] , and ms [ 112 , 113 ] . leptin stimulates human breast cancer cell lines , whereas adiponectin acts protectively , inhibiting the growth of these cell lines [ 57 , 108 , 114 ] . third , insulin has been shown to have a mitogenic effect upon breast cancer cells in vitro through several mechanisms . moreover , low serum hdl - c concentrations indicate higher circulating bioactive estrogen levels , which in turn may stimulate target breast tissue . the increasing prevalence of ms and its association with breast cancer , among other comorbidities , point toward the critical need to develop public health strategies to manage ms . given the increasingly large global burden of metabolic risk factors , risk assessment tools can be developed which incorporate ms as a risk factor for breast cancer . healthcare providers will then be better equipped to identify high - risk women for primary and secondary prevention . this study has several strengths . first , to the best of our knowledge , this is the first systematic review and meta - analysis examining the association between ms and risk of breast cancer in all adult women . the overlapping meta - analysis on metabolic syndrome and breast cancer was confined to postmenopausal women only . second , a number of other analyses were performed which strengthened the robustness of findings . third , the results of this study provide direction for future research on this topic . these include ( 1 ) the different methods used to assess exposure , identify cancer , control for confounders , and define ms , ( 2 ) limiting studies to those published in english , which may have inflated the results , ( 3 ) the relatively small number of studies that met the inclusion criteria , ( 4 ) the inability of some studies to provide raw data for calculating the rr , ( 5 ) the different study designs employed , and ( 6 ) the varied populations studied , including those with diabetes and/or taking medications for diabetes . most notably and with respect to controlling for adiposity , a potential confounder , two of the included studies controlled for bmi [ 48 , 62 ] but no information was available from the other studies with respect to controlling for bmi or any other obesity - related measures , including such measures of central obesity as waist circumference and waist - to - hip ratio [ 13 , 14 , 43 , 51 , 63 , 64 ] . given the potential association between breast cancer and adiposity , it may be prudent for future studies to control for this potential confounder . to this point , kabat et al . suggested that some , but not all , studies have reported an association between increased central adiposity and an increased risk for postmenopausal breast cancer . the inclusion of such information in future studies may be important , given the potential differences in risk according to exposure and disease subtype . in order to inform and undergird a biological rationale for the observed positive association between ms and breast cancer risk in adult females , future research should comprise analyses based on a standard definition of ms and employ objective and standard biomarkers for assessing each ms component . . it would be helpful if future studies examined the relationship between ms and breast cancer risk separately in perimenopausal and premenopausal women since breast cancer in women may be estrogen - independent . along those lines , not all studies adjusted for hormone replacement therapy , a potential confounder . furthermore , they need to examine in situ and invasive cancers separately in relation to metabolic syndrome . finally , a focus on obese women with respect to ms and breast cancer seems appropriate . in conclusion , the overall results of this meta - analysis suggest that there is a modest positive association between ms and risk of breast cancer in adult females .

background . although individual metabolic risk factors are reported to be associated with breast cancer risk , controversy surrounds risk of breast cancer from metabolic syndrome ( ms ) . we report the first systematic review and meta - analysis of the association between ms and breast cancer risk in all adult females . methods . studies were retrieved by searching four electronic reference databases [ pubmed , cumulative index to nursing and allied health literature ( cinahl ) , web of science , and proquest through june 30 , 2012 ] and cross - referencing retrieved articles . eligible for inclusion were longitudinal studies reporting associations between ms and breast cancer risk among females aged 18 years and older . relative risks and 95% confidence intervals were calculated for each study and pooled using random - effects models . publication bias was assessed quantitatively ( trim and fill ) and qualitatively ( funnel plots ) . heterogeneity was examined using q and i2 statistics . results . representing nine independent cohorts and 97,277 adult females , eight studies met the inclusion criteria . a modest , positive association was observed between ms and breast cancer risk ( rr : 1.47 , 95% ci , 1.151.87 ; z = 3.13 ; p = 0.002 ; q = 26.28 , p = 0.001 ; i2 = 69.55% ) . no publication bias was observed . conclusions . ms is associated with increased breast cancer risk in adult women .

hadrontherapy is an innovative form of radiotherapy , based on high - technology equipment using proton or carbon ions beams to destroy tumours [ 1 , 2 ] . this treatment method enables significantly higher ballistic precision to be achieved , compared to photons ( x - rays ) with , as expected therapeutic benefit , an improvement of quality of life and chances of recovery . carbon ions are also specifically characterised by superior biological efficacy ( relative biological effectiveness from 1.5 to 3 ) , overcoming the radiation resistance of certain cancers to photons and even protons . indeed , carbon ion beams when compared to x - rays represent a distinct advantage for the treatment of highly radiation - resistant tumours . an initial study assessing recruitment potential for proton therapy was conducted in 1998 in italy , showing an incidence of 10 825 cases / year . one year later , a second study was carried out in the context of the medaustron project for the construction of a carbon ions therapy centre in austria . considering patients living in austria and neighbouring countries , the patient recruitment potential ( proton and carbon ) was estimated with an incidence of 13 145 cases / year . in france , in 2002 , a third study was conducted in the context of the etoile medical project . this study revealed an annual incidence of approximately 5320 cases / year for carbon ions . concomitantly , a fourth study was conducted , again in the context of the medaustron project , estimating the incidence of cases eligible for hadrontherapy in austria at 2044 cases / year . finally , again in 2002 , in the context of the project for the construction of the cnao centre in italy , an epidemiological study was conducted , estimating the incidence of cases eligible for carbon therapy at 3694 cases / year and proton therapy at 1885 cases / year . . since 2004 , no other epidemiological studies have been published for hadrontherapy . in 2011 , however , the japanese team from the national institute for radiological sciences ( nirs ) , pioneer in carbon therapy , published the results of its 1994 to 2011 clinical activity . patients were included in clinical trials and the number of patients receiving carbon therapy was 6157 . this value is the incidence of treated cases , over 17 years without any notion of recruitment potential and with a rather slow ramp up of the activity . a list of priority indications that can be treated by hadrontherapy ( table 1 ) was drawn up by the gcs etoile medical group , in collaboration with national and international experts , based on published data ranked using an evidence - based medicine this literature review was based on provided or expected medical service criteria ( survival and quality of life ) . according to the classification used by the european survey group of rare diseases , the eligible tumours that can be treated by hadrontherapy conform to the criteria of rare diseases based on the following thresholds of epidemiological indicators : prevalence 50 cases/100 000 persons within a given population and incidence 6 cases/100 000 persons / year . indeed , for all tumor types identified during the previous hadrontherapy epidemiological studies [ 58 ] , the calculated and cumulative incidences are all below these thresholds ( table 2 ) . amongst the hadrontherapy indications , 25 are tumours listed in the orphanet international database of rare diseases ( cf . the exact incidence of each disease eligible for hadrontherapy is difficult to determine from the available resources . indeed , the published data [ 513 ] pertain to series of patients for which the aim was not to assess the level of demand for treatment . these were rather series of patients who had received the therapy , but whose recruitment had not been accounted for in tumour registries . considering the scarcity and specific nature of the tumours concerned by hadrontherapy , it is difficult to extrapolate and assess the treatment demand from these results . hadrontherapy indications are defined according to anatomical location of the tumours , clinical stage , pathology , therapeutic alternatives ( e.g. , surgical contraindications ) , and patient characteristics ( general condition and comorbidities ) . french cancer registries record data required to estimate cancer incidence but fail to provide sufficient detail of the tumour stage or of therapeutic data . moreover , the registries cover only approximately 15% of the french population and mainly for the most frequent cancers . therefore , these registries do not allow the incidence of these tumours to be estimated . this regional study will provide us with more detailed figures concerning the potential number of new cases eligible for hadrontherapy , allowing us to better adapt the future care offer delivered by the national hadrontherapy centre . this survey should also provide us with information on certain medical indicators to assist in the design of future hadrontherapy clinical trials ( detailed incidence data , patient population characteristics , tumour stages , recruitment pools , etc . ) . finally , this epidemiological field study is also eventually intended to be used to organise a network of highly specialised cares . according to the first epidemiological study carried out in france , in association with ( i ) the results of the literature review that defined the list of hadrontherapy indications and ( ii ) the indications chosen for proton therapy in france , the number of cases in the rhne - alpes region eligible for hadrontherapy is estimated at 200 per year ( the rhne - alpes region is home to 10% of the french population ) . the main goal is to asses , within the rhne - alpes region , the incidence of cancers with hadrontherapy indication . the secondary goals are ( 1 ) to characterise the affected population , ( 2 ) to describe the characteristics of the observed cancers , and ( 3 ) to describe the characteristics of the treatments implemented when hadrontherapy is unavailable . this is a prospective , multicentre incidence study , lasting 24 months , conducted with the rcp ( runion de concertation pluridisciplinaire multidisciplinary tumor board ) groups of the rhne - alpes region in france , that offer a therapeutic strategy for cancers for which hadrontherapy is an alternative . this study required the involvement of a part - time consultant epidemiology engineer , who totalled one woman - year of work to provide ( 1 ) phone canvassing to identify and involve regional cancer research players ; ( 2 ) the protocol construction and testing phase , along with the data collection tools ; ( 3 ) meetings with 3c ( cancer coordination centres ) coordinators and rcps ; ( 4 ) investigator centres inclusion meetings . these included year of birth ; gender ; patient 's department of residence ; world health organization ( who ) performance status , patient 's therapeutic status : initial stage or recurrence ; initial staging ( uicc tnm classification : international union against cancer ) ; treatments received before recurrence ; time to recurrence ; staging at time of current tumour management ; icd-10 ( international classification of diseases ) ; icdo ( international classification of diseases for oncology ) histological type ; site of surgery and margin quality ; postsurgical histopathological stage ( ptnm stage ) ; prior radiotherapy ; proposed treatment . the criteria included adults and children , no age limit , and patients presenting with hadrontherapy indications listed in table 1 , whose medical files have been discussed by experts during an rcp . compatible general and psychological condition refers to patients not suffering from life - threatening comorbidities ( no acute or chronic diseases whose short - term lethal risk is dominant relative to the cancer ) , capable of adhering to a disease monitoring protocol and of understanding and accepting a complex treatment requiring close cooperation and staying for several days away from home . tumour inclusion criteria . these include unresectable tumour , belonging to a known radiation - resistant pathological group , mainly in a locoregional development stage , recurrent or local relapse , with low metastatic potential or m+ dissemination ( presence of one or more remote metastases at the time of diagnosis ) presenting a low threat ( slow development and/or metastases that can be readily treated ) . these inclusion criteria are the results of a consensus synthesis of a systematic literature review performed during 8 years , pathology by pathology with the participation of about one hundred of european medical , surgical , and radiation oncologists , managed by physicians trained in hadrontherapy ( mainly protontherapy ) . the review was carried out by an academic laboratory specialized in clinical trial setup and analysis and was made according to the evidence - based medicine definition of tumour inclusion criteria radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique sfro ) , and the french society for medical physics ( socit franaise de physique mdicale sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie afppe ) ) . unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means . this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example , nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) . these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique sfro ) , and the french society for medical physics ( socit franaise de physique mdicale sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie afppe ) ) . unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means . this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example , nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) . these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . they are the description of the tumours and population characteristics , along with the treatments proposed and implemented by the rcps . those were healthcare establishments ( private and public ) in the rhne - alpes region hosting rcp groups and having radiotherapy departments . they consisted of groups of oncology specialists with expertise in the following diseases : musculoskeletal sarcomas and tumours , head and neck ( h and n ) , gastroenterology , paediatrics , dermatology , endocrine and central nervous system tumours . case screening is performed continuously by the physicians and clinical research technician in light of the list of indications ( table 1 ) , during each rcp . the questionnaire , along with the organisation of the investigation , was assessed beforehand by a sample population of investigators ( 3c coordinators and cras ) via a semiguided telephone interview . for the questionnaire , the evaluation focused on general understanding , the form and length of the questionnaire , time required for completion , amount of data to collect , and the relevance of the selected criteria . the evaluation of the investigation method pertained to the choice of individuals involved in data collection organisation , the data recording method , the organisation for data recording , and the identification source for clinical cases . variables are encoded in a uniform and standard manner in an investigator 's guide book , which contains detailed information concerning the definitions of each indicator to collect . dual computer data input is performed , along with checks for missing or improbable data ( encoding errors , date inconsistencies ) . statistical methods . the incidence of cancers is expressed as the number of cases reported from the demographic figures of the rhne - alpes region over the studied period and expressed per 100,000 inhabitants . the study involves 13 clinical research technicians , 50 rcp coordinating physicians and 12 cancer coordination centres ( see table 3 ) . the study mobilises 27 rhne - alpes region healthcare centres , of which 8 university hospitals , 1 regional cancer centre , 1 public - private cancer institute , 12 general hospitals , the lyon paediatric oncology hospital institute , and 4 private clinics . this led to a sense of ownership and motivation on the part of the centres . as a whole , the study covered the region 's 11 hospital areas and involved 52 groups , out of some sixty listed , of cancer specialists with expertise in the diseases that were to identify . during the study , mean patient age was 43 years [ 289 ] , and men were more numerous ( or = 1.52 ) . the highest number of cases ( 39.6% ) was in the 218 years age group . the number of patients with recurrence was slightly lower ( 45.3% ) than the number of patients receiving initial care ( 54.7% ) . carcinomas , sarcomas ( table 4 ) , and paediatric tumours ( table 5 ) were the most frequently identified diseases , and their locations are varied . during the rcp , radiotherapy , chemotherapy , and surgery were prescribed , respectively , in 58% , 57% , and 57% of cases . previous treatments were chemotherapy , surgery , or conventional radiotherapy in 62.5% , 58.3% , and 58.3% of cases , respectively . the number of cases identified during the study was approximately four times lower than the potential estimate . ( i.e. , 2.5 cases / rcp ) , and the rhne department identified 56.6% ( i.e. , 1.2 cases / rcp ) . isre identified twice as many cases as rhne , even though it only has nine expert groups , that is , 2.6 times less than in rhne , which has 24 . the geographic areas not covered by the study correspond to the location of 15 private healthcare centres and to the medical activity of six general and four specialist rcp groups . the first six groups had no recruitment potential according to the statement of their coordinators . this preliminary data collection , conducted over a one - year period , reported 53 cases of hadrontherapy indications , compared to the estimated cap of 200 cases . the estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010 , regardless of disease , is of 8.5/100,000 inhabitants . the diseases to identify in this study are little known , their distribution has not been studied , and we are unaware of any epidemic outbreaks . moreover , considering that the cancers to identify are rare , it is likely that the number of cases to identify per investigator centre will be very low ( estimated incidence of at the most 1 to 5 cases / year ) . finally , the primary goal of the rcp groups is to provide an immediate therapeutic solution . case identification for an observational study is thus unusual in the context of an rcp . because of this , sustaining team vigilance and motivation for case identification is difficult to obtain and requires regular refreshing throughout the study . this step also served to circulate hadrontherapy information and to facilitate adhesion , ownership , and mobilisation around the project . the individuals involved in study organisation were in charge of systematically directing each cancer patient to the elective experts . patients suffering from diseases eligible for hadrontherapy were frequently in relapse condition and rather at the end of their therapeutic options . this is due to the fact that these diseases have usually a slow and long evolution ; therefore , this study had more chance to catch them somewhere in an advanced stage than at the beginning . it can also be considered that the lack of satisfactory treatment means as hadrontherapy makes these populations have more chance to show more advanced disease than it will be expected to be seen in the future . the identification and decision to include a patient in the study were made by the rcp groups including these experts . under these conditions , we have confidence in the case orientation and identification system . selection criterion is one of the criteria used to identify patients eligible for hadrontherapy . in our current state of knowledge this can be explained in part by the fact that it is easier to create multidisciplinary expert groups in public hospitals and to set up clinical research . due to their structure , these establishments benefit from human resources with more varied medical specialities than in private radiotherapy practices . in all likelihood , cases eligible for hadrontherapy could have been treated by these private centres during the study and ignored . one should be very cautious when comparing the results of this study with those of published incidence studies . the methodologies employed in these previous studies are heterogeneous , and/or their flow is insufficiently documented . moreover , their goal was to estimate a recruitment potential based on the extrapolation of the results . the methodologies used were not intended to provide information concerning an exhaustive incidence , as proposed by our study . finally , the published studies were conducted in attractive institutions , specialising in cancer care and selected for this reason ; this may bias the assessment of prevalence . under these conditions , it is easy to confuse incidence and prevalence . these elements could , in part , explain the observed difference between the preliminary results obtained in our study and the estimate calculated from the epidemiological results published in the literature . only the publication of results obtained from other exhaustive epidemiological studies conducted in the same areas could support this hypothesis . it is unlikely that the lack of case recording associated with the medical activity of the four groups not involved in this data collection could alone explain the observed difference between the calculated incidence estimates and the preliminary results obtained . for several clinicians involved in the study , the prognosis and risk of metastatic development were difficult to estimate considering the lack of knowledge for these rare diseases . moreover , the novelty of hadrontherapy , which can potentially modify usual treatment strategies , could have led to the omission of this indication in some cases , in particular conditions unusually treated by radiotherapy as hepatic and biliary tumours . these difficulties , along with the more sustained vigilance effort made by some groups relative to others , could account for the nonhomogeneous data collection and the observed difference between predictions and obtained results , despite the high motivation of the investigators . furthermore , the reorganisations ( case of two major rcp clusters ) , changes of persons involved and the low frequency of cases to report , lead to a constant erosion of vigilance , requiring continuous action by the organiser . however , some important institutions with their own recruitment targets did not wish to participate , which reduces the scope of our results . about the detailed conditions of the recruited cases , there are a large proportion of recurrent diseases as explained above , and some metastatic diseases have also been included according to defined characteristics : ( i ) the number of metastasis was limited ( 1 to 3 metastasis ) ; ( ii ) their very slow growing speed was not immediately threatening ; ( iii ) the possibility of effective treatment of these pauci - metastatic conditions , essentially by ablative procedures . actually , the principle to treat , with curative intend , patients in such situation is more and more accepted by expert oncologists . the estimated incidence of cancers , irrespective of type and location , eligible for treatment by hadrontherapy , for the study , that is , 8.5/100,000 individuals , is mildly higher than the reference incidence threshold of 6 cases/100,000 individuals , as defined in the orphanet international database of rare diseases . this figure can be obviously explained by the fact that the incidence was calculated by adding the five major cancer categories and 34 different locations . if one was to calculate the incidence for each of the disease types , their respective incidence would be significantly below the reference threshold of 6 cases/100,000 . indeed , for our study , the minimum number of cases listed per disease is of 1 case and the maximum is of 7 cases . table 4 . considering all of the difficulties and limitations of this preliminary phase of the study , the yield of approximately 25% notification , compared to the expected maximum , appears satisfactory . in the perspective of a ( necessary ) ramp up of a future hadrontherapy centre , there is thus a sufficient population to initiate operation and to progressively establish recruitment for this centre , without immediately generating an insufficient offer effect . future epidemiological studies will benefit from focusing on the characterisation of the metastatic development of diseases eligible for hadrontherapy . all cancers have their own development pattern , and , in this respect , the literature provides few elements concerning diseases eligible for hadrontherapy . this information should serve both to improve diagnosis procedures , medical care , and the surveillance of these rare diseases . this study of hadrontherapy healthcare decision networks in the rhne - alpes region , able to identify areas not properly covered by rcps , should enable us to analyse the potential effect of this lack on the outcome of the patients of these areas . they shed no light on posttreatment medical activity : follow - up visits , medical imaging , and other care beyond the irradiated activity per se . one of the most valuable outcomes of this study has been to make the existence of some particularly rare diseases known and to provide information concerning an innovative treatment : hadrontherapy , whose existence will doubtless have a favourable effect on the very knowledge of these diseases . an additional benefit of this approach has been to open the way for a new treatment system in a region of france that is home to approximately 10% of the country 's population . at last , these elective indications will have to be validated by health authorities to register them as part of the good professional practices of oncology and give an equal chance to each patient to receive hadrontherapy through the well - established decision process of the rcps .

context . hadrontherapy is an innovative form of radiotherapy using beams of protons or carbon ions able to destroy some radio - resistant tumours . because these tumours are highly specific amongst all cancerous tumours , it is impossible to determine the incidence of these diseases from surveillance registries . goal . to assess , within the rhne - alpes region , the incidence of cancers being hadrontherapy indications . method . prospective , multicentre continuous data collection during 1 year , by practitioners participating to multidisciplinary tumor board . tumours are inoperable , radio resistant , at primary stage of development , or locally recurrent , with low metastatic potential . results . study involved 27 healthcare centres , 52 groups of specialist practitioners . the estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010 , that is , for 34 locations in all , is of 8.5/100 000 inhabitants . appraisal of the low potential of metastatic progression is impeded , because these are rare diseases , whose outcome is unfamiliar to investigators . conclusion . future epidemiological studies will need to focus on prognosis and on the metastatic progression rate of these diseases . indeed , there are few information available on this subject in the literature that could be used to improve preventive measures , medical care , and the surveillance of these rare cancers .

craniovertebral junction ( cvj ) abnormalities constitute an important group of treatable neurological disorders , especially in certain ethnic groups like indian subcontinent . these are one of the major causes of spinal cord , vascular and nerve compression , and hydrocephalus . hence , in every patient presenting with these features , cvj abnormalities should be excluded . cvj is a complex region that incorporates the occiput as well as the c1 and c2 vertebrae , while the stability and flexibility to cvj is provided by the bony as well as ligamentous nature of atlantooccipital and atlantoaxial joints . variety of abnormalities can affect the cvj with their diagnostic dilemmas and few studies have been conducted in this regard . this study is an effort to systematically classify various cvj abnormalities according to their etiological group and to define the importance of precise diagnosis for pretreatment evaluation with multidetector computed tomography ( mdct ) and/or magnetic resonance imaging ( mri ) . this study presents the data of 62 patients referred to our department between october 2012 and september 2014 . written informed consent for the study all patients clinically suspected to have a cvj disorder were included in the study , from all age groups and both genders . the abnormalities involving cvj had been grouped as developmental or congenital , traumatic , degenerative , infective , inflammatory , neoplastic , and unknown . all patients were subjected to mdct and/or mri and contrast was given if required in patients with normal serum creatinine ( normal up to 1.3 mg% ) . ct was performed in neutral , flexion , and extension positions ; except in situations where patient was not in a condition to perform flexion and extension and in cases of trauma . after thin axial images were obtained , these were isotropically reconstructed into coronal and sagittal planes . mri was done on 3 t philips machine and the following sequences were obtained : t1-weighted spin echo ( t1w se ) and t2-weighted turbo spin echo ( t2w tse ) in the sagittal and axial planes.t1w se and short tau inversion recovery ( stir ) in coronal plane.t2w tse in flexion and extension whenever required.post gadolinium administrationt1w fat saturated ( fs ) images were obtained in all three planes . t1-weighted spin echo ( t1w se ) and t2-weighted turbo spin echo ( t2w tse ) in the sagittal and axial planes . post gadolinium administrationt1w fat saturated ( fs ) images were obtained in all three planes . typical parameters used for t1w se sequence were a repetition time ( tr ) of 598 ms and an echo time ( te ) of 27 ms ; for t2w tse sequence were tr of 4,100 ms and te of 100 ms ; for stir sequence were tr of 2,300 ms , te of 60 ms , and inversion time ( ti ) of 150 ms . these sequences were done with slice thickness of 3 mm with an inter - slice gap of 0.3 mm . subsequently , in cases where contrast was required , gadolinium enhanced ( administered in a dose of 0.1 mmol / kg ) t1w fs sequences were obtained in all three planes . craniometric measurements used in radiologic assessment of cvj abnormalities include chamberlain 's line , mcgregor line , mcrae line , wackenheim clivus line , fishgold digastric line , fishgold bimastoid line , welcher basal angle , and atlantooccipital joint axis angle . statistical analysis was done using ratio and percentages for gender distribution and percentage distribution of each disease affecting the cvj . statistical analysis was done using ratio and percentages for gender distribution and percentage distribution of each disease affecting the cvj . in our study of 62 patients , 39 were males and 23 were females with male to female ratio of 1.6:1 . most common age group was 2140 years ( 19 patients , 30.64% ) followed by older age of more than 60 years ( 17 patients , 27.41% ) . the most common presenting complaint in these group of patients was neck pain ( 26 patients , 41.93% ) followed by limb weakness ( 17 patients , 27.41% ) ; with others being headache , tingling numbness , restriction of neck movements , ataxia , giddiness , and scoliosis . developmental anomalies were the most common etiology group followed by traumatic , degenerative , infective , inflammatory , and neoplastic , and no cause found in one patient in our study [ table 1 ] . distribution of patients according to etiological group among the developmental anomalies , congenital atlantoaxial dislocation ( aad ; 56.45% ) [ figure 1a and b ] , basilar invagination ( 37.10% ) [ figure 2 ] , and occipitalization of atlas ( 20% ) were the common radiological features and often exist with each other in varying combinations . other developmental anomalies were os odontoideum ( nine patients , 14.51% ) , chiari malformation ( eight patients , 12.90% ) [ figure 3 ] , platybasia ( five patients , 8.06% ) , klippel - feil syndrome , and unassimilated rudimentary atlas ( one patient each , 1.61% ) . 1 = flexion , 2 = neutral , 3 = extension of neck ct cvj region bone window image sagittal reconstruction showing tip of the odontoid process invaginated into the foramen magnum and was above the chamberlain line ( longer line ) and mcrae 's line ( shorter line ) in basilar invagination . ct = computed tomography , cvj = craniovertebral junction mri cvj region stir midsagittal image showing syringomyelia and peg - like tonsils 8.2 mm below the foramen magnum . mri = magnetic resonance imaging , stir = short tau inversion recovery out of the 10 patients in traumatic group , five patients had type 2 fracture of the dens [ figure 4 ] , two had type 3 fracture of the dens , one patient had tear drop fracture of anterioinferior aspect of the axis body , and one each had bilateral atlantooccipital dislocation and type 3 atlantoaxial rotatory dislocation . ct cvj region bone window coronal reconstruction showing type ii dens fracture degenerative changes affecting the cvj commonly mimic cvj anomalies . amongst the infective etiologies , tuberculosis ( tb ) [ figure 5a and b ] was seen in four patients . inflammatory pathology like rheumatoid arthritis ( ra ) [ figure 6a and b ] was seen in three patients . the neoplasms detected were , one meningioma [ figure 7 ] , another was chordoma of axis which was also involving c3 vertebra , and a nerve sheath tumor was found at c2 vertebral level . only two patients had a syndromic association , one was klippel - feil syndrome and other was down 's syndrome . mri cvj region ; ( a ) midsagittal t1w and ( b ) axial t2w images . hyperintense soft tissue in the predental and left paravertebral space extending through c1 c2 foramen with erosions of dens and left lateral anterior arch of c2 , which was to be of tuberculous origin . ( a ) postcontrast soft tissue window midsagittal reconstruction showing enhancing soft tissue pannus surrounding the dens . ( b ) precontrast bone window coronal reconstruction showing erosion and thinning of the dens suggestive of ra . ra = rheumatoid arthritis mri cvj region postcontrast t1w midsagittal image showing strongly enhancing anterior dural - based cvj mass lesion with dural tail sign suggestive of meningioma the craniovertebral junction is a complex region formed by the occipital condyles , atlas ( c1 ) , axis ( c2 ) vertebrae , and their articulations . cvj has intricate relationship with the major neurovascular structures which can lead to medullary - cervical cord compression , cranial or spinal nerve compressions , vertebral artery compression , and obstructive hydrocephalus . therefore , treatment of various types of cvj abnormalities poses many challenges which are critically affected by the precise diagnosis of the abnormality as shown in our series by classifying the abnormalities into etiological group with their precise diagnosis . the clinical manifestations are often delayed into the 2 and 3 decade because they are usually subtle and easily missed in children unless looked for specifically . in our study of 62 patients , male to female ratio was 1.6:1 ( 39 males and 23 females ) and the most common age group was 2140 years ( 19 patients , 30.64% ) , which correlated well with the study by jawalkar et al . , and sankhe and kumar . in our study , most common presenting symptom was neck pain , followed by limb weakness , which correlated well with most other studies . moreover , the radiological picture is confusing as the ossification of the bones is completed only by 9 years of age . the incidence of different types of abnormalities varies with the demographic environment and has ill - defined genetic factors . the indian subcontinent with very high population density of varying socioeconomic strata , high incidence of infectious diseases , and lack of healthcare awareness among the population shows wide spectrum of cvj abnormalities and in its late stages . these different types of abnormalities with a complex pathological bony anatomy need precise diagnosis by experienced radiologists for individual management decision tailored for that particular case , which is why this study was conducted for categorizing the abnormalities etiologically , making precise diagnosis for pretreatment evaluation , and ruling out mimickers . plain radiographs of cvj show overlap of many soft tissue structures . due to anatomic complexities of the cvj and high frequency of craniocervical trauma with muscle spasm , , the cross - sectional imaging used was mdct and mri , as mdct is ideal modality for evaluation of complex osseous anatomy associated with cvj abnormalities while mri , with its multi - planar capabilities and high soft tissue contrast resolution , has become the mainstay in radiological evaluation of the cvj . the craniometry of the cvj uses a series of lines , planes , and angles to define the normal anatomic relationships of the cvj namely chamberlain 's line , mcgregor line , mcrae line , wackenheim clivus line , height index of klaus , welcher basal angle , clivus canal angle , atlantooccipital joint axis angle ( schmidt angle ) , fishgold digastric line , and fishgold bimastoid line . also ct with its sagittal and coronal reconstruction confirms the diagnosis and helps precisely to know the occipitalization of atlas , hypoplastic posterior arch of atlas , and c1-c2 instability . enhancing soft tissue pannus at cvj in cases of ra , retropharyngeal abscess , and lytic lesions in the vertebrae in cases of tuberculosis were precisely diagnosed with ct . hence , knowing the underlying cause of the abnormality helps in better prognostication and treatment of the patient 's condition . in our study , developmental anomalies were the most common etiology group , followed by traumatic , degenerative , infective , inflammatory , and neoplastic , and no cause found in one patient [ table 1 ] . study out of 159 cases of cvj anomaly , developmental anomalies were the most common affecting 147 patients , neoplasm was found in 10 patients . another study of 189 cases by kale and colleagues showed developmental cause as the most common followed by traumatic and tuberculosis in decreasing order . in clinicoradiological practice , congenital aad accounted for 51.568% of all cvj anomalies , which correlated with our study . in our study , os odontoideum was found in nine patients with male to female ratio of 2:1 ( 6 males:3 females ) which correlated with study done by dai et al . , and spiering and braakman ; one patient presented with bilateral atlantooccipital dislocation and left lateral atlantoaxial subluxation after road traffic accident . one patient had traumatic type iii atlantoaxial rotatory dislocation , which is very uncommon in adult patient . till date , a total of 16 patients have been reported in the literature . in our four patients of cvj tb , osteolytic destruction of the anterior arch of atlas was seen in three patients and in another three ; erosions were noted in the dens . basilar invagination was seen in one case and atlantoaxial dislocation was seen in two patients . in one patient , active pulmonary tuberculosis was present , while in other three patients there was past history of tuberculosis for which they had completed treatment . chest radiograph in two of three patients showed sequelae of old tuberculosis . in our three patients of ra , the history of ra was long standing , more than 5 years in all patients . all the three patients had erosion of the odontoid process and enhancing soft tissue pannus along the dens . syringomyelia was found in five patients , scoliosis in two , and platybasia in two . peg - like tonsillar herniation of more than 8 mm was seen in all eight patients . one patient of chiari i had multiple osseous abnormalities such as aad , platybasia , fusion of c2 and c3 vertebrae , and hemivertebra at c4 . these results were comparable in order of frequency with the study carried out in ayub teaching hospital , abbottabad between july 2008 and july 2010 with a prospective cohort of 60 symptomatic patients . these were meningioma which was anterior dural based , another was chordoma of the c2 body and spinous process and also involving the c3 vertebral body , and one c2 nerve sheath tumor causing aad . in three patients in the age group of 7581 years , who presented with complains of neck pain , walking difficulty , and imbalance ; we found aad and basilar invagination . age - related degeneration of ligaments and bones caused the patients to present with above symptoms . cvj abnormalities constitute an important group of treatable neurological disorders , especially in certain ethnic groups , and are approached with much caution by clinicians . thus , it is essential that radiologists should be able to make a precise diagnosis of cvj abnormalities , classify them into the appropriate etiological group , and rule out important mimickers on mdct and/or mri ; as this information ultimately helps determine the management of such abnormalities , prognosis , and quality of life of patients .

background : craniovertebral junction ( cvj ) abnormalities constitute an important group of treatable neurological disorders with diagnostic dilemma . their precise diagnosis , identification of probable etiology , and pretreatment evaluation significantly affects prognosis and quality of life of patients.aims:the study was to classify various craniovertebral junction disorders according to their etiology and to define the importance of precise diagnosis for pretreatment evaluation with multidetector computed tomography ( mdct ) and magnetic resonance imaging ( mri).materials and methods : this is a prospective observational study of 62 patients referred to our department between october 2012 and september 2014 . all patients suspected to have a craniovertebral junction disorder were included in the study , from all age groups and both genders . detailed clinical history was taken . radiographs of cervical spine were collected if available . all patients were subjected to mdct and/or mri.results:in our study of 62 patients ; 39 were males and 23 were females , with male to female ratio of 1.6:1 . most common age group was 2nd -3rd decade ( 19 patients , 30.64% ) . developmental anomalies ( 33 patients , 53.22% ) were the most common etiology group followed by traumatic ( 10 patients , 16.12% ) , degenerative ( eight patients , 12.90% ) , infective ( four patients , 6.45% ) , inflammatory and neoplastic ( three patients each , 4.8% ) , and no cause found in one patient.conclusions:cvj abnormalities constitute an important group of treatable neurological disorders , especially in certain ethnic groups and are approached with much caution by clinicians . thus , it is essential that radiologists should be able to make a precise diagnosis of craniovertebral junction abnormalities , classify them into etiological group , and rule out important mimickers on mdct and/or mri , as this information ultimately helps determine the management of such abnormalities , prognosis , and quality of life of patients .

the mucoadhesive polymer containing oral drug delivery systems has the capacity to prolong residence time of drugs at the absorption site and facilitate intimate contact with underlying absorptive surface to enhance bioavailability . polymers used in the mucoadhesive formulations include natural , semisynthetic , and synthetic ones . in recent years , a growing interest has been identified in the development of natural polymer - based drug delivery systems due to their biodegradability , biocompatibility , aqueous solubility , swelling ability , easy availability , and cost - effectiveness . amongst various natural polymers , alginates have been widely used in the development of drug delivery applications [ 36 ] . it is composed of linear copolymers of two monomeric units , that is , -d - mannuronic acid and -l - guluronic acid . sodium alginate ( sa ) undergoes ionotropic - gelation by ca to form calcium alginate due to an ionic interaction between carboxylic acid groups of alginate chain and ca . sodium alginate has mucoadhesive property ; however , the cross - linked alginates are usually fragile [ 9 , 10 ] . therefore , blending of different mucoadhesive polymers is one of the most popular approaches to formulate ionotropically cross - linked alginate - based mucoadhesive beads [ 9 , 11 , 12 ] . again , blending with suitable polymers , may improve the drug encapsulation , which is found comparatively lower in alginate - based beads prepared by ionotropic - gelation method . ispaghula ( plantago ovata f. ) husk is an indigenous product of south asia and is widely used herbal product both in traditional and modern medicines . the ispaghula husk mucilage ( ihm ) is white , hydrophilic in nature , and forms a colorless gel in presence of water [ 1416 ] . ihm contains a high amount of highly branched neutral arabinoxylan ( arabinose 22.6% , xylose 74.6% , and traces of other sugars ) and about 35% of nonreducing terminal sugar residues . few investigations had been carried out to formulate mucoadhesive beads as drug delivery matrices using both untreated ispaghula husk and alkaline treated ispaghula husk directly as polymeric blend with sodium alginate [ 1 , 17 , 18 ] . however , no attempt has been taken to formulate mucoadhesive alginate - based beads using isolated ihm as polymeric blend for the use in drug delivery . in the current investigation , the utility of isolated ihm , as a possible natural polymeric - blend with sa for the development of new ihm - blended alginate beads containing glibenclamide through ionotropic - gelation , glibenclamide is a sulfonylurea used in the treatment of non - insulin - dependent diabetes mellitus ( niddm , type - ii ) [ 2 , 19 ] . its plasma half - life is 46 hours , which makes multiple dosing to maintain the therapeutic blood level . therefore , it would be beneficial to develop a mucoadhesive system of glibenclamide using ihm - alginate for oral use , which might facilitate an intimate contact with the mucous membranes ( i.e. , mucoadhesion or bioadhesion ) in the gastrointestinal tract , and thus the gastric residence could be prolonged to release glibenclamide at the target site at controlled rate over an extended period to maximize the therapeutic effect . in the development of any pharmaceutical formulation , an important issue is to design a formulation with optimized quality in a short time period and minimum number of trials [ 20 , 21 ] . traditionally , pharmaceutical formulators develop various formulations by changing one variable at a time while keeping others fixed . however , many experiments do not succeed in their purpose because they are not properly thought out and designed , and even the best data analysis can not compensate lack of planning . therefore , it is essential to understand the influence of formulation variables on the quality of formulations with a minimal number of experimental trials and subsequent selection of formulation variables to develop an optimized formulation using established statistical tools [ 5 , 2224 ] . factorial designs , where all the factors are studied in all possible combinations , are considered the most efficient in estimating the influence of individual variables and their interactions performing minimum numbers of experiments . a computer - aided optimization technique based on 3 ( two factors and three levels ) factorial design and response surface methodology was employed to investigate the effects of two independent process variables ( factors ) , that is , sa : ihm and cross - linker ( cacl2 ) concentration on the properties of glibenclamide - loaded ionotropically gelled ihm - alginate beads such as drug encapsulation and drug release . india ) , sa ( central drug house , india ) , ispaghula husk ( shree baidyanath ayurved bhawan pvt . the mucilage from ispaghula ( plantago ovata f. ) husk was extracted according to the previously reported method [ 26 , 27 ] . an amount of 10 ml of 0.1 m hcl was heated to boil in a 100 ml flask and 1 gram of plantago ovata f. husk was added . after total change of color , the flask was finally removed from heat and the solution was filtered through a clean muslin cloth while still hot . in order to separate residual traces of mucilage , the seeds were washed twice with 5 ml of hot water and the solution obtained each time was filtered . the combined filtrate , containing the dissolved mucilage , was mixed with 60 ml of 95% ethyl alcohol , stirred , and allowed to stand for 5 hours . finally , the supernatant liquid was decanted and the precipitate in the beaker was dried in an oven at 50c . the dried ihm cake was grounded with a mortar and passed through a sieve ( 0.15 mm mesh size ) . the isolated ihm powders were packed in a plastic bag and kept airtight desiccators until further use . the glibenclamide - loaded ihm - alginate beads were prepared by ionotropic - gelation technique using calcium chloride ( cacl2 ) as cross - linker . briefly , sa and ihm aqueous dispersions were prepared separately using distilled water . these dispersions were well mixed with stirring for 15 min at 1000 rpm using a magnetic stirrer ( remi motors , india ) . the ratio of drug to polymer was maintained 1 : 2 in all formulations . the final mixtures were homogenized for 15 min at 1000 rpm using a homogenizer ( remi motors , india ) and ultrasonicated for 5 minutes for debubbling . the added droplets were retained in the cacl2 solution for 15 min to complete the curing reaction and to produce rigid beads . the wet beads were collected by decantation and washed two times with distilled water and dried at 37c for 24 h. the dried glibenclamide - loaded ihm - alginate beads were stored in a desiccator until used . a 3 factorial design was employed for optimization with sa : ihm ( x1 ) and concentration of cacl2 ( x2 ) as the prime selected independent variables , which were varied at three levels , low ( 1 ) , medium ( 0 ) , and high ( + 1 ) . the drug encapsulation efficiency ( dee , % ) and 10 hours ( r10 h , % ) were used as dependent variables ( responses ) . the matrix of the design including investigated responses , that is , dee and r10 h , is shown in table 1 . the effects of independent variables upon the all measured responses were modelled using the following quadratic mathematical model generated by 3 factorial design : ( 1)y = b0+b1x1+b2x2+b3x1x2+b4x12+b5x22 , where y is the response , b0 is the intercept , and b1 , b2 , b3 , b4 , b5 are regression coefficients . x1 and x2 are individual effects ; x1 and x2 are quadratic effects ; x1x2 is the interaction effect . one - way anova was applied to estimate the significance ( p < 0.05 ) of generated models . the response surface methodology was applied to analyze the effect of independent factors ( sa : ihm and cacl2 concentration ) on the measured responses ( dee and r10 h ) . accurately weighed , 100 mg of beads were taken and were crushed using pestle and mortar . the crushed powders of drug containing beads were placed in 500 ml of phosphate buffer , ph 7.4 , and kept for 24 hours with occasionally shaking at 37 0.5c . after the stipulated time , the mixture was stirred at 500 rpm for 15 min on a magnetic stirrer . the polymer debris formed after disintegration of bead was removed filtering through whatman filter paper ( no . 40 ) . the drug content in the filtrate was determined using a uv - vis spectrophotometer ( shimadzu , japan ) at 228.5 nm . the dee of beads was calculated using this following formula : ( 2)dee ( % ) = actual drug content in beadstheoretical drug content in beads100 . particle size of 100 dried beads from each batch was measured by optical microscopic method for average particle size using an optical microscope ( olympus ) . samples were gold coated by mounted on a brass stub using double - sided adhesive tape and under vacuum in an ion sputter with a thin layer of gold ( 3 ~ 5 nm ) for 75 seconds and at 20 kv to make them electrically conductive , and their morphology was examined by scanning electron microscope ( zeiss evo 40 , japan ) . samples were reduced to powder and analyzed as kbr pellets by using a fourier transform - infrared ( ftir ) spectroscope ( perkin elmer spectrum rx i , usa ) . spectral scanning was taken in the wavelength region between 4000 and 400 cm at a resolution of 4 cm with scan speed of 1 cm / second . the release of glibenclamide from various ihm - alginate beads was tested using a dissolution apparatus usp ( campbell electronics , india ) . the baskets were covered with 100-mesh nylon cloth to prevent the escape of the beads . glibenclamide - loaded ihm - alginate beads equivalent to 30 mg glibenclamide were taken in 900 ml of dissolution medium ( 0.1 n hcl , ph 1.2 for first 2 hours and phosphate buffer , ph 7.4 for next 8 hours ) . an amount of 5 ml of aliquots was collected at regular time intervals , and the same amount of fresh dissolution medium was replaced into the dissolution vessel to maintain sink condition throughout the experiment . the collected aliquots were filtered and suitably diluted to determine absorbance using a uv - vis spectrophotometer ( shimadzu , japan ) at 228.5 nm against appropriate blank . in order to predict and correlate the in vitro release behaviour of glibenclamide from formulated glibenclamide - loaded ihm - alginate beads , it is necessary to fit into a suitable mathematical model . the in vitro drug release data were evaluated kinetically in different mathematical models [ 5 , 6 ] : ( i)zero - order model : q = kt + q0 , where q represents the drug released amount in time t , and q0 is the start value of q ; k is the rate constant;(ii)first - order model : q = q0e , where q represents the drug released amount in time t , and q0 is the start value of q ; k is the rate constant;(iii)higuchi model : q = kt , where q represents the drug released amount in time t , and k is the rate constant;(iv)korsmeyer - peppas model : q = kt , where q represents the drug released amount in time t , k is the rate constant , and n is the diffusional exponent , indicative of drug release mechanism . zero - order model : q = kt + q0 , where q represents the drug released amount in time t , and q0 is the start value of q ; k is the rate constant ; first - order model : q = q0e , where q represents the drug released amount in time t , and q0 is the start value of q ; k is the rate constant ; higuchi model : q = kt , where q represents the drug released amount in time t , and k is the rate constant ; korsmeyer - peppas model : q = kt , where q represents the drug released amount in time t , k is the rate constant , and n is the diffusional exponent , indicative of drug release mechanism . the korsmeyer - peppas model was also employed in the in vitro drug release behaviour analysis of these formulations to distinguish between competing release mechanisms [ 5 , 6 ] : fickian release ( diffusion - controlled release ) , non - fickian release ( anomalous transport ) , and case - ii transport ( relaxation - controlled release ) . when n is 0.43 , it is fickian release . the mucoadhesivity of optimized glibenclamide - loaded ihm - alginate beads was evaluated by ex vivo wash - off method [ 11 , 12 ] . freshly excised pieces of goat intestinal mucosa ( 2 2 cm ) ( collected from slaughterhouse ) were mounted on glass slide ( 7.5 2.5 cm ) using thread . fifty beads were spread onto the wet tissue specimen , and the prepared slide was hung onto a groove of disintegration test apparatus . the tissue specimen was given regular up and down movement in a vessel containing 900 ml of 0.1 n hcl ( ph 1.2 ) and phosphate buffer ( ph 7.4 ) , separately , at 37c . after regular time intervals , the machine was stopped and the number of beads still adhering to the tissue was counted . in vivo studies were performed in alloxan - induced diabetic albino rats of either sex ( weighing 266342 grams ) . the experimental protocol was subjected to the scrutiny of the institutional animal ethical committee and was cleared before starting . the animals were handled as per guidelines of committee for the purpose of control and supervision on experimental animals ( cpcsea ) . all efforts were made to minimize both the suffering and number of animals used . the rats were made diabetic by intraperitoneal administration of freshly prepared alloxan solution at a dose of 150 mg / kg dissolved in 2 mm citrate buffer ( ph 3.0 ) . after one week of alloxan administration , alloxanized rats with fasting blood glucose of 300 mg / dl or more were considered diabetic and were employed in the study for 12 hours . the alloxan - induced diabetic rats were divided randomly into 2 groups of 6 rats each and treated as follows . group a was administered with pure glibenclamide in suspension form , and group b was administered with optimized formulation of glibenclamide - loaded ihm - alginate beads , both at a dose equivalent to 5 mg glibenclamide / kg body weight using oral feeding needle . blood samples were withdrawn ( 0.1 ml ) from tail tip of each rat at regular time intervals under mild ether anesthesia and were analyzed for blood glucose by oxidase - peroxidase method using accu - chek sensor comfort ( roche diagnostics , germany ) test strips . statistical optimization was performed using design - expert version 8.0.6.1 software ( stat - ease inc . the in vivo data were tested for significant differences ( p < 0.05 ) by paired samples t - test . all other data was analyzed with simple statistics . the simple statistical analysis and paired samples t - test ihm was isolated from ispaghula ( plantago ovata f. ) husk , and the average yield of ihm was found 39.86% w / w . for the 3 factorial design , a total of 9 trial formulations were proposed by design - expert version 8.0.6.1 software . according to this trial proposal , various glibenclamide - loaded ihm - alginate beads were prepared by ionotropic - gelation technique . when various dispersion mixtures containing polymer - blend ( sa and ihm ) and glibenclamide were dropped into the solutions containing calcium ions , gelled glibenclamide - loaded ihm - alginate beads were formed instantaneously due to electrostatic interaction between negatively charged alginate ions and positively charged calcium ions present in the cross - linking solutions . overview of matrix of the design including investigated responses ( dee and r10 h ) was presented in table 1 . the values of dee and r10 h , measured for all trial formulations , were fitted in the 3 factorial design to get model equations . the design - expert version 8.0.6.1 software provided quadratic model equations involving individual main factors and interaction factors for all response parameters . the results of the anova indicated that these models were significant for all response parameters ( table 2 ) . the model equation relating dee ( % ) as response became dee ( % ) = 87.24 5.22x1 2.49x2 0.06x1x2 + 0.41x1 + 0.35x2 ( r = 0.9999 ; f value = 10398.24 ; p < 0.05 ) . the model equation relating r10 h(% ) as response became r10 h ( % ) = 83.94 + 2.37x1 + 0.01x2 + 0.34x1x2 0.13x1 0.23x2 ( r = 0.9981 ; f value = 309.07 ; p < 0.05 ) . model simplification was carried out by eliminating nonsignificant terms ( p > 0.05 ) from previously mentioned model equations , giving dee ( % ) = 87.24 5.22x1 2.49x2 0.06x1x2 + 0.41x1 + 0.35x2 and r10 h(% ) = 83.94 + 2.37x1 + 0.01x2 + 0.34x1x2 0.23x2 . linear correlation plots between the actual , the predicted response variables are presented in figures 1 and 2 , and their corresponding residual plots showing the scatter of the residuals versus predicted values are presented in figures 3 and 4 . the influences of main effects ( factors ) on responses ( here , dee and r10 h ) were further elucidated by response surface methodology . response surface methodology is a widely proficient approach in the development and optimization of drug delivery devices [ 5 , 8 , 28 ] . response surface methodology encompasses the generation of model equations of the investigated responses over the experimental domain to determine optimum formulation ( s ) . the three - dimensional response surface plot is very useful in learning about the main and interaction effects of the independent variables ( factors ) , whereas two - dimensional contour plot gives a visual representation of values of the response . the three - dimensional response surface plot relating dee ( figure 5 ) indicates the increment of dee with the lowering of sa : ihm ( x1 ) and increasing of cacl2 concentration ( x2 ) . however , an increment in r10 h values with the increasing of sa : ihm ( x1 ) and lowering of cacl2 concentration ( x2 ) is indicated by the three - dimensional response surface plot relating r10 h ( figure 6 ) . all the two - dimensional contour plots relating measured responses ( figures 7 and 8) showed nonlinear relationships between independable variables , investigated for this study . numerical optimization technique using the desirability approach was employed to develop optimized formulations with desired response ( optimum quality ) . the desirable ranges of the independable variables ( factors ) were restricted to 1.00 x1 1.50 and 9.50 x2 11.50 , whereas the desirable ranges of responses were restricted to 95.00 dee 100.00% and 60.00 r10 h 65.00% . the optimal values of responses were obtained by numerical analysis using the design - expert version 8.0.6.1 software based on the criterion of desirability . the desirability plot indicating desirable regression ranges for optimal process variable settings was presented in figure 9 , and overlay plot indicating the region of optimal process variable settings was presented in figure 10 . in order to evaluate the optimization capability of these models generated according to the results of 3 factorial design , optimized glibenclamide - loaded ihm - alginate beads were prepared using one of the optimal process variable settings proposed by the design ( prediction r = 1 ) . the selected optimal process variable setting used for the formulation of optimized formulation was x1 = 1.35 and x2 = 10.99 . the optimized beads containing glibenclamide ( f - o ) were evaluated for dee ( % ) and r10 h(% ) . table 3 lists the results of experiments with predicted responses by the mathematical models and those actually observed . the optimized glibenclamide - loaded ihm - alginate beads ( f - o ) showed dee of 94.43 4.80% and r10 h of 65.78 3.44% with small error values ( 0.94 , and 3.69 , resp . ) , indicating that mathematical models obtained from the 3 factorial design were fitted well . the dee ( % ) of all these glibenclamide - loaded ihm - alginate beads was within the range between 68.03 1.77 and 94.43 4.80% w / w ( tables 1 and 3 ) . it was observed that dee ( % ) was increased with the lowering of sa : ihm in polymer - blend , which may be due to increase in viscosity of the polymeric solution by the ihm addition as polymeric - blend with sa . this might have prevented drug leaching to the cross - linking solution and the elevation of cross - linking by cacl2 . again , the dee of these beads was increased with increasing cacl2 concentration in cross - linking solutions , due to the high degree of cross - linking by the concentrated calcium ions . the glibenclamide - loaded ihm - alginate beads prepared using lower cacl2 concentration might have larger pores due to insufficient cross - linking , and drug leaching may occur through the pores that may result in lower drug encapsulation . the average bead size of glibenclamide - loaded ihm - alginate beads was within the range of 0.80 0.06 to 1.47 0.12 mm ( table 4 ) . increase in the average size of beads was found with the increasing incorporation of ihm as a polymer - blend with sa . this could be attributed due to the increase in viscosity of polymer - blend solution with incorporation of ihm in increasing ratio that in turn increased the droplet size of polymer - blend solutions to the cross - linking solutions during preparation . again , the decrease in average size of these formulated ihm - alginate beads was observed , when concentrated cacl2 solution was used for cross - linking , which might be due to shrinkage of polymeric gel by higher degree of cross - linking . the surface morphological analysis of glibenclamide - loaded ihm - alginate beads was visualized by sem and presented in figure 11 . their surface morphologies appeared to have rough with characteristic large wrinkles and cracks , as it was evident from the sem photographs . these cracks and wrinkles might be caused by partly collapsing the polymeric gel network during drying . the ftir spectra of pure glibenclamide , glibenclamide - loaded ihm - alginate beads , and ihm - alginate beads without drug are shown in figure 12 . the ftir spectrum of pure glibenclamide and the principal absorption peaks appeared at 3314 cm due to the nh stretching , 3116 cm for aromatic hydrogen absorption , and a peak at 1717 cm occurs due to c = o absorption peak . in the ftir spectrum of glibenclamide - loaded ihm - alginate beads , this indicates that glibenclamide maintained its identity after formulation of ihm - alginate beads through ionotropic - gelation technique . in both the ftir spectra of glibenclamide - loaded ihm - alginate beads and ihm - alginate beads without drug , the strong and broad absorption band peaks had been observed between 36003200 cm due to oh stretching along with some complex bands in the region 12001030 cm due to c o and c o c stretching vibrations , which are the characteristic of the natural polysaccharides . in addition , absorption bands in the regions 930820 cm and 785730 cm were also observed due to vibrational modes of pyranose rings of polysaccharides . the presence of strong asymmetric stretching absorption band between 1650 cm and 1620 cm and weaker symmetric stretching band near 1420 cm supported the presence carboxylate anion of alginate structure . the ftir analysis confirmed the compatibility of the glibenclamide with sa and ihm used to prepare the glibenclamide - loaded ihm - alginate beads by ionotropic - gelation technique . the in vitro glibenclamide release studies were carried out for glibenclamide - loaded ihm - alginate beads in the 0.1 n hcl ( ph , 1.2 ) for first 2 hours and then in phosphate buffer ( ph , 7.4 ) for next 8 hours . glibenclamide release from these ihm - alginate beads in the acidic ph was found slow due to the shrinkage of alginate at acidic ph . the trace amount of drug release at the initial stage of the dissolution study could probably be due to the surface adhered drug . after that , glibenclamide release was observed faster in phosphate buffer ( ph , 7.4 ) comparatively , due to the higher swelling rate of these beads in phosphate buffer . the cumulative drug released from these formulated beads containing glibenclamide in 10 hours ( r10 h , % ) was within the range of 65.78 3.44% to 92.07 4.05% , and this was found to be higher with the decreasing sa to ihm ratio in the polymer - blend and increasing cacl2 concentration in cross - linking solution . in case of comparatively higher ihm containing beads , the more hydrophilic property of ihm could bond better with water to form viscous gel - structure . this might blockade the pores on the surface of beads and sustain drug release profile . again , the glibenclamide release from ihm - alginate beads prepared using higher cacl2 concentration was comparatively sustained than the beads formulated with that of lower concentration . the higher concentration of cacl2 ( cross - linker ) could produce high degree of cross - linking and thereby slower the drug release from highly cross - linked glibenclamide - loaded ihm - alginate beads . the in vitro drug release data from various glibenclamide - loaded ihm - alginate beads were evaluated kinetically using various mathematical models like zero - order , first - order , higuchi , and korsmeyer - peppas models . the result of the curve fitting ( r ) into various mathematical models is given in table 5 . when the respective r of glibenclamide - loaded ihm - alginate beads was compared , it was found to follow the zero - order model ( r = 0.992 to 0.997 ) over a period of 10 hours . this was also observed to be closest to korsmeyer - peppas model ( r = 0.985 to 0.994 ) . the best fit of zero - order model indicated that the glibenclamide release from these ihm - alginate beads followed controlled - release pattern . the values of diffusional exponent ( n ) determined from korsmeyer - peppas model ranged from 1.025 to 1.115 , indicating the drug release from these glibenclamide - loaded ihm - alginate beads following the super case - ii transport mechanism controlled by swelling and relaxation of polymeric - blend ( sa - ihm ) matrix . this could be attributed due to polymer dissolution and polymeric chain enlargement or relaxation . the ex vivo wash - off behavior of optimized glibenclamide - loaded ihm - alginate beads ( f - o ) using goat intestinal mucosa was found faster in intestinal ph ( 7.4 ) than that in gastric ph ( 1.2 ) . in gastric ph , the percentage of beads adhered onto the goat intestinal mucosal tissue varied from 64.88 5.06% over 10 hours , whereas this was 30.47 3.86% in intestinal ph ( figure 14 ) . thus , the results of the ex vivo wash - off test indicated that the newly developed optimized glibenclamide - loaded ihm - alginate beads had good mucoadhesivity . in alloxan - induced diabetic rats , the comparative in vivo blood glucose level and the mean percentage reduction in blood glucose level after oral administration of pure glibenclamide and optimized glibenclamide - loaded ihm - alginate mucoadhesive beads ( f - o ) are presented in figures 15 and 16 , respectively . in case of the group treated with pure glibenclamide ( group a ) , a rapid reduction in blood glucose level was observed within 2 - 3 hours of administration , and after that , the blood glucose level recovered rapidly towards the normal level . in case of the group ( group b ) treated with optimized glibenclamide - loaded ihm - alginate mucoadhesive beads , the reduction in blood glucose level was found slower than that of the group treated with pure glibenclamide ( group a ) up to 3 hours . significant differences ( p < 0.05 ) were found between the blood glucose levels after administration of pure glibenclamide and optimized glibenclamide - loaded ihm - alginate mucoadhesive beads ( f - o ) at each time point measured . however , the reductions in glucose level were increased gradually with the increment of time in case of group b ( treated with optimized glibenclamide - loaded ihm - alginate mucoadhesive beads ) and were sustained over 10 hours . a 25% reduction in glucose level is considered a significant hypoglycemic effect . therefore , the significant hypoglycemic effect by the optimized glibenclamide - loaded ihm - alginate mucoadhesive beads ( f - o ) was observed over 10 hours . in this investigation , glibenclamide - loaded ihm - alginate mucoadhesive beads were successfully developed and optimized . these developed optimized mucoadhesive beads demonstrated high drug encapsulation , good mucoadhesivity with the biological membrane , sustained drug release profile at a controlled rate , and significant antidiabetic activity in alloxan - induced diabetic rats over prolonged period after oral administration . therefore , these glibenclamide - loaded ihm - alginate mucoadhesive beads were found suitable for prolonged systemic absorption of glibenclamide through sustained drug release and mucoadhesive properties after oral administration maintaining tight blood glucose level and improved patient compliance in the management of non - insulin - dependent diabetes mellitus . moreover , the technique for the preparation of these beads was found simple , economical , and consistent . this type of beads can also be exploited for drug delivery of other drugs to improve their bioavailability and therapeutic efficacy .

the current study deals with the development and optimization of ispaghula ( plantago ovata f. ) husk mucilage- ( ihm- ) alginate mucoadhesive beads containing glibenclamide by ionotropic gelation technique . the effects of sodium alginate ( sa ) to ihm and cross - linker ( cacl2 ) concentration on the drug encapsulation efficiency ( dee , % ) , as well as cumulative drug release after 10 hours ( r10 h , % ) , were optimized using 32 factorial design based on response surface methodology . the observed responses were coincided well with the predicted values by the experimental design . the optimized mucoadhesive beads exhibited 94.43 4.80% w / w of dee and good mucoadhesivity with the biological membrane in wash - off test and sustained drug release profile over 10 hours . the beads were also characterized by sem and ftir analyses . the in vitro drug release from these beads was followed by controlled release ( zero - order ) pattern with super case - ii transport mechanism . the optimized glibenclamide - loaded ihm - alginate mucoadhesive beads showed significant antidiabetic effect in alloxan - induced diabetic rats over prolonged period after oral administration .

several attempts have been made to address this problem such as diet changes , lowering carbohydrate intake , efficient oral hygiene measures and rinsing fluoride mouthwashes . application of sealants , fluoride releasing bonding agents , elastic modules impregnated with fluoride and fluoride varnishes are the most commonly used preventative methods by orthodontists . despite all these efforts , we still witness white spot lesions following bracket removal . if a void exists between the adhesive and enamel surface at the margin of brackets , this could lead to microleakage and accumulation of cariogenic bacteria in inaccessible areas . these lesions occur in about 45% of orthodontic patients and according to a previous study , they have a higher incidence in males . they are unsightly and appear as enamel opacity , involving at least one - third of the labial surface . corrosion and craters in the stainless steel bracket base initiate at the gap developed between the brackets and adhesives . investigated the amount of microleakage following the application of different adhesive systems for bonding ceramic and metal brackets . in their study , the amount of microleakage was reported to be higher beneath the metal brackets at both interfaces regardless of the adhesive system used . uysal et al , also reported higher amount of microleakage at the gingival in comparison to the occlusal margin ( light curing was done at the occlusal margin for both adhesive systems ) . although use of light activated composites has become popular in orthodontics , prevention of light penetration by metal brackets , the most commonly used attachments in fixed orthodontics , is a major concern . also , the stress due to the polymerization shrinkage is among the most important problems associated with light - activated composites . if the contracting forces of curing resins overcome the bond of adhesive , marginal failure and microleakage may result . to overcome this side effect , the suggested three - sided light curing technique was based on the assumption that contraction of photo - activated composite resin is directed toward the light source . in our study , the transillumination technique was used to assess its efficacy for bonding of metal brackets . this method was initially suggested for cementation of acid - etched fixed partial dentures . in this technique , some previous studies have investigated the bond strength of visible light - cured composites using transillumination as a curing technique [ 1518 ] . in 2013 , kumar et al . showed that 90% of light intensity was lost when using transillumination technique for bonding brackets . however , they found that both the conventional method and transillumination technique yielded similar results in terms of bond strength of metal brackets . also , in 2013 heravi et al . concluded that to achieve an acceptable bracket bond strength for the posterior teeth , doubling the curing time from 40 to 80 seconds and increasing the light intensity to 800 mw / cm during transillumination technique were required . from the previous studies it can be concluded that transillumination is an applicable technique for improving the bond strength ; however , marginal seal is also an important factor to prevent marginal corrosion and bacterial attacks , and inappropriate marginal seal can lead to white spot lesions or bracket bond failure during the course of treatment . to the best of our knowledge , no study has evaluated the effect of transillumination as a curing technique on the microleakage beneath metal orthodontic brackets . the aim of this study was to evaluate the effect of transillumination as a light curing technique on the amount of microleakage , in comparison to the conventional technique of curing , using two methods of enamel conditioning . according to a study by arhun et al , 120 freshly extracted bovine deciduous mandibular incisors were collected for this study . the teeth were examined to be free of surface developmental defects and cracks under direct light . the soft tissue remnants and the debris were removed and the specimens were polished with pumice paste and rubber cups for 10 seconds each , and were then stored in distilled water for one month . before the onset of the bonding process , all the specimens were disinfected by immersion in 1% thymol solution for one week . the middle - third of the enamel surface of each tooth was prepared using two enamel preparation methods . the first method included the use of 37% phosphoric acid gel ( unitek , monrovia , ca ) for enamel conditioning and transbond xt primer ( 3 m , unitek , monrovia , ca ) as a sealant . in the second method , transbond plus ( 3 m , unitek , monrovia , ca ) was applied as self - etching primer . the stainless steel twin maxillary central incisor brackets ( .022-in - daynalock series , 3 m , monrovia , ca ) were bonded on the middle third of the buccal surface in all samples with transbond xt ( 3 m , unitek , monrovia , ca ) , a light - cured orthodontic resin , using a light curing unit ( coltolux 75 , coltene / whaledent gmbh , langenau , germany ) with a light intensity of 530 mw / mm . in order to maintain a fixed distance and angle of light curing , a holder was designed and used to set the position of the samples at 5 mm distance from the tip of the light - curing unit . the device held the samples vertically in such a way that the tip of the light - curing unit was perpendicular to the long axis of the samples on both sides depending on the group of samples ( fig . the samples were randomly divided into four groups of 30 teeth and were prepared as follows : group i : the teeth were etched with 37% phosphoric acid for 30 seconds , and then rinsed and dried for 20 seconds with oil - free air spray . the brackets were bonded using transbond xt primer as the sealant and transbond xt as the adhesive . finally , the mesial and distal margins of the brackets were light cured for 10 seconds each.group ii : the same methods of enamel preparation and bracket bonding were applied as in group i ; but transillumination ( applying the light source to the middle third of the lingual surface ) was used for 50 seconds as the technique of resin polymerization.group iii : transbond plus self - etching primer was applied for 35 seconds with mild pressure of the brush according to the manufacturer s instructions , followed by a gentle burst of air for 12 seconds . then , the brackets were bonded using transbond xt and 10 seconds of light curing was performed at the mesial and distal margins.group iv : the same method of enamel preparation as in group iii was applied ; but transillumination for 50 seconds from the lingual side was used as the method of curing . thermal cycling in deionized water was performed at 5 2 c 55 2 c for 500 cycles with a dwell time of 30 seconds and transfer time of 5 seconds . then , the samples were stored in distilled water at room temperature and a dark environment for three months for the aging process . group i : the teeth were etched with 37% phosphoric acid for 30 seconds , and then rinsed and dried for 20 seconds with oil - free air spray . the brackets were bonded using transbond xt primer as the sealant and transbond xt as the adhesive . finally , the mesial and distal margins of the brackets were light cured for 10 seconds each . group ii : the same methods of enamel preparation and bracket bonding were applied as in group i ; but transillumination ( applying the light source to the middle third of the lingual surface ) was used for 50 seconds as the technique of resin polymerization . group iii : transbond plus self - etching primer was applied for 35 seconds with mild pressure of the brush according to the manufacturer s instructions , followed by a gentle burst of air for 12 seconds . then , the brackets were bonded using transbond xt and 10 seconds of light curing was performed at the mesial and distal margins . group iv : the same method of enamel preparation as in group iii was applied ; but transillumination for 50 seconds from the lingual side was used as the method of curing . thermal cycling in deionized water was performed at 5 2 c 55 2 c for 500 cycles with a dwell time of 30 seconds and transfer time of 5 seconds . then , the samples were stored in distilled water at room temperature and a dark environment for three months for the aging process . for evaluation of microleakage in all margins , each group was randomly divided into two subgroups of 15 samples in order to perform tooth sectioning in two different directions . in subgroup one , the teeth were sectioned from the middle of the brackets in incisogingival direction while in subgroup two , the teeth were sectioned in mesiodistal direction to evaluate the margins that were directly light cured as instructed by the manufacturer . the apices of all teeth were sealed with sticky wax and then all surfaces were coated with two layers of nail varnish except for 1 mm around the bracket margins . in the next step , the samples were immersed in 0.5% basic fuchsine solution for 24 hours at room temperature . after removal from the solution , the teeth were rinsed with distilled water , the superficial dye was removed with a brush and the teeth were left to dry . the samples were embedded in epoxy resin blocks according to the direction of sections using an index in the heavy putty impression ( fig . one sample fixed in the holding chuck of the cutting machine ( struers , denmark ) before sectioning ( a ) and after sectioning ( b ) sectioning was carried out using a low - speed diamond saw ( accutom-50 , struers , denmark ) . all samples were numbered before sectioning according to their group allocation and were examined randomly under a stereomicroscope ( motic , xiamen , china ) at 40 magnification . the microleakage scores were directly recorded using an electronic digital caliper ( guanglu measuring instrument co. ltd , shanghai , china ) by a single blinded observer ( sh.a ) . half of the samples were randomly examined blindly for the second time under the same stereomicroscope by the same observer ( sh.a ) after a week to assess the intra - observer error of measurements . the incisogingival and mesiodistal dimensions of each section were examined at enamel - adhesive and adhesive - bracket interfaces ( in each side ) and scored based on the amount of microleakage ( fig . stereomicroscopic views ( a and b ) of a sample sectioned in mesiodistal direction ( black arrow : enamel - adhesive interface , white arrow : adhesive - bracket interface ) for data analysis , the mean and standard deviation values of all groups were obtained using spss version 15.0 ( microsoft , il , usa ) . to compare the sides and interfaces within each group ( dependent samples ) , the non - parametric wilcoxon singed rank test was used . the kruskal - wallis ( independent non - parametric test ) and the mann - whitney u tests with bonferroni correction were used to compare the groups . for evaluation of microleakage in all margins , each group was randomly divided into two subgroups of 15 samples in order to perform tooth sectioning in two different directions . in subgroup one , the teeth were sectioned from the middle of the brackets in incisogingival direction while in subgroup two , the teeth were sectioned in mesiodistal direction to evaluate the margins that were directly light cured as instructed by the manufacturer . the apices of all teeth were sealed with sticky wax and then all surfaces were coated with two layers of nail varnish except for 1 mm around the bracket margins . in the next step , the samples were immersed in 0.5% basic fuchsine solution for 24 hours at room temperature . after removal from the solution , the teeth were rinsed with distilled water , the superficial dye was removed with a brush and the teeth were left to dry . the samples were embedded in epoxy resin blocks according to the direction of sections using an index in the heavy putty impression ( fig . one sample fixed in the holding chuck of the cutting machine ( struers , denmark ) before sectioning ( a ) and after sectioning ( b ) sectioning was carried out using a low - speed diamond saw ( accutom-50 , struers , denmark ) . all samples were numbered before sectioning according to their group allocation and were examined randomly under a stereomicroscope ( motic , xiamen , china ) at 40 magnification . the microleakage scores were directly recorded using an electronic digital caliper ( guanglu measuring instrument co. ltd , shanghai , china ) by a single blinded observer ( sh.a ) . half of the samples were randomly examined blindly for the second time under the same stereomicroscope by the same observer ( sh.a ) after a week to assess the intra - observer error of measurements . the incisogingival and mesiodistal dimensions of each section were examined at enamel - adhesive and adhesive - bracket interfaces ( in each side ) and scored based on the amount of microleakage ( fig . stereomicroscopic views ( a and b ) of a sample sectioned in mesiodistal direction ( black arrow : enamel - adhesive interface , white arrow : adhesive - bracket interface ) for data analysis , the mean and standard deviation values of all groups were obtained using spss version 15.0 ( microsoft , il , usa ) . to compare the sides and interfaces within each group ( dependent samples ) , the non - parametric wilcoxon singed the kruskal - wallis ( independent non - parametric test ) and the mann - whitney u tests with bonferroni correction were used to compare the groups . the overall intra - observer agreement for each group was high ( kappa value of 0.792 ) . in case of disagreements , the mean of measurements was reported . all groups showed microleakage at the incisal and gingival margins ; but in comparison between conventional and transillumination groups , significant differences were observed only between incisal and gingival margins in the transillumination group ( p<0.001 ) . comparison of mesial and distal margins revealed no statistically significant differences in any group ( p>0.05 ) . when direct illumination and transillumination were compared as the two methods of curing , the amount of microleakage was significantly higher at the gingival margins compared to the incisal margins in both interfaces in the transillumination group irrespective of the method of enamel preparation ( p<0.05 ) . at the mesial and distal margins comparison of groups based on the method of enamel conditioning revealed no significant differences at the incisal and gingival margins in any of the interfaces ; but at the mesial and distal margins , only group seven ( self - etching primer + direct illumination ) showed significantly lower microleakage score in comparison with group five ( acid - etching + direct illumination ) ( p=0.001 ) . comparison of microleakage scores ( mm ) between enamel - adhesive and adhesive - bracket interfaces in buccolingual sections n indicates sample size ; sd , standard deviation ; s , significant ; ns , not significant group 1 : acid etching + direct illumination ; group 2 : acid etching + transillumination ; group 3 : self etching primer + direct illumination ; group 4 : self etching primer + transillumination . comparison of microleakage scores ( mm ) between enamel - adhesive and adhesive - bracket interfaces in mesiodistal sections n indicates sample size ; sd , standard deviation ; s , significant ; ns , not significant group 5 : acid etching + direct illumination ; group 6 : acid etching + transillumination ; group 7 : self etching primer + direct illumination ; group 8 : self etching primer + transillumination multiple comparisons among all groups are shown in tables 3 and 4 with bonferroni correction . multiple comparisons of microleakage scores among groups at incisal and gingival margins in enamel - adhesive and adhesive - bracket interfaces n indicates sample size ; sd , standard deviation ; s , significant ; ns , not significant ; group 1 : acid etching + direct illumination ; group 2 : acid etching + transillumination ; group 3 : self etching primer + direct illumination ; group 4 : self etching primer + transillumination . multiple comparisons of microleakage scores among groups at the mesial and distal margins in enamel - adhesive and adhesive - bracket interfaces n indicates sample size ; sd , standard deviation ; s , significant ; ns , not significant ; group 5 : acid etching + direct illumination ; group 6 : acid etching + transillumination ; group 7 : self etching primer + direct illumination ; group 8 : self etching primer + transillumination . this process occurs rapidly and mineral loss has been reported even within a few months of treatment initiation . the aim of the current study was to compare the amount of microleakage of an orthodontic adhesive following the use of two different methods of enamel conditioning and light curing . in this study , we used bovine incisors as available substitutes for human incisors . bovine deciduous lower incisors have nearly the size of permanent human maxillary central incisors , which are the most ideal for testing bonding properties since they provide flat bonding surfaces . these two types of teeth have been compared in a number of previous studies [ 15,20 , 21 ] . the assumption that the contraction of photo - activated composite resins is directed toward the light source , and also the problem of not being able to directly cure the composite resin under metal brackets led to the idea of evaluating transillumination as a method of curing in this study . behrents et al , also supported the use of this technique for bonding of lingual attachments due to its practical application in the oral environment . in the current study , one reason for less microleakage following the use of self - etching adhesive systems at margins cured directly could be the simultaneous penetration of etchant and monomer and the identical length of primer tags in the etched enamel . in contrast to the conventional method of enamel conditioning , self - etching primers produce a uniform and more conservative etched pattern providing uniform adhesive penetration and less aggressive enamel demineralization . in a study conducted by vicente et al , on the effect of thermocycling on the microleakage beneath brackets bonded to bovine incisors , they found that microleakage increased significantly at the enamel - adhesive interface when using transbond xt as the bonding agent . in the current study , this may explain the greater amount of microleakage at all margins in comparison with some other studies on this topic [ 7 , 24 ] . in 2013 , sabzevari et al . demonstrated that thermocycling did not significantly increase the microleakage when a self - etching primer was used as a conditioner . however , comparison of different methods of bonding after thermocycling showed no significant differences between self - etching primers and the conventional method of conditioning . surface conditioning causes leakage of fluid and bacteria beneath the orthodontic brackets ; thus , a deeper etching pattern with acid etchant can not guarantee an interface free of microleakage . on the other hand , less penetrated resin tags in these systems may not resist the contracting forces of resin shrinkage . however , this is acceptable in restorative dentistry when a bulk of composite is placed in a prepared cavity . orthodontic adhesive layers are very thin and the free , floating brackets are pulled closer to the enamel surface by the shrinkage . the amount of microleakage reported in our study was lower than that in some previous studies ; this finding is in agreement with less microleakage reported for transbond xt in an in vitro study by sener et al . less microleakage at the margins of specimens in self - etching primer groups cured directly is similar to the results of a study by uysal et al , which showed significantly higher scores at the gingival compared to the occlusal margins ( where the tip of the light curing unit was positioned ) . another factor that should be taken into account regarding the microleakage scores is a phenomenon called percolation . the linear coefficient of thermal expansion for enamel , metal brackets and the adhesive is not the same ( =12 for enamel , =16 for stainless steel brackets and =2055 ppm / c for composite resin ) . these materials expand and contract at different rates when hot and cold foods are consumed ; thus , the fluids are sucked in and pushed out at the margins of the brackets bonded to the teeth in both tooth - adhesive and adhesive - bracket interfaces . the greater the light energy received by the composite , the greater the polymerization ; therefore , transillumination must provide greater light energy than direct curing . since pulpal temperature should not exceed 56 c , extending the exposure time should be done with caution . with 1 mm of dentine between the composite and the pulp , the temperature increases to 6c with 40 seconds of continuous exposure . in orthodontics , oesterle and shellhart reported comparable bond strength in the group using transillumination for 50 seconds with the group cured for 40 seconds at the margins . however , heravi et al . concluded that 80 seconds of curing with high power mode of blue phase c8led light curing unit with transillumination technique resulted in a clinically acceptable shear bond strength of brackets to posterior teeth with no risk of pulpal damage . as reported by yazici et al , and haiduc et al , led units cause significantly lower rise of pulp temperature in comparison with halogen light curing units . in the current study , the amount of microleakage in the transillumination groups was comparable to that in the conventional curing groups at most of the margins of brackets except for the gingival margin . this may be due to the reflection of light from the bracket base as was pointed out by oesterle and shellhart . showed that even though the amount of light intensity lost in transillumination technique was significant , there were no differences in the shear bond strength of brackets bonded by transillumination technique or conventional method of curing at the mesial and distal margins . in our study , only at the gingival margin of the samples , irrespective of the method of enamel conditioning , a significant amount of microleakage was observed . this may be explained by the gradual increase of buccolingual width from the incisal toward the gingival side . consequently , although some studies reported adequate bond strength by transillumination , microleakage should be a concern especially in teeth with greater thickness . as concluded by heravi et al , to achieve an acceptable bracket bond strength to the posterior teeth , doubling the curing time from 40 to 80 seconds and increasing the light intensity to 800 mw / cm during the transillumination technique should be done . in studies by ramuglu et al , and uysal et al , light was irradiated from the occlusal surface and a significant amount of microleakage was reported at the gingival margin . they reasoned that this result might be due to the degradation of light intensity and insufficient polymerization of composite . other studies in which the samples were cured according to the manufacturer s instructions did not score the microleakage at the directly cured margins ( mesial and distal margins ) . in our study , the manufacturers instructions were followed in all groups and evaluation of all margins showed that the amount of microleakage at the mesial and distal margins was lower than that at the incisal and gingival margins and these differences were statistically significant in groups which received self - etching primer for enamel conditioning . polymerization starts in areas of resin closest to the light source . even for metal brackets the best result will be gained if light is irradiated from all four sides of brackets . however , a well - designed study on this technique of curing may better elucidate this topic . it is impossible to extrapolate the results of an in vitro study to the actual oral environment ; thus , future studies are necessary for further assessment of results . within the limitations of this study , use of self - etching primer and conventional light curing method caused less microleakage compared to the transillumination technique . the transillumination technique , irrespective of the method of enamel preparation , caused greater microleakage in both interfaces at the gingival margin and thus should not be used as the method of curing in orthodontic treatment .

objectives : this study aimed to compare the microleakage beneath metallic brackets following two different methods of enamel preparation and light curing.materials and methods : a total of 120 bovine deciduous lower incisors were randomly divided into four groups of 30 teeth . the preparations were as follows : group i : acid etching + transbond xt primer + direct illumination , group ii : acid etching + transbond xt primer + transillumination , group iii : transbond xt self - etching primer + direct illumination and group iv : transbond xt self - etching primer + transillumination . dye penetration was used as the method of microleakage evaluation . sections made at the enamel - adhesive and adhesive - bracket interfaces were evaluated under a stereomicroscope . the kruskal - wallis and mann - whitney u tests were used for statistical analysis . the level of significance was set at p<0.05.results : all groups showed greater microleakage at the gingival in comparison to the incisal margin and the differences were significant among groups with transillumination ( p<0.001 ) . no significant differences were observed in the microleakage scores at the gingival and incisal margins in any of the interfaces ( p>0.05 ) . mesiodistal margins of the self - etching group with direct illumination showed significantly lower scores in comparison with acid etched group ( p<0.05).conclusion : use of self - etching primers for bonding of orthodontic brackets yields acceptable results if all bracket margins are cured directly .

subjective cognitive complaints ( scc ) are quite prevalent among older adults , with some estimates suggesting that between 25% and 50% of all older adults have self - perceived memory impairment [ 1 , 2 ] . in clinical practice , it is often difficult to assess the veracity and severity of subjective cognitive complaints , primarily because such complaints vary widely from individual to individual . as a result , clinicians and caregivers perhaps do not consider subjective complaints to have the same weight as objective findings . however , studies have shown that subjective complaints may be valid indicators of current and future cognitive impairment . a recent study by amariglio and colleagues showed that certain subjective complaints , such as i have trouble finding my way around familiar streets , are correlated with impairment in delayed recall , naming , and semantic fluency . a review conducted by jonker and colleagues showed that memory complaints may be predictive of dementia or alzheimer 's disease onset within two to four years , especially in individuals with a diagnosis of mild cognitive impairment ( mci ) . subjective cognitive complaints also have clinical implications that are outside the realm of cognitive function . a review by mol and colleagues found that scc correlated with depression , anxiety [ 4 , 5 ] , and low level of well - being , even in the absence of objective cognitive impairment . scc and objective cognitive function in older patients with and without major depression and found no changes in objective cognitive function between the two groups , but significantly more scc in the depressed group . scc have also been correlated with decreased functional ability , even when depression is controlled for . although the direction of causality between many of these variables remains unclear , scc should be considered in a clinical setting because of their associations with objective impairment , dementia , depression , low quality of life , and functional ability . previous research in the area of scc has largely focused on memory complaints [ 13 , 7 , 8 ] . in the current study , we have broadened the scope of scc in order to examine other areas of cognition that have shown impairment following complaints . the most common method appears to be a single , dichotomous , yes or no assessment of complaints ( e.g. , do you find that you have trouble with your memory ? ) [ 713 ] , or a combination of similar yes or no questions [ 3 , 14 , 15 ] . furthermore , most of the existing literature on scc examines community - dwelling , healthy , and older populations [ 3 , 7 , 9 , 12 , 16 ] . subjective cognitive complaints , in addition to predicting the onset of a neurodegenerative process , may yield important information about areas of impairment , specifically in patients with mild disease . two such disorders are mild cognitive impairment ( mci ) and its vascular equivalent , vascular mild cognitive impairment ( vamci ) . differences in functional impairment between individuals with mci and those with vamci , which may be overlooked by objective cognitive testing , may be predicted by an analysis of significance of scc . in the current study , we compared the profile of scc in individuals with a diagnosis of mci to those with vamci using the neuropsychological impairment scale ( nis ) . the nis is a comprehensive scale - based questionnaire , which may elicit differences in presence and severity of scc between the two groups . we hypothesized that the severity of subjective cognitive complaints would not differ between the two groups , because objective cognitive functioning is similar . however , we hypothesized that the specific types of scc might differ between mci and vamci , due to the differing pathologies of these two disorders . twenty patients with mci and twenty patients with vamci matched on demographic characteristics ( age , education ) were recruited from the memory disorders clinic at st . patients are referred to the clinic by their primary or secondary care physicians , for assessment and diagnosis of cognitive impairment . patients typically undergo a standardized history and a standardized cognitive exam as part of the workup . in addition , routine blood work including tsh , b12 , and rbc folate is done to rule out reversible causes of dementia . as well , most patients receive structural imaging ( either computed tomography ( ct ) or magnetic resonance imaging ( mri ) ) and in some cases functional imaging ( single photon emission computed tomography ( spect ) ) . for this study , only patients clinically diagnosed with either mci or vamci were recruited . the diagnostic criteria for mci describe cognitive impairment which is more severe than normal aging but less severe than dementia and typically not associated with serious impairment in everyday functions . vamci is described as the vascular equivalent of mci , thereby indicating a similar level of objective cognitive performance despite possible differences in mechanism and etiology . currently , there are no specific neuroimaging or vascular criteria that are necessary for a diagnosis of vamci . some patients underwent mri scans instead of ct scans as part of their clinical visit . as we used different imaging modalities we could not compare degree of white matter disease across study subjects in a quantifiable manner . rather , patients were assigned to the vamci group if scans showed evidence of white matter lesions , lacunar infarcts , and/or moderate to severe microangiopathic change . cognitive testing and data intake occurred during the patients ' initial visits to the clinic . follow - up visits to the memory disorders clinic were not considered for this cross - sectional analysis . subjective cognitive complaints were assessed using the neuropsychological impairment scale ( nis ) [ 20 , 21 ] . the nis is a questionnaire consisting of 95 complaints , such as i am forgetful and i am easily distracted . subjects rated these statements on a five - point likert scale according to applicability and intensity . when scored , the nis divides complaints into seven domains : critical items ( e.g. , head injury , stroke , and dizziness ) , cognitive efficiency ( e.g. , confusion , mental slowness ) , and attention , memory , frustration tolerance , verbal learning , and academic skills ( e.g. , counting change , learning new tasks ) . the nis also includes three scores that give a general , comprehensive picture of complaints : the global measure of impairment ( gmi ) score is a simple sum of all domain subscores ; the total items circled ( tic ) score indicates how many complaints are reported ; and the symptom intensity measure ( sim ) score indicates the average intensity of complaints . the nis also includes three validity checks : defensiveness , inconsistency , and affective disturbance . thus , the nis can detect if subjects are overly defensive or inconsistent ; and the affective disturbance validity check controls for levels of depression and other emotional disturbances . the mini - mental state examination ( mmse ) was used to measure cognitive performance from a general perspective . the bna consists of five subcategories : memory , attention , naming , visuospatial function , and executive function . functional ability was objectively assessed using the older american resources and services ( oars ) activities of daily living questionnaire . this questionnaire assesses fourteen activities of daily living ( adls ) : seven basic adls ( eating , dressing and undressing , grooming , walking , getting in and out of bed , bathing , and going to the bathroom ) and seven instrumental adls ( using the telephone , traveling , shopping , preparing meals , doing housework , taking medications , and handling money ) . subjects were asked about their own ability to perform the above adls ; where possible , informants who knew the subjects well were asked to supplement and verify responses . mean scores for each group ( mci and vamci ) were compared via an independent samples t - test . age , education , and other demographic factors were also compared between the two groups . the mci and vamci groups were matched on demographic measures such as age , years of education , and hollingshead two - factor index of social position ( table 1 ) . functional ability , as measured by the oars questionnaire , was also similar between the two groups ( p = 0.919 ) ( table 2 ) . objective cognition function was measured by the mmse and the bna ( table 2 ) . mmse scores were similar between the two groups ( p = 0.330 ) , which implies similar levels of general cognitive function . overall bna scores were also similar between the mci and vamci groups ( p = 0.177 ) . scores from subcategories of the bna were also compared between groups to look for specific areas of cognitive impairment ( table 2 ) . performance on all subcategories of the bna ( attention , memory , naming , visuospatial function , and executive function ) was similar between mci and vamci groups . performance on the individual tasks which comprise the above five subcategories was also similar between groups , with the exception of the explaining proverbs task ( p = 0.034 ) . on this task , which falls into the subcategory of executive function , mci subjects averaged a better score than vamci subjects ( 3.15 compared to 2.30 ) . despite similar performance on objective functional and cognitive measures ( oars , mmse , and bna ) , the mci and vamci groups achieved markedly different results when subjective cognitive complaints were assessed by the nis . in general , vamci subjects had significantly higher scores on the nis than mci subjects ( table 3 ) ; higher scores indicate greater scc . the nis global measure of impairment ( gmi ) score gives a broad , comprehensive view of complaints . the vamci group averaged a gmi score of 117.0 , compared to just 79.3 for the mci group ( p = 0.050 ) . a qualitative analysis of the total items circled ( tic ) scores shows that vamci subjects had a greater number of complaints than mci subjects ( 52.8 compared to 44.2 ) ; however this difference was not statistically significant ( p = 0.136 ) . the symptom intensity measure ( sim ) scores reveal that vamci subjects ' complaints were greater in severity than those of mci subjects ( 2.0 compared to 1.7 ) ; however , again , this difference was not statistically significant ( p = 0.065 ) . thus , we can not attribute the difference in gmi scores to just one cause . rather , it is likely that difference in gmi scores stems from differences in both complaint quantity and complaint severity . quantitatively , the vamci group had higher scores in all seven domains of the nis ( table 3 ) . four of these domains yielded differences in scores that were statistically significant : critical items ( p = 0.017 ) , cognitive efficiency ( p = 0.043 ) , memory ( p = 0.046 ) , and verbal learning ( p = 0.027 ) . the nis also includes three validity checks to test for defensiveness , affective disturbance , and inconsistency . in our sample of subjects , there was no statistical difference in any of the above variables between the mci and vamci groups ( table 3 ) , confirming that the complaints were valid . although vamci subjects had qualitatively higher scores on the three validity measures , their scores were not significantly different from those of the mci group . mci and vamci are clinical conditions that precede the development of neurodegenerative disorders including alzheimer 's disease and vascular dementia , respectively . apart from differences on neuroimaging there has been very little study of how these two clinical conditions differ in terms of their subjective and objective clinical profiles . taken altogether , our findings from the mmse , bna , oars , and nis show that despite similar levels of functional ability and objective cognitive function , vamci subjects have significantly more scc than mci subjects . our findings support the existing view of mci and vamci as disorders with similar presentations with regard to objective function , but ultimately different etiologies . as previously discussed , mci and vamci subjects were matched on age , education , and social status and achieved statistically similar scores on the mmse and bna . the one component of the bna that differed between the two groups was the explaining proverbs task ; however , this seeming disparity may have resulted from differences in cultural background . in our sample of patients from the diverse city of toronto , 17 out of 40 subjects ( unfamiliarity with english proverbs certainly confounded this result and may explain this difference observed on the bna . leaving aside this single anomaly , our findings show that mci and vamci subjects had comparable levels of objective cognitive function . however , our findings from the nis questionnaire show that vamci subjects had more subjective cognitive complaints than their mci counterparts . this discrepancy suggests that vamci patients are exhibiting particular cognitive deficits , which are not identified by the bna , or that vamci patients are more sensitive to and aware of mild deficits in these particular areas . the nature of vamci - specific deficits may be determined from the domains in which vamci patients report greater complaints : critical items , cognitive efficiency , memory , and verbal learning . the critical items domain references events in a patient 's history that are evident predictors of cognitive difficulty : dizziness , concussion , head trauma , stroke , and so forth . vamci subjects reported greater complaints in this domain than mci subjects ; this finding may be explained by simply considering the diagnostic criteria of vamci compared to those of mci . as mentioned above , history of clinical stroke can differentiate vamci from mci . a vamci patient is more likely than an mci patient to have experienced stroke or stroke - like symptoms , so he / she is more likely to have complaints in the critical items domain . vamci subjects ' greater complaints in the memory , verbal learning , and cognitive efficiency domains may be explained by examining the pathology of vamci , which differs from that of mci upon neuroimaging . a study by vannorsdall et al . found that white matter hyperintensities in both periventricular and subcortical regions correlated with poorer working memory . found that deep white matter hyperintensities correlated with decreased performance on verbal fluency tasks , while periventricular hyperintensities were not associated with any particular domain . white matter lesions have also been correlated with slowed information processing [ 25 , 27 ] . recent research has demonstrated that this decrease in processing speed may be specific to white matter lesions in the anterior thalamic radiation . our analysis may be hampered by the limitations of our cognitive testing methods . in this study , the mmse and bna may have suffered from ceiling effects , which would have masked any differences in cognitive function between the mci and vamci groups . however , the advantages of the mmse and bna are numerous and informed our decision to use these tests . in addition , more sensitive cognitive testing would not have been possible at the study facility , given the amount of time allotted for a clinical visit . the differences we have found in scc between mci and vamci groups may suggest a need for alternate cognitive testing methods , which , unlike the mmse or bna , would elicit different results between the two groups and would not suffer from ceiling effects . our study has demonstrated that vamci patients may be exhibiting particular cognitive deficits in memory , verbal learning , and cognitive efficiency , separate from those exhibited by mci patients . these deficits may be explained by the greater white matter burden in vamci patients , as compared to age - matched mci patients . further research should be done to elaborate on the nature of vamci - specific deficits , particularly in the domains we have identified in this study . as well , vamci - specific deficits should be related to neuroimaging findings , in order to associate deficits with particular locations and severities of white matter disease . finally , tools for more sensitive cognitive testing must be developed , so that we may identify vamci - specific cognitive deficits .

objective . vascular mild cognitive impairment ( vamci ) is differentiated from mild cognitive impairment ( mci ) by the presence of vascular events such as stroke or small vessel disease . typically , mci and vamci patients present with subjective complaints regarding cognition ; however , little is known about the specific nature of these complaints . we aimed to create a profile of subjective cognitive complaints in mci and vamci patients with similar levels of objective cognitive performance . methods . twenty mci and twenty vamci patients were recruited from a memory disorders clinic in toronto . subjective cognitive complaints were assessed and categorized using the neuropsychological impairment scale . results . mci and vamci patients achieved similar scores on measures of objective cognitive function ( p > 0.100 ) . however , the vamci group had more subjective complaints than the mci group ( p = 0.050 ) , particularly in the critical items , cognitive efficiency , memory , and verbal learning domains of the neuropsychological impairment scale . conclusions . our findings support the idea that vamci and mci differ in their clinical profiles , independent of neuroimaging . vamci patients have significantly more subjective cognitive complaints and may be exhibiting particular deficits in memory , verbal learning , and cognitive efficiency . our findings promote the need for further research into vamci - specific cognitive deficits .

disability has been assigned as the umbrella term to include impairments , activity limitations or participation restrictions by the international classification of functioning ( icf ) disability and health . the older concept of disability along with the terms impairment and handicap , as described by icf , have been subsumed under the concept of activity and participation. neuropsychiatric disorders continue to be leading contributors to the global burden of disease . as per the world health report , 2001 mental illnesses accounted for 25% of total disability and 16% of the total burden . among women aged 15 - 44 years , mental disorders constitute three of the 10 leading causes of disease burden in low - and middle - income countries , and 4 of the leading 10 in high - income countries . as per the census of india , 2001 there are an estimated 2,263,821 ( for a population of 1,028,610,328 ) people suffering from disability due to mental illness in the country . mental disorders rank third among the five leading contributors to disability in india . however , a recent community - based study in india found mental disability to be the most common type of disability accounting for 36.7% of total disability . schizophrenia has been identified as one of the most disabling condition known to mankind . as per the estimates for global burden of a disease study , it usually starts during young age and is frequently associated with deterioration from the previous level of functioning . this deterioration is reflected in various functional deficits leading to social isolation and poor occupational functioning . it is also being increasingly recognized that disability is one of the outcome indices for chronic illnesses such as schizophrenia . the indian literature on disability in schizophrenia has focused on patients seeking treatment in out - patient setting , in patient setting and community setting . however , till recently there was no published literature on disability certification seeking behavior of patients diagnosed with schizophrenia . a recent study from karnataka explored the utilization of disability benefits by those who have been issued disability certificates . the current study aimed at understanding the profile of patients diagnosed with schizophrenia seeking disability certification for a tertiary level multispecialty hospital in india . the findings of this study are expected to provide valuable information on various socio - demographic and illness related variables of these individuals . the current study aimed at assessing the profile of subjects diagnosed with schizophrenia seeking disability certification . additionally it aimed at assessing the various socio - demographic and illness related variables among these individuals . the study was carried out at the psychiatry department for a tertiary care hospital in india . we here present the findings form review of cases seeking disability certification over a 5 year period . the evaluation of the subjects was carried out in the presence of a primary care giver . detailed history taking and mental status examination was carried out as part of this evaluation process . the evaluation board comprised of three qualified psychiatrists with at least 3 years of post - senior residency experience in psychiatry . first , the diagnosis was established using icd-10 ( international statistical classification of diseases and related health conditions ) . subsequently , the disability was assessed using indian disability evaluation assessment scale ( ideas ) . socio - demographic information from all the subjects was collected using a pre - approved semi - structured proforma . ideas are approved by the government of india for documenting the level of disability due to mental illness . it assesses disability under four domains : self - care , interpersonal activities ( social relationships ) , communication and understanding , and work . each item is scored between 0 and 4 , i.e. , from no to profound disability , adding scores on 4 items gives the total disability score. global disability score is calculated by adding the total disability score and mi2y score ( months ill in 2 years- a score ranging between 1 and 4 , depending on the number of months in the last 2 years the patient exhibited symptoms ) . global disability score of 0 corresponds to no disability , a score between 1 and 7 corresponds to mild disability , and a score of 8 - 13 corresponds to severe disability , and a score of 20 corresponds to profound disability. an ideas is a well - validated instrument and is being used across the country for disability evaluation in psychiatric disorders . the alpha value for the scale has been found to be 0.8682 , indicating good internal consistency between the items . criterion validity of the scale has been established by comparing ideas with schedule for the assessment of psychiatric disability which has been standardized in india . data were analyzed using spss ( statistical package for social sciences ) software version 17 . categorical variables were analyzed using chi - square test and continuous variables were analyzed using parametric tests . these included independent sample t - test for in - between group differences for male and female subjects ; and family history positive / negative subjects . further , correlation between different illness variables and ideas scores was carried out using pearson 's correlation coefficient . conditions of anonymity and confidentiality as recommended were strictly adhered to during the course of the study and data reporting . socio - demographic information from all the subjects was collected using a pre - approved semi - structured proforma . ideas are approved by the government of india for documenting the level of disability due to mental illness . it assesses disability under four domains : self - care , interpersonal activities ( social relationships ) , communication and understanding , and work . each item is scored between 0 and 4 , i.e. , from no to profound disability , adding scores on 4 items gives the total disability score. global disability score is calculated by adding the total disability score and mi2y score ( months ill in 2 years- a score ranging between 1 and 4 , depending on the number of months in the last 2 years the patient exhibited symptoms ) . global disability score of 0 corresponds to no disability , a score between 1 and 7 corresponds to mild disability , and a score of 8 - 13 corresponds to severe disability , and a score of 20 corresponds to profound disability. an ideas is a well - validated instrument and is being used across the country for disability evaluation in psychiatric disorders . the alpha value for the scale has been found to be 0.8682 , indicating good internal consistency between the items . criterion validity of the scale has been established by comparing ideas with schedule for the assessment of psychiatric disability which has been standardized in india . data were analyzed using spss ( statistical package for social sciences ) software version 17 . categorical variables were analyzed using chi - square test and continuous variables were analyzed using parametric tests . these included independent sample t - test for in - between group differences for male and female subjects ; and family history positive / negative subjects . further , correlation between different illness variables and ideas scores was carried out using pearson 's correlation coefficient . conditions of anonymity and confidentiality as recommended were strictly adhered to during the course of the study and data reporting . a total of 169 subjects seeking disability certification over the study period of 5 years were diagnosed with schizophrenia . out of 169 subjects 132 ( 78.1% ) were male and 37 ( 21.9% ) were female . the mean age of male subjects and female subjects were 36.89 ( standard deviation ( sd ) 9.67 ) years and 39.56 ( sd11.79 ) years , respectively . majority of the study subjects were literate [ table 1 ] . only around 5.3% of males and 10% of females were illiterate . majority of the study subjects ( 93.9% males and 86.4% females ) were from an urban setting . in - between group differences for male and female subjects for socio - demographic variables there was a statistically significant difference ( chi - square=25.27 , df=4 , p<0.05 ) in the marital status of the male and female study subjects . while the majority of male subjects were unmarried ( 62.1% ) and an equal proportion of female subjects were married and unmarried ( 35.1% each ) . also , 18.9% of the female subjects were either separated or divorced . in comparison , there was a statistically significant difference in the employment status of the male and female subjects ( chi - square=7.84 , df=2 , p=0.02 ) . while 17.4% of male subjects were employed , none of the female subjects was currently employed . around 56.7% of the female subjects were from a lower socio - economic background . the rest belonged to middle socio - economic status . in comparison , 15.9% of the male subjects belonged to lower socio - economic status , with the rest belonging to middle socio - economic status . this difference was not statistically significant ( chi - square=3.23 , df=1 , p=0.07 ) . a significantly higher ( chi - square=8.99 , df=2 , p=0.01 ) percentage of male subjects ( 10.6% ) were the primary earning member of the family as compared to female subjects ( none ) . family history of psychiatric illness was positive in 9.8% of male subjects and 10.8% of female subjects [ table 2 ] . of these , around 76.4% of the family members were suffering from schizophrenia . in - between group differences for male and female subjects for illness related variables there was no significant difference between male and female subjects for the duration of illness ( t=1.20 , p=23 , 95% ci-4.62 - 1.12 ) and duration of being on treatment ( t=0.86 , p=38 , 95% ci-4.03 - 1.57 ) . the mean global score on ideas were 12.29 sd2.67 and 11.78 sd2.82 for male and female subjects , respectively [ figure 1 and table 3 ] . * statistically significant difference at p<0.05 in - between group differences for male and female subjects for age , illness related and ideas variables male and female subjects did not differ significantly on the ideas global score ( t=1.01 , p=31 , 95% ci-0.48 - 1.51 ) , personal care ( t=0.68 , p=0.49 , 95% ci-0.20 - 0.41 ) , interpersonal interaction ( t=0.35 , p=0.72 , 95% ci-0.33 - 0.22 ) , and understanding and communication ( t=0.16 , p=0.87 , 95% ci-0.33-.28 ) domains of ideas . the two groups differed significantly on the work domain ( t=3.92 , p<0.05 , 95% ci-0.43 - 1.30 ) . the disability on work domain was higher for male ( mean=3.38 sd1.21 ) as compared to female ( mean=2.51 sd1.09 ) subjects . around 59.8% of male subjects and 56.7% of female subjects were suffering from moderate level of disability . severe disability was found in 34.8% of male subjects and 35.1% of female subjects [ table 2 ] . there was a significant positive correlation between total duration of illness and global ideas score ( r=0.287 , p=0.001 ) and duration for seeking treatment and global ideas score ( r=0.242 , p=0.005 ) for male subjects [ table 4 ] . correlation between socio - demographic , illness variables and ideas score for the male subjects however , no such correlation was observed for female subjects [ table 5 ] . correlation between socio - demographic , illness variables and ideas score for the female subjects additionally , the subjects with or without a family history of psychiatric illness did not differ on various domains of ideas scale as well as global ideas score [ table 6 ] . in - between group differences for the study variables for subjects with and without a family history of psychiatric illness the current study aimed at assessing the profile of patients with schizophrenia seeking disability certification at a tertiary care multispecialty hospital in india . additionally it aimed at assessing the various socio - demographic and illness related variables among these individuals . the existing indian literature on disability among patients diagnosed with schizophrenia has focused on patients seeking treatment in out - patient , in - patient and community settings . we analyzed a total of a total of 169 subjects seeking disability certification over the study period of 5 years was diagnosed with schizophrenia . schizophrenia remains the most common diagnosis among previous indian studies on disability among treatment seeking psychiatric patients . reported 65.3% of the subjects to be diagnosed with schizophrenia in their total sample of 285 over a 3 year period . however , affective disorders have been found to be the most common cause of disability among not treatment seeking individuals in community based studies . in spite of comparatively lower prevalence than other mental disorders schizophrenia continues to be over represented among treatment seekers in hospitals . in the current study , an overwhelming majority of study subjects were male . in a community based epidemiological survey for disability in rural karnataka , females constituted 68% of individuals with mental disability . in a recent study from karnataka , females constituted 49.1% of all disability certificate seeking individuals over a 3 year period . similarly , in a study among long stay patients diagnosed with schizophrenia 67.86% were male . in another study from assam , 31.6% of the study subjects were female . while previous indian studies have reported relatively later help seeking for female patients suffering from schizophrenia , the same is not reflected in the findings of the current study . in the present study male and female subjects did not differ significantly with regards to age at presentation , total duration of illness and duration since seeing treatment . the study by kujur et al . also observed a significant difference in marital status of male and female patients with schizophrenia with majority ( 57.7% ) of women were separated from their husbands . a significantly higher proportion of female subjects were divorced or separated in the current study . previous studies from the west have documented moderate disability in most of patients diagnosed with schizophrenia irrespective of the setting . in the current study indian studies have revealed the disability among patients with schizophrenia to moderate to severe as early as 2 - 5 years after illness onset . in a study from assam , 64% of patients suffering from schizophrenia and 30% of those suffering from bipolar affective disorder had severe disability as per the ideas . however , an indian study among non - treatment seeking individuals in a community setting found mild disability as the most common . in the present study , the highest disability for both male and female subjects was observed in the work domain of ideas . previous indian studies have also reported occupational disability as the most disabling of all the categories . additionally , the least disability observed for personal care ( for both male and female subjects ) was also in keeping with similar observations in previous studies . however , another indian study conducted in a mental hospital setting found the highest level of disability for understanding and communication domain of world health organisation disability assessment schedule ii ( whodas ii ) . disability in schizophrenia has been found to be affected by characteristics like age of onset and duration of illness among other factors . in a previous indian study from ranchi it was found that duration of illness has a significant correlation with personal areas of disability and age of onset has significant positive correlation with personal and occupational area of disability . however , in the current study a significant positive correlation between total duration of illness and global ideas score was observed only for the male subjects . a previous study by thara et al . found a significantly higher global disability and occupational disability among male patients suffering from schizophrenia as compared to the female patients . however , the two did not differ for disability on other domains including self - care , social withdrawal and social contact . in the present also the male and female subjects differed significantly only on the work domain of ideas . no significant differences were observed for personal care , interpersonal interaction and understanding and communication domains . also , there was no significant difference on the global ideas score for male and female subjects . stigma , poor knowledge about the ideas , fear of misuse of certificates , discomfort to approach government hospitals , time constraints , rigid negative thinking about legal issues , denial of disability have been specified as some of reasons for underutilization of disability certification . lack of education among disabled is an important barrier for effective delivery of services . according to national sample survey organization , 2002 gender has been found to be an important determinant of help seeking for mental disorders . women with common mental disorders were more likely to have sought some form of help than men in studies from western setting . however , the situation is different in developing countries such as india . due to socio - cultural factors women researchers has revealed that those closest to the individual play an influential role in whether or not an individual seeks mental health services when experiencing distressing symptoms . multi - ethnic studies have reported that asian patients with psychiatric illness tend to show the longest delay in help seeking . more intensive , extended and persistent family involvement is a possible reason for the delay in seeking help by these individuals . the present study offers some interesting and important insights in to disability certification seeking behavior of patients diagnosed with schizophrenia . there is a relatively long gap between the onset of illness and disability certification . in spite of regulatory threshold of 2 years , also , all the female subjects in the present study were not gainfully employed and were financially dependent on the family . also , a significant greater proportion of them were either divorced or separated . the findings also suggest a relatively early stabilisation of disability among females as the duration of illness was not found to be correlated with global ideas score . however , there was a significant correlation between these two variables for male subjects . relatively later impact of disorder on employment status is a likely possible reason for this observation . lack of impact of presence of psychiatric illness in another family member does not impact the disability seeking behavior of the study subjects . this is a worrisome finding and it reflects poor education of family with regard to the provisions of disability certification and disability benefits available . there is a need to disseminate information related to impact on disabled , community mobilization , opportunity for education , opportunity for work , transfer skills to community level , program activities , and involvement of disabled people . also , research with respect to services , fund allocation , cost - effectiveness , manpower , training , and technical aid of disabled people should be strengthened . a special emphasis should be placed on providing rehabilitation services and disability benefits to the unreached persons with disabilities living in rural areas and small towns of the country . positive impact of disability assessment camps on disability certification seeking behavior has been documented in india . even those who have received disability certification do not utilize the disability benefits offered by the government . the underutilization is even more prominent in rural areas with disability pension being the most utilized benefit . we included all the subjects diagnosed with schizophrenia seeking disability certification over the study period . assessment was carried out using a standardized indian instrument that has been used previously in indian settings . however , there are certain limitations as well . also , the findings need to be replicated in multisite studies before they could be generalized . also , it would be informative to review these subjects over time to assess the stability of disability level . additionally , information on the utilization of disability benefits subsequent to certification should also be assessed in future studies . we included all the subjects diagnosed with schizophrenia seeking disability certification over the study period . assessment was carried out using a standardized indian instrument that has been used previously in indian settings . also , the findings need to be replicated in multisite studies before they could be generalized . also , it would be informative to review these subjects over time to assess the stability of disability level . additionally , information on the utilization of disability benefits subsequent to certification should also be assessed in future studies . majority of patients with schizophrenia seeking disability certificate continue to be male . however , male and female subjects tend to differ very little on various socio - demographic and illness related variables . there are significant difference in the likely impact of schizophrenia on marital status of males and females . however , the work related disability is relatively higher among males and females continue to be financially dependent on the family members .

background : schizophrenia has been identified as one of the most disabling condition known to mankind . until recently there was no published literature on disability certification seeking behavior of patients diagnosed with schizophrenia . the current study aimed at understanding the profile of patients diagnosed with schizophrenia seeking disability certification for a tertiary level multispecialty hospital in india.materials and methods : the study was carried out at the psychiatry department for a tertiary care hospital in india . the evaluation of the subjects was carried out in the presence of a primary care giver . first , the diagnosis was established using international statistical classification of diseases and related health conditions ( icd)-10 . subsequently , the disability was assessed using indian disability evaluation assessment scale ( ideas ) . data were analyzed using spss ( statistical package for social sciences ) software version 17.results:a total of 169 subjects seeking disability certification over the study period of 5 years were diagnosed with schizophrenia . out of 169 subjects 132 ( 78.1% ) were male and 37 ( 21.9% ) were female . there was a statistically significant difference in the marital status of the male and female study subjects . family history of psychiatric illness was positive in 9.8% of male subjects and 10.8% of female subjects . there was no significant difference between male and female subjects for the duration of illness and duration of being on treatment . male and female subjects did not differ significantly on the ideas global score , personal care , interpersonal interaction , and understanding and communication domains of ideas . the two groups differed significantly on the work domain.conclusions:majority of patients with schizophrenia seeking disability certificate continue to be male . however , male and female subjects tend to differ very little on various socio - demographic and illness related variables . the levels of disability are also comparable among males and females . however , the work related disability is relatively higher among males and females continue to be financially dependent on the family members .

from now on , molecular study regarding female sexuality has focused on female sexual hormone , like estrogen receptor ( er ) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin ( 5-ht ) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug . in clinical stage 2a as antidepressant , flibanserin could not prove its effectiveness , but almost no sexual function disorder was reported in subjects . for this reason , the arizona sexual experiences scale ( asex ) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression , but in answering this question " how intense is your sexual desire ? " in the asex , flibanserin turned out to be more excellent in both placebo and active - comparator . since then , drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder ( hsdd ) . hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual - text revision ( dsm - iv - tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life . sexual function disorder is not explained well by other axis 1 disorders ( except other sexual function disorders ) and also it is not that it is caused only by direct physiological effects of materials ( i.e. , drug overuse , drug ) or overall medical status . the dsm-5 completed in 2013 addresses new illness called as female sexual interest / arousal disorder ( fsiad ) , which has both properties of hsdd and another illness of dsm - iv known as female sexual arousal disorder . flibanserin underwent new drug application ( nda ) as original drug in 2009 . the two central phase 3 studies ( violet6 , daisy7 ) all used e - diary and did not show big statistical improvement compared to placebo in the pre - specified co - primary efficacy endpoint that assessed daily sexual desire . in both violet6 and daisy7 studies , flibanserin showed an increase in satisfying sexual events ( sse ) and female sexual function index ( fsfi ) and reduction in female sexual distress scale - revised ( fsds - r ) , but did not show a meaningful increase in e - diary scores compared to placebo . in both studies , the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness ( 11.8% ) , vertigo ( 10.5% ) , and fatigue ( 10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo . applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi , but most advisory committees did not agree to alteration in the e - diary evaluation method set as the one designed to evaluate sexual desire . the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug - drug interactions ( ddis ) and flibanserinalcohol interactions . another central stage 3 study is begonia,8 which set sse , fsfi , fsds - r as major evaluation methods rather than using e - diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds - r compared to placebo . however , many problems with safety were brought up in the second submission , too . vertigo , drowsiness , and vomiting are the most commonly reported adverse effects , and these occurred 2% to 3% , respectively if using placebo during stage 3 placebo - control premenopausal hsdd , whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime . events matching central nervous system depression ( fatigue , drowsiness , and vertigo ) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group . flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 ( cyp3a4 ) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure . and ethanol administered in combination with flibanserin greatly increases the risk of drowsiness , fatigue , orthostatic hypotension , and swoon . additionally , these studies were limited to general healthy women who did not take benzodiazepines , sleep aid , narcotics and many other drugs . the effectiveness was not so big in general , and considering these concerns , fda refused to approve it again and recommended that additional safety studies are required . according to the latest fda review , there is no data indicating new efficacy . instead , flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving , data comparing the commercialized products and the side effects profile of these products , and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies , fda product review is not a fundamental comparison , in fact , and so comparison of safety between individual products may be misunderstood . in particular , alcohol interaction study was conducted in 25 healthy volunteers as a sample , among which only two were female . since the refusal from fda in 2013 , a civic group called as even the score was formed to support what was called as " gender equality " in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved . this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone . despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies , fda 's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval . sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others . the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved ( although none were based on sexual desire ) . among all new molecular entities and biological permit applications approved by fda from 2006 to 2010 , gnanasakthy et al.12 found that among which , 17% ( 20 in 116 ) used the results reported by acknowledged patients as primary outcome in approval labels . the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time . fda raised some concerns about optimizing fsfi as desire assessment tool , including the validity of contents and the period of recall , but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi . the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees . despite little reliable estimate of hsdd prevalence , this product will be used obviously in women in a broader sense than had been studied so far like others , many will not satisfy the official diagnostic criteria of hsdd , and many will increase the risk of adverse effects or be administering other drugs . the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin , but at the same time , it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold . on june 4 , 2015 , fda called a committee meeting to review the efficacy and safety of flibanserin , and after the meeting , the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily . despite the presence of many problems with flibanserin , fda finally approved it under the condition that risk evaluation and mitigation strategies ( rems ) should be enforced for flibanserin . in the future , flibanserin is expected to gradually increase in amount with changes from off - label to on - label . if prescription with flibanserin is determined as necessary , before using it , it is important to check prescription criterion , possible adverse effects after prescription , and precautions during administration . to get a prescription with flibanserin , the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop ( straw ) criteria . the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone ( fsh ) . in addition , it should be prescribed if primary diagnosis is hsdd according to dsm - iv - tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd . besides , its prescription is likely to be considered if the fsds - r ( range , 0 - 52 ) score13 used in clinical studies is 15 points or higher . in addition , in case that sexual desire disorder comes from disease , psychiatric problems , and interpersonal problems or in case that it does so from medications or other substances taken in conjunction , these cases are not the diseases for which medicine is efficacious . besides , postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415 the adverse effects most common in prescribing flibanserin are reported as vertigo , fatigue , vomiting , insomnia , and mouth dryness . precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 ( cyp2c19 ) which are associated with drug metabolism , for example , administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor , which are likely to increase the risk of orthostatic hypotension side effects . furthermore , selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors ( ssri / snri ) may cause anxiety , drowsiness , fatigue , insomnia , and vertigo , if used . so carefulness is required . alcohol is contraindication because if administered with alcohol , it is highly likely to cause central nervous system depression ( fatigue , drowsiness , and vertigo ) , low blood pressure , and swoon . hormonal contraceptives are known as weak cyp3a4 inhibitors , and likely to cause side effects such as vertigo , drowsiness , and fatigue if coadministered with flibanserin . triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin . in addition , if flibanserin is administered to patients with decline in liver failure , increase in concentration of drug in blood is observed and thus it 's considered that adverse effects are likely to occur . besides , taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast - feeding is contraindication . as we discussed above , flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods . most common side effects were central nervous system depression , like fatigue , drowsiness , and vertigo . ddis with fluconazole , ethanol , and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data . with the additional profile and analysis which can prove their safety compared with other products , on june 4 , 2015 , fda finally approved it on the grounds that rems should be enforced , and soon we can get flibanserin on - label under the strict prescription . flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle , but if liver function fails , if breast - feeding , and if drinking alcohol , the use of flibanserin is contraindication . also , its administration with fluconazole , ketoconazole , ssri / snri , triptans , hormonal contraceptives , herb extracts , and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects . we are waiting for patients ' reaction , considering the cost and effectiveness , in countries where flibanserin will be on sale , and more agents like unicenta ( uncnt ) and melsmon , which proved as having a efficacy of menopausal symptoms , have to be studied as treating for female sexual dysfunction.16

there have been several products developed for male sexual dysfunction . however , developing agents for female sexual dysfunction is lagging behind for various reasons . sildenafil citrate ( viagra ) and tadalafil ( cialis ) , which have been prescribed for male sexual function disorders , are known to act on vessels.[1 ] on the other hand , flibanserin is thought to act on brain . flibanserin has been approved by u. s. food and drug administration ( fda ) for treatment of hypoactive sexual desire disorder ( hsdd ) of premenopausal women in 2015 , and is expected to be released in south korea soon . authors wrote this article to acknowledge flibanserin to sexologists for females or physicians for menopausal medicine , so that this agent can be safely used for females who have hsdd .

cementoblastoma in the current world health organization classification of odontogenic tumor , is in the category of tumors of mesenchyme and/or odontogenic ectomesenchyme with or without odontogenic epithelium . it generally occurs in young persons , comprises < 1 - 6.2% of all odontogenic tumor and is characterized as being attached to the roots , most frequently associated with first permanent molar . the majority of these tumor are radiopaque , but radiolucent tumor may occur in rare instances . histologically , it presents as a well - circumscribed tumor composed of cementum like tissue surrounded by a fibrous capsule . this was a case report of a 16-year - old male patient who reported to the department of oral medicine , gdc , ahmedabad with the chief complaint of swelling and mild pain in the right side of the jaw since 6 months , with a history of extraction of 46 because of pain before 6 months in a private dental clinic . on clinical examination , there was diffuse bony hard swelling present in the right body of mandible with normal overlying skin . intra - orally , there was diffuse bony hard swelling in 46 region with normal overlying mucosa and expanded buccal and lingual cortical plates [ figure 1 ] . adjacent teeth were immobile and undisplaced . radiological examination revealed a well - defined round radiopacity with radiolucent rim in the right body of mandible [ figure 2 ] . he had pre - extraction intraoral periapical radiography ( iopa ) radiograph of 46 region , which showed a well - defined radiopacity surrounded by radiolucent rim attached to roots of 46 [ figure 3 ] . the typical radiographic presentation of radiopacity attached to root with radiolucent rim suggested of benign cementoblastoma with differential diagnosis of hypercementosis , osteoblastoma , periapical cemental dysplasia and condensing osteitis . histopathologically it showed a well - circumscribed tumor composed of cementum like tissue surrounded by a fibrous capsule [ figure 4 ] . intra - oral view showing diffuse swelling in right lower quadrant with missing 46 and expansion of buccal and lingual cortical plates right lateral oblique of mandible showing well defined radiopacity surrounded by radiolucent zone in body of mandible with missing 46 pre - extraction radiograph showing radiopaque lesion attached to roots of 46 microphotograph showing sheet of cementum like tissue with intervening loose fibrovascular connective tissue stroma the male to female ratio of the prevalence has been reported to be 2.1:1 with a mean age of 20.7 years . benign cementoblastoma is also reported in the maxillary sinus and associated with deciduous and unerupted permanent tooth and multiple teeth . clinical sign and the radiographic appearance of benign cementoblastoma is well - defined radiopacity with radiolucent zone . though it is a benign tumor , but some cases reported in the literature exhibited signs of local aggressiveness and destruction , including bony expansion , erosion of cortical plates , displacement of adjacent teeth , maxillary sinus involvement , invasion of the pulp chamber and root canals and extension to and incorporation of adjacent teeth . expansion , pain and erosion or perforation of bony cortex were seen in a higher percentage of recurrent tumors , but were also seen in non - recurrent tumors . the differential diagnosis for a periapical radiopacity include cementoma , osteoblastoma , periapical cemental dysplasia , condensing osteitis and hypercementosis . histologically , this tumor presents sheets of cementum like tissue , which may contain a large number of reversal lines with active cementoblasts . cementoblastoma and osteoblastoma are essentially identical histologically and the only distinguishing feature is attachment of cementoblastoma to the root of a tooth . histologically , the cementoblast in cementoblastoma may be plump with pleomorphic and hyperchromatic nuclei , however , mitotic figures are not seen in cementoblastoma . in contrast to osteoblastoma , the cementoblastoma is an odontogenic tumor that recapitulates cementum deposition similar to that during root formation in the later stages of odontogenesis . furthermore , the cementoblastoma is continuous with the cemental layer of the apical third of the tooth root and remains separated from bone by a continuation of the periodontal ligament , all of which supports an odontogenic origin . whereas osteoblastoma arises in the medullary cavity of bones . the treatment of choice is complete removal of the lesion with extraction of associated tooth , followed by thorough curettage and peripheral ostectomy . cases have been also reported of endodontically preserving the tooth while surgical removal of benign cementoblastoma is done . we presented a rare case of benign cementoblastoma in mandible and it should be considered in differential diagnosis of bony swellings of mandible . when extraction is attempted in such cases leaving the lesion behind makes the clinical diagnosis difficult . though the patient had pre - extraction records , which helped in formulating the diagnosis .

we present a rare case of radiopaque lesion with radiolucent rim in the right body of mandible , with history of extraction of involved tooth , which made diagnosis confusing unless pre - extraction intraoral periapical radiography radiograph was recovered , finally diagnosed as benign cementoblastoma . it was surgically managed , with no recurrence of the lesion more than 2 years of follow - up . benign cementoblastoma is a rare , benign tumor of odontogenic ectomesenchymal origin , usually associated with roots of first mandibular molar .

in the previous issue of critical care , rhodes and colleagues report on significantly increased levels of circulating dna in patients admitted to the intensive care unit ( icu ) in comparison with healthy controls . they show plasma dna levels to be an independent predictor of mortality and the development of sepsis in these patients . in sepsis and trauma , circulating nucleosomal dna is positively correlated with disease severity and adverse outcome . in cancer , interestingly , in systemic lupus erythematosus , an autoimmune disease in which nucleosomal dna functions as autoantigenic target , no correlation of circulating nucleosomal dna with disease severity can be found ; instead there is a correlation with anti - nucleosomal dna antibodies . dna in plasma most probably circulates bound to proteins in the form of mononucleosomes and/or oligonucleosomes and is released after the cleavage of easily accessible linkage sites of cellular dna by endonucleases after cell death . a mononucleosome consists of a core particle composed of an octamer of two copies each of histones h2a , h2b , h3 and h4 , around which a stretch of helical dna 146 base pairs in length is wrapped . oligonucleosomes are composed of variable amounts of mononucleosomes connected by intact linker dna with a variable length of 15 to 100 base pairs containing a ' linker ' histone h1 . once released into the circulation , nucleosomes seem to be protected by their structure from further degradation by endonucleases . in healthy individuals , the concentration of circulating dna is low , because dead cells are removed efficiently from circulation by phagocytes . circulating dna has a short half - life ( 10 to 15 minutes ) and is removed mainly by the liver . accumulation of dna in the circulation can result from an excessive release of dna caused by massive cell death , inefficient removal of the dead cells or a combination of both . rhodes and colleagues demonstrate that increased circulating dna not only predicts the development of sepsis but also mortality in patients admitted to the icu . moreover , they show that patients requiring renal support have significantly higher values of circulating dna than patients with sufficient renal function . unfortunately , the authors provide no information on liver function , because most nucleosomal dna is efficiently cleared by the liver and only a small fraction is eliminated by the kidney . our recent study in patients with sepsis showed that nucleosomal dna increased with disease severity , but we found no difference in nucleosome levels in patients with severe renal insufficiency and normal renal function , respectively . other studies in patients with trauma and stroke showed that increased circulating dna levels were correlated with morbidity and mortality . hence , assessment of circulating dna offers a useful tool for predicting mortality and morbidity of patients admitted to the icu . further studies on circulating dna in icu patients , including more patients and other scoring systems for illness severity such as saps ii ( simplified acute physiology score ii ) , logistic organ dysfunction and apache ii ( acute physiology and chronic health evaluation ii ) scores , are needed to establish circulating dna as a predictor for mortality and morbidity in patients admitted to the icu . quantification of circulating nucleosomes can be assessed either by real - time quantitative pcr ( rq - pcr ) or immunological assays . however , contamination of a sample with nucleated cells can affect the apparent concentration of circulating dna . chiu and colleagues showed that a two - step procedure of sample centrifugation ( 800 g or 1,600 g ) followed by either high - speed centrifugation or filtration was superior to a single centrifugation step only . nevertheless , a 13.5-fold variation in circulating dna levels over 3 days can be detected in female volunteers . therefore , even though an appropriate sample preparation protocol may be used , notable variation requires a careful interpretation of circulating dna levels . nucleosomal dna can also be assessed by elisa technique as recently described by different groups . in our laboratory we developed an elisa with the use of a mouse monoclonal anti - histone 3 antibody ( clb - ana/60 ) as a catching antibody and a monoclonal mouse antibody recognizing an epitope exposed on complexes of histone h2a , histone h2b and double - stranded dna , present only on nucleosomes , as a detection antibody . this technique renders quantitative determinations reliable and reproducible . also with elisa , careless blood withdrawal and delayed centrifugation can result in false positive results , and insufficient storage conditions can lead to false negative results . moreover , sandwich elisas are vulnerable to false positive results resulting from xenoantibodies , c1q , rheumatoid factors and anti - nucleosome antibodies ( l aarden , unpublished work ) . a comparison of rq - pcr and elisa methods revealed a high concordance in the quantification of circulating dna in plasma and serum . quantification should preferably be performed in plasma because , probably as a result of the clotting process , higher levels of circulating dna can be measured . however , determination of circulating dna by rq - pcr seems to be more sensitive than by elisa . the lowest circulating dna level measured by rq - pcr in the present study was 14 ng / ml , which corresponds to 2,121 genome - equivalents / ml ( assuming a dna content of 6.6 pg per cell ) . however , circulating dna can be detected by rq - pcr up to 2 genome - equivalents / ml . in our recent study on nucleosome levels assessed by elisa , we reported a detection limit of 35 units / ml , which corresponds to 3,500 cells / ml . further improvement of the assay improved the detection limit to 1,000 cells / ml . fully automated systems in dna isolation , pcr mixture preparation and rapid thermal cycling profile offer a quick and sensitive tool for quantifying circulating dna in plasma . however , these systems require considerable amounts of plasma : reliable dna extraction for rq - pcr requires at least 200 l of plasma , whereas only 25 l suffices for elisa . assessment of circulatory dna is a useful tool for predicting morbidity and mortality in patients admitted to the icu . elisa = enzyme - linked immunosorbent assay ; icu = intensive care unit ; lod = logistic organ dysfunction ; rq - pcr = real - time quantitative polymerase chain reaction .

in various diseases , such as cancer , autoimmune disease , sepsis or myocardial infarction , elevated levels of circulating dna can be measured . however , its predictive value is under debate . circulating dna in plasma is protein - bound ( nucleosomal ) dna . quantification of circulating dna can be performed by real - time quantitative pcr or immunological methods such as elisa . the diagnostic value of both methods can be impaired by inappropriate handling of the samples . assessment of circulating dna in patients admitted to the intensive care unit offers a tool for predicting morbidity and mortality .

unilateral neglect ( un ) refers to the neuropsychological condition in which patients experience reduced orientation and concentration and response to stimuli on the contralesional side owing to behavioral problems that occur after brain injury , although these patients having sensorimotor abilities1 . un is caused by various forms of brain damage and has an especially high incidence in patients with stroke in the right inferior parietal lobe2 . it is negatively associated with the rehabilitation period because patients with un show relatively poor functional recovery compared with those without un3 . in addition , these patients experience difficulties in activities of daily living ( adl ) , including maintaining personal hygiene , eating , and dressing , and also are at increased risk of falling because of gait disturbance4 . therefore , having an appropriate treatment and strategy are important while treating this condition because it is essential to reduce un to achieve effective rehabilitation in these patients . we proposed a new treatment method for un by modifying current treatment methods and combining them with mental practice and electromyogram - triggered electrical stimulation ( mp - emg es ) . mp - emg es is a new , state - of - the - art rehabilitation treatment technology based on neurologic theories . mp - emg es involves only repetitive mental practice ( for example , imagination of intense movement of the upper limbs on the neglected side ) , not voluntary muscular contraction . when the generated electric potential reaches a preset threshold , an electric stimulus is generated , resulting in actual muscular contraction and cortical activation5 . in other words , mp - emg es can produce a synergistic effect by combining two treatments ( mental practice plus electrical stimulation ) with proven efficacy in the alleviation of neglect . the purpose of this study was to identify the effects of this novel training method mp combined with emg es over a 6-week period of daily training in patients showing right brain damage with un . the participants for this study were recruited from the rehabilitation center of a local university hospital . the inclusion criteria for participation were : ( 1 ) onset duration of > 6 months , ( 2 ) right hemisphere stroke with un , ( 3 ) mini - mental status examination score > 24 , ( 4 ) ability to imagine ( average score on the vividness of movement imagery questionnaire < 3 ) , and ( 5 ) active wrist muscle strength > 2 on the medical research council ( mrc ) scale . exclusion criteria were : ( 1 ) an implanted cardiac pacemaker , ( 2 ) skin lesion on the affected side or hypersensitivity at the electrode site , ( 3 ) a history of seizure or epilepsy , and ( 4 ) unstable medical conditions . in total , thirty - three stroke patients with neglect were eligible for this study . all participants provided written informed consent , and this study was approved by the inje university institution review board . this study was designed as a single - blind randomized control study and was scheduled to last for a total 6 weeks . eligible participants were randomly allocated to the two groups by block randomization using opaque envelopes containing a code specifying the group . the experimental group ( n=16 ) received mp - emg es in addition to conventional rehabilitation therapy ( crt : physical therapy and occupational therapy ) , whereas the control group ( n=17 ) received cyclic es in addition to the same crt . mentamove ( mentamove deutschland gmbh , munich , germany ) was used to apply mp - emg es in the experimental group . surface electrodes were attached to the wrist extensor muscle and a reference electrode was attached at the lateral side of the forearm . the site of electrode placement was marked using a permanent marker throughout the intervention . during motor imagery training , when the potentials reached a preset threshold , the induced electrical stimulation would contract the targeted muscle . the mentamove process consisted of three stages : ( 1 ) motor imagery ( approximately 12 s ) , ( 2 ) electrical stimulation ( approximately 6 s ) , and ( 3 ) relaxation ( approximately 12 s ) . the motor imagery used in this study was a vigorous waving of the affected whole arm . this imagery was selected because the emg was not able to detect electrical stimulation induced by motor imagery of simple extension of the wrist or elbow . the emg pick - up threshold was set afresh for each subject in every session . if the subject repeatedly reached the threshold during the mp - emges , the threshold was automatically increased slightly . imagine that your left arm moves rapidly and intensely when you see motor imagery in the tool window . if your performance is successful , you will experience an electrical stimulation in your forearm . cyclic es ( mendel gmbh , germany ) without the emg function was used to apply electrical stimulation in the control group . electrodes also were attached to the wrist extensor muscle . in either instrument , biphasic pulses with a frequency of 35 hz and pulse width of 200 s were applied for 12 s. stimulation intensity led to a clear extension of the wrist ( average 2030 ma ) . over a period of 6 weeks , both groups were treated 30 times in two 30-min sessions . the line bisection test ( lbt ) , star cancellation test ( sct ) , and catherine bergego scale ( cbs ) were used to quantify the severity of un . twenty lines were drawn on an a4 sheet parallel to the long axis , and 18 of these lines were organized into 3 set of 6 : 1 set lay primarily on the left , 1 at the center , and 1 on the right side of the sheet . the evaluator initially demonstrated the procedure by marking the 2,150-mm lines at the top and bottom of the sheet . patients were asked to mark the center points of each of the 18 lines in order . distances from the left of each line to patient s marks and to true line centers were measured . deviations were measured using the formula : percent deviation ( % ) = [ ( marked left side distance - true half length)/true half length ] 100 . in sct , the left and right halves of the sheet each contained 27 stars , and patients were asked to mark all of the stars . the scores range from 0 to 27 , with lower scores indicating more severe un . assessment using cbs involved evaluating the performance of patients by directly observing them perform activities ( e.g. , dressing , grooming ) in real - life situations , and consisted of 10 items . each item was scored on a four - point scale as follows : 0 : no neglect 110 : mild neglect , 1120 : moderate neglect , and 2130 : severe neglect7 . participant characteristics were analyzed using a statistical software program ( spss statistics 20 ) and descriptive statistics were presented as mean sd . the shapiro - wilk test was used to check normality of the outcome variables . to evaluate the intervention effects , measures before and after the intervention in each group an independent t - test was used to compare changes in outcome measures between the two groups . all demographic and clinical characteristics of the participants were comparable and are summarized in table 1table 1.baseline characteristics of the subjects included in the studyexperimental group ( n=16)control group ( n=17)gendermale10 8 female69age ( years)67.510.362.210.2time post stroke ( months)3.31.33.51.6stroke typehemorrhage45infarction1212 . the experimental group showed a significant improvement in the lbt ( p<0.01 ) , cbs , and sct ( p<0.05 ) scores after the intervention compared with the values obtained before the intervention ( table 2table 2.clinical parameters before and after the treatment ( n=33)experimental group control groupbeforeafterbeforeaftercbs12.14.210.14.612.63.811.24.1lbt30.58.924.110.332.69.127.710.6sct10.65.213.05.5 9.25.211.04.6the values are mean standard deviation . cbs : catherine bergego scale , lbt : line bisection test , sct : star cancellation test . * p<0.05 by paired t test between the initial and final scores in the group . on the other hand , no significant difference was observed in the control group , except in the lbt score ( p<0.05 ) . no significant differences were observed in the lbt , cbs , and sct scores between the two groups after treatment . cbs : catherine bergego scale , lbt : line bisection test , sct : star cancellation test . * p<0.05 by paired t test between the initial and final scores in the group . mp - emg es is a recently developed method that produces tiny electrical signals in the brain through mental practice , rather than through actual physical movements , and this method is used to induce muscle contraction in the upper extremity ( u / e ) on the neglected side . in other words , the aim of mp - emg es is to form sensorimotor circuits for movement through a repeated cycle of brain signal transmission muscle movement , which can be used to produce functional changes in the central nervous system and the body . the continual activation of these circuits helps reorganize the damaged areas of the cerebral cortex8 . it is thought that mp - emg es works by inducing neural and peripheral changes through activation of the cerebral cortex via mental practice and afferent stimulation of the neglected u / e via electrical stimulation . hong et al.5 reported mp - emg es is more effective than cyclic es for improving motor function on the affected side and improves cerebral glucose metabolism in supplementary motor , precentral and postcentral gyri on the contralesional side . however , there is little evidence about the effects of this treatment in cases of un . in this study , we investigated the effects of mp - emg es on neglect in comparison with those of cyclic es . cbs , lbt and sct scores significantly improved after treatment in the experimental group . the control group received various crt interventions that were the same as the experimental group . this control treatment was important because cyclic es has been proven to alleviate neglect to some extent by applying afferent stimulation to the neglected side . therefore , while the control group only showed a significant improvement in lbt , they also showed a slight improvement in scores for cbs and sct , and as a result , no significant differences were demonstrated between the two groups in any assessments . some studies reported that mental practice is effective for rehabilitation in patients with contralesional neglect , but it is difficult to verify whether patients imagine the specific movement well9 . mp - emg es seems to remedy the deficiencies of mental practice alone . in this study , patients were instructed to imagine a large and powerful movement of the neglected u / e during the mental practice stage of mp - emg es treatment . when imagining the movement of the neglected u / e , the muscles in the u / e show dynamic regulation and changes5 . in this regard , when a participant imagined a complex and powerful movement instead of a simple movement , the motor - evoked potential and the excitation in the corticospinal tract increased10 . this study showed that mp - emg es is an effective method for reducing unilateral neglect in stroke patients . however , several factors need to be considered when applying this method . because mp - emg es requires the patient to be capable of imaging movement of the body on the neglect side

[ purpose ] the aim of this study was to investigate the effect of mental practice combined with electromyogram - triggered electrical stimulation on neglect and activities of daily living in stroke patients with unilateral neglect . [ subjects and methods ] thirty - three stroke patients with unilateral neglect were recruited from a local university hospital , and were divided into two groups . the experimental group received an intervention consisting of mental practice combined with electromyogram - triggered electrical stimulation on the neglected side , while the control group received cyclic electrical stimulation at the same site . in addition , both groups received an identical intervention of conventional occupational and physical therapy . [ results ] after the intervention , the experimental group showed a statistically significant improvement in the line bisection test result , star cancellation test result , and catherine bergego scale scores . the control group showed a significant improvement only in the line bisection test result . [ conclusion ] these data suggest that mental practice combined with electromyogram - triggered electrical stimulation is an effective , novel treatment for reducing unilateral neglect in stroke patients .

long - acting beta-2 agonists such as salmeterol and formoterol are important controller medications in the maintenance therapy of chronic stable asthma , used as an add - on drug to inhaled corticosteroid . however , their usefulness in the management of acute attacks of asthma has recently been recognized , and they are approved for use as reliever medication in europe . short - acting 2 agonists are the preferred drugs as the initial bronchodilator for acute asthma because of their rapid onset of action . however , due to short duration of action they require frequent administration . formoterol is a unique bronchodilator having rapid onset and long duration of action with a favorable safety profile . so it can be an ideal alternative to short - acting 2 agonists in the management of acute asthma exacerbation by providing rapid bronchodilatation and reducing the need for frequent administration . onset of action of formoterol is similar to salbutamol ( 13 min ) , and 8090% of brochodilatation occurs by 510 min of inhalation . duration of action is up to 12 h. however , duration of systemic effects with formoterol is found to be as short as salbutamol . arformoterol ( [ r , r ] formoterol ) is a single isomer form of formoterol , which is available in india recently . this study compares the efficacy and safety of arformoterol and salbutamol delivered by nebulization in the management of acute non - severe asthma . a total of 50 patients attending the emergency room or chest opd of a tertiary care hospital with acute non - severe asthma over a 6-month period from january 2010 to june 2010 were selected for this study . the inclusion criteria were as follows : ( a ) age > 18 years ; ( b ) british thoracic society definition of acute non - severe asthma ; and ( c ) ability to perform forced expiratory maneuver . the following patients were excluded from the study : ( a ) patients presenting with acute severe / acute life - threatening / near - fatal asthma ; ( b ) patients with a previous diagnosis of chronic obstructive pulmonary disease ( copd ) ; ( c ) history of hypersensitivity to 2 agonists ; and ( d ) pregnant or lactating women . the study was approved by the institutional ethics committee , and informed consent was obtained from each patient . the age , sex , duration of asthma symptoms , and present controller medications were recorded . the pulse rate , respiratory rate , and spo2 ( measured with fingertip pulse oximeter , model 6500 , nidek medical , kolkata , india ) were also recorded at the time of initial assessment . baseline peak expiratory flow rate ( pefr ) was measured with a peak flow meter ( peak flow master , cipla ltd . , patients were then assigned by random number allocation to either of the two groups , that is , salbutamol or arformoterol group according to the medication given to them . in the salbutamol group , 5-mg salbutamol respules were administered through oxygen driven ( 6 l / min ) nebulizer ( pulmo mist ii nebuliser , nidek medical , kolkata , india ) at 20-min intervals for 1 h amounting to a total of 15 mg . in the arformoterol group , total 45 g of the drug was administered as 15 g respules every 20 min through the same nebulizer . the drugs were administered in a double - blind manner . to eliminate the effects of other drugs on the treatment outcome pefr was also measured 5 min after each dose . at each time , best of the three pefr measurements was recorded . differences in mean pefr between the two groups were analyzed by unpaired t - test . one - sample test was used to compare pefr before and after each dose of the drug to determine whether there was significant improvement in either group . among 50 patients satisfying the inclusion criteria , 25 patients received salbutamol and 25 patients received arformoterol therapy . baseline characteristics of the patients ( n = 50 ) receiving salbutamol and arformoterol therapy from the table , it is evident that the demographic profile and baseline pefr in the two groups were comparable . in both the groups , pefr showed significant increase over the baseline values and the increase was evident after each dose of the drug . the increases in pefr after the first and the second dose were significantly more with arformoterol than with salbutamol , but the increase in pefr after the third dose was similar in these two groups . the comparison of improvement following the therapy with salbutamol and arformoterol is shown in table 2 . improvement in pefr after each dose of bronchodilator when compared with the baseline both drugs were well tolerated ; no major adverse effect was noted in either group . as arformoterol is the only long - acting 2 agonist available in india as a nebulizing solution , we have used this drug considering the fact that the clinical pharmacology will be similar with formoterol . our study has shown that both salbutamol and arformoterol are equally effective as a reliever medication as nebulizing solution in acute non - severe asthma . improvement in pefr was demonstrated in both the groups and following each dose of the drugs . the absolute increase in the pefr after the first and the second dose were more with arformoterol than with salbutamol , but the increase in the pefr after the third dose was similar with these two drugs . relatively , hydrophilic drugs such as salbutamol have a rapid onset of action due to their ability to reach the 2 receptor from the aqueous phase . the aqueous portion rapidly activates the 2 receptor , whereas the lipophilic portion is taken up into the cell membrane from which it diffuses slowly to stimulate 2 receptor over a prolonged period . several studies have shown the efficacy of formoterol in acute non - severe asthma , acute severe asthma , exercise induced bronchospasm , childhood asthma , and copd.[1317 ] most of the studies have used formoterol with terbuhaler , aerolizer , or metered dose inhaler . however , studies using formoterol nebulization are lacking . although few studies have shown clinically significant improvement in lung function ( fev1 ) with formoterol when compared with salbutamol , most studies have shown a comparable efficacy . safety of formoterol is also well documented even at high doses in patients with asthma and copd . although it is a long - acting drug , systemic effects with formoterol are as short as salbutamol . in few studies , the impact of high - dose formoterol on heart rate , blood pressure , serum potassium , electrocardiogram changes , and arterial blood gas were assessed which showed identical changes comparable with salbutamol we did not study these parameters but evaluated only the side effects reported by the patients . the side effects were very few , non - severe , and comparable between these two drugs . dryness of mouth was the most reported side effect of formoterol inhalation in one study , but we found oral irritation and headache as common side effects . the sample size is relatively small which limits our ability to detect small but potentially significant differences . this study is restricted to patients of acute non - severe asthma only ; efficacy and tolerability of arformoterol in other acute settings such as acute severe asthma and acute exacerbation of copd are yet to be explored . the side effect profile studied by us was only on the basis of the self - reported symptoms of the patients , and objective assessment and laboratory abnormalities were not assessed . we also did not follow the patients after discharge ; so long - term efficacy and safety were not studied . further studies are thus needed to clarify the efficacy , actual dose , dose frequency , cost - effectiveness , and long - term safety of arformoterol in acute exacerbations of asthma and copd with different levels of severity .

arformoterol , a long - acting beta-2 agonist , has a rapid onset and long duration of action . its role as rescue medication in acute asthma attack is undetermined . to compare the efficacy and tolerability of arformoterol with salbutamol nebulization , a study was conducted among 50 patients with acute non - severe asthma . patients were randomly assigned to group 1 ( n = 25 ) and group 2 ( n = 25 ) who received three doses of salbutamol and arformoterol nebulization , respectively , at 20-min intervals . the peak expiratory flow rate ( pefr ) was measured at the baseline and 5 min after each dose . the demographics and baseline characteristics were comparable between the two groups . the mean pefr significantly increased in both these groups when compared with the baseline . the increases in the pefr in two groups were similar after the third dose . the adverse effects in both these groups were minor . arformoterol was as effective and safe as salbutamol in acute non - severe asthma .