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recently , a large number of cardiovascular risk factors have been detected , but the role of new risk factors added to traditional ones in predicting or preventing cardiovascular diseases and mortality is still unknown . homocysteine and lipoprotein a are two new risk factors of heart diseases and may cause plaque formation on vessel walls . however , the pathogenic role of these risk factors in the developing cardiovascular disease is still debating . homocysteine is a nonessential sulfur containing amino acid mediating metabolic path of nonessential methionine amino acid . it is well documented that in humans and animals an encoding in homocysteine metabolism gene enzyme results in increased homocysteine and causes several metabolic disorders such as atherosclerosis , clotting in vessels and endothelial malfunctions . epidemiological studies have proved the positive relationship between homocysteine concentration and risk of cardiovascular disease . randeva et al . examined the effect of a 6 month exercise program on homocysteine levels . duncan et al . examined the effect of severity and frequency of exercise sessions per week on homocysteine levels in middle age and observed a small increase in homocysteine levels . it exacerbates atherosclerosis and its structure is similar to low density lipoprotein ( ldl ) , but with a glycoprotein called apoprotein an attached to it and its concentration being genetically determined . research findings indicate that increased lipoprotein a < 30 mg / d lit increases cardiovascular diseases risk 3 - 10 times depending on the existence of other factors . there are two mechanisms , which may explain the relationship between lipoprotein a and cardiovascular disease . first , it is argued that lipoprotein a , like ldl , plays a role in initiating the progression of atheromatous plaque as a particle . second , it is suggested that this particle competes with the plasminogen particles in preventing thrombolytic process . zafari examined the effects of regular exercise on lipoprotein a levels in middle age men and observed that lipoprotein a levels in active and sedentary individuals are less than coronary artery disease patients . almeida et al . studied the effects of 12 week of aerobic and strength training on rats and found no significant differences in lipoprotein a levels . surveyed the effect of 8 week walking on lipoprotein a levels in women and observed no significant difference . physical fitness reduces cardiovascular risk up to 50% in middle aged and is accompanied by preventing the developments of triglyceride ( tg ) , cholesterol and ldl risk factors and increasing high density lipoprotein ( hdl ) levels . from general health point of view , aerobic training is one the most effective ways to reduce the risk of cardiovascular diseases . aerobic training is a key factor either alone or in combination with appropriate lifestyle in improving blood lipid profile . aerobic training is recommended for improving lipid and lipoprotein levels as a low cost way for middle age . researches have reported significant improvements in lipid profile and lipoprotein levels as a result of aerobic training positive . however , several paradoxes have been reported . indicated the effect of 16 week of progressive aerobic training with moderate intensity on lipid profile in middle aged men . as research shows , significant improvements will be observed in tg , cholesterol , ldl and hdl levels after aerobic training . akcakoyun investigated the changes in lipid profile after moderate intensity training programs in middle aged men . the results showed that levels of serum tg decreased , and hdl increased in the experimental group after the training program . in a study banitalebi et al . investigated the effects of 12 week of progressive aerobic exercise on blood lipids and lipoproteins in 23 - 49 years old men . no significant changes in levels of tg , cholesterol , ldl and hdl after 12 week were observed between the two groups . long term studies show that physical inactivity in middle aged increases the risk of cardiovascular diseases up to 50% , in this study , we examined the effect of 12 week of aerobic training on homocysteine , lipoprotein a , and lipid profile levels in sedentary middle age men . regarding controversies in previous studies findings and limitation of study about the influence of aerobic training on homocysteine and other lipoproteins this study seem necessary . objectives , procedure , as well as possible risks of training programs were described to the subjects and their written consents were obtained . subjects were selected according to their age , > 30 body mass index , physical health , not attending in regular exercises since 6 month before the study , normal lipid profile status and lack of a specific diet , medication , and smoking . the primary criterion for evaluating subjects health was to check the physical examination done by a physician and review the health questionnaire . none of the subjects were affected to chronic autoimmune , systemic , cardiovascular , hepatic or other diseases , which can continuously change the lipid status . also at the preliminary session the participants were asked not to change their diet and lifestyle during the 12 week . qualified individuals were randomly assigned into two aerobic ( n = 12 ) and control ( n = 12 ) groups . participant characteristics of the aerobic training and control group the subjects performed training sessions using a treadmill 3 times a week ( on nonconsecutive days ) and 20 min / session ( 60% of maximum heart rate ) to 60 min ( 75% of maximum heart rate ) . subjects height and weight were recorded using a medical scale with stadiometer ( seca : 220 , germany ) . body compositions were measured using a body composition analyzer ( inbody 3.0 , south korea ) . blood samples were collected after 12 h fasting at pretest and 48 h after the last training session at posttest . after collecting blood samples at each step , axis homocysteine eia reagent kit ( axis - shield co. , dundee , uk ) . kit ( made in england ) was used to measure homocysteine levels and drg elisa total human lipoprotein ( a ) ( drg international inc . pars azmoon kit ( pars azmoon co. , tehran , iran ) and enzymatic method were used for the measurement of plasma levels of tg , total cholesterol and hdl ; and ldl was calculated by friedwald equation ( ldl = tc ( hdl + tg/5 ) in terms of milligrams per deciliter . statistical analysis was performed using spss for windows software , version 18 ( spss inc . paired t - test was used for within group comparison and independent t - test was used for between groups comparison . the subjects performed training sessions using a treadmill 3 times a week ( on nonconsecutive days ) and 20 min / session ( 60% of maximum heart rate ) to 60 min ( 75% of maximum heart rate ) . subjects height and weight were recorded using a medical scale with stadiometer ( seca : 220 , germany ) . body compositions were measured using a body composition analyzer ( inbody 3.0 , south korea ) . blood samples were collected after 12 h fasting at pretest and 48 h after the last training session at posttest . after collecting blood samples at each step , axis homocysteine eia reagent kit ( axis - shield co. , dundee , uk ) . kit ( made in england ) was used to measure homocysteine levels and drg elisa total human lipoprotein ( a ) ( drg international inc . pars azmoon kit ( pars azmoon co. , tehran , iran ) and enzymatic method were used for the measurement of plasma levels of tg , total cholesterol and hdl ; and ldl was calculated by friedwald equation ( ldl = tc ( hdl + tg/5 ) in terms of milligrams per deciliter . statistical analysis was performed using spss for windows software , version 18 ( spss inc . , paired t - test was used for within group comparison and independent t - test was used for between groups comparison . research findings indicate that there were statistically significant differences in the levels of homocysteine , lipoprotein a , tg , cholesterol , ldl and hdl in posttest compared to pretest ( p = 0.002 , 0.003 , 0.008 , 0.024 , 0.019 , and 0.017 , respectively ) . table 3 shows the comparison of the variables between the aerobic training and control groups following 12 week . the table 's results show significant differences between the values of homocysteine , lipoprotein a , tg , cholesterol , ldl and hdl between the two groups ( p = 0.005 , 0.001 , 0.006 , 0.015 , 0.022 and 0.004 , respectively ) . the results of paired t test in aerobic training and control group before and after the intervention comparison of changes in measured variables following 12-week in two groups this study was designed to investigate the effect of 12 week of aerobic training on homocysteine , lipoprotein a and lipid profile levels of sedentary middle age men . the findings revealed that there was a significant decrease in homocysteine levels at posttest compared to pretest , and following 12 week of aerobic training significant differences between aerobic and control groups . . one of the mechanisms associated with homocysteine function in cardiovascular diseases is due to a decrease in hdl levels . homocysteine decreases hdl concentration in plasma by inhibiting hepatic synthesis of apoprotein a existing in hdl . decreased hdl concentration and increased homocysteine levels may increase the risk of cardiovascular diseases . decreased homocysteine due to participating in regular training probably increase available apoprotein a existing in hdl . three major factors influencing homocysteine levels except for regular training are mode , intensity and duration of the exercises . the reason for inconsistency might be different mode , intensity and duration of the training programs in mentioned studies . the findings revealed that there were significant decreases in lipoprotein a levels at posttest compared to pretest , and following 12 week of aerobic training significant differences were observed between aerobic and control groups . these results were consistent with zafari but did not match almeida et al . and firozeh et al . recent data from cross sectional studies have shown that regular training with moderate intensity is effective in reducing the concentration of lipoprotein a , but intense training does not induce this effect . transient increase of lipoprotein a levels immediately after severe or prolonged exercise is because of the role of lipoprotein a in repairing tissues , which have been damaged due to free radicals and severe and prolonged training . one reason for the reduction in lipoprotein a is an anti oxidant effect of aerobic training , because free radicals oxygen increase expression of inflammatory mediators and attaching molecules . the reason for this difference is probably related to differences in the subjects of this study as well as the intensity and duration of training sessions . furthermore , in mentioned studies due to low exercise capacity of subjects , training intensity was lower than this study . the findings revealed that there were significant decrease in tg levels at posttest compared to pretest , and following 12 week of aerobic training between aerobic and control groups . these results were consistent with martin et al . and akcakoyun but did not match banitalebi et al . aerobic training reduces the risk of cardiovascular diseases , and based on research evidence ; sedentary individuals are exposed to these risk factors . one mechanism related to reducing the risks of cardiovascular diseases could be due to reducing tg levels . lipoprotein lipase is an enzyme catalyzing tg and causing the release of free fatty acids from tg in order to provide energy during aerobic training . thus , a high correlation exists between lipoprotein lipase enzyme activity and blood tg removal . probably , aerobic training increases the activity of lipoprotein lipase enzyme and the enzyme activity lead to reducing the levels of tg at the posttest . present research findings have indicated that cholesterol levels decreased significantly posttest compared to pretest in an aerobic group , and also significant differences were observed after 12 week of aerobic training between the control and aerobic groups . these results were consistent with szymanska et al . but did not match banitalebi et al . one of the first and the most obvious changes observed during aerobic training is reduced plasma insulin . therefore , probably one of the factors that may influence plasma cholesterol is plasma insulin levels . and it can be stated that reduction in plasma insulin leads to activation of lipolysis from fat tissue and increasing the concentration of free fatty acids in plasma . and both hormones lead to increased activity of ketogenesis which then changes the cholesterol precursor buildup . research findings showed that there were statistically significant decreases in an aerobic group in ldl levels at posttest compared to pretest , and following 12 week of aerobic training significant differences were observed between aerobic and control groups . these results were consistent with monda et al . but did not match banitalebi et al . from physiological and molecular mechanisms involved in the reduction of ldl levels , it can be stated that exercising leads to increased lipoprotein lipase activity . because of the effect of this enzyme increase on cholesterol rich lipoprotein catabolism , the amount of ldl reduces due to physical activity . since the increase in serum lipid is of cardiovascular diseases risk factors , it can be acknowledged that increased aerobic training reduces some of risk factors such as ldl levels . research findings showed a significant increase in hdl levels in posttest compared to pretest in an aerobic group , and following 12 week of aerobic training significant differences were observed between aerobic and control groups . hence , one of the reasons for an increase in hdl levels can be its increased production by the liver and alterations in several enzymes such as increased activity of lipoprotein lipase , lecithin cholesterol acyl transferees activity , and decreased activity of hepatic lipase . the results showed that 3 sessions / week of aerobic training induce a positive influence on homocysteine , lipoprotein a and lipid profile levels in sedentary middle aged men .

employment in cotton processing industry is associated with exposure to broken cotton fibers , bracts , and pericarps as well as to bacteria and fungi that may grow on cotton products . the classical occupational lung disease associated with cotton industry is byssinosis , an acute and reversible response to cotton dust characterized by chest tightness on working days and remitting on off - days only to relapse upon re - exposure . other chronic effects of cotton industry - associated exposure have received much less attention include obstructive airway diseases , interstitial lung disease , and hypersensitivity pneumonitis . bronchiolar disorders are a group of diseases of diverse etiology predominantly involving the membranous and the more distal airways . we diagnosed a case of obliterative bronchiolitis in a nonsmoker male with 4 decades of employment in cotton processing industry . the case is reported in view of the absence of any previous report of such an association . a 63-year - old nonsmoker male presented with 10-year complaints of recurrent dry cough and progressive exertional dyspnoea , limiting his activities severely at present . there was no history of hemoptysis , joint pains , fever , chest pain , or any constitutional symptoms . he had worked in the textile industry processing raw cotton for 35 years , but had quit following the development of symptoms . chest radiography showed bilateral hyperinflated lung fields with low flat diaphragms [ figure 1 ] . the electrocardiogram showed a north - west axis with a right bundle branch block and poor progression of r waves . lung function study revealed severe airflow limitation with hyperinflation and air trapping and impaired diffusion capacity . the forced vital capacity ( fvc ) was 1.64 l ( 68% of predicted ) , forced expiratory volume in 1 second ( fev1 ) was 0.41 l ( 21% of predicted ) with an fev1 /fvc ratio of 25% . post - bronchodilator , the fvc showed a 300 ml and 18% increase over the baseline and fev1 increased by 110 ml and 26% . lung volumes measurement by closed - circuit multiple - breath helium dilution showed a residual volume ( rv ) of 2.74 l ( 148% of predicted ) , functional residual capacity ( frc ) of 3.68 l ( 140% of predicted ) , and a total lung capacity ( tlc ) of 4.49 l ( 105% of predicted ) . single breath diffusion capacity for carbon monoxide ( dlco ) was 14.3 ml / min / mmhg ( 69% of predicted ) . arterial blood gas analysis showed moderate hypoxaemia and mild metabolic alkalosis ( ph 7.45 , pco2 45.9 mmhg , po2 58.6 mmhg and hco3 33.6 a six - minute walk test resulted in further desaturation to 84% within 1 minute . serum rheumatoid factor , antinuclear antibodies and cross - reactive protein ( crp ) were in the normal range . chest radiograph ( pa view ) showing bilateral hyperinflated lung fields with low flat diaphragms a high resolution computed tomogram ( hrct ) of the chest showed bilateral centrilobular nodules with tree - in - bud pattern and areas of air trapping [ figure 2 ] . a diagnosis of cotton exposure - associated obliterative bronchiolitis was established based on clinical features and occupational history , a highly characteristic hrct picture and supportive evidence on lung function testing . the patient was advised treatment with inhaled corticosteroids , inhaled long acting bronchodilators ( formoterol and tiotropium ) and pulmonary rehabilitation . high resolution computed tomogram ( hrct ) of the chest showing bilateral centrilobular nodules with tree - in - bud pattern ( arrow ) and areas of air trapping ( arrow ) the occupational safety and health administration ( osha ) of the united states of america has designated cotton dust exposure as one of the five occupational health hazards . the most well - known disease caused by cotton dust is byssinosis though chronic obstructive pulmonary disease ( copd ) is also known to occur with occasional reports of diffuse pulmonary fibrosis . changes in both reactivity and airways caliber have led to the suggestion that small airways maybe affected by cotton dust exposure . an inflammatory response in the small airways may be due , in part , to the contamination by gram - negative bacterial products . the specific effect of exposure may depend on the site of deposition in the tracheobronchial tree , duration of exposure , action of specific components of the dust , as well as the presence of other confounding causative agents such as smoking . obliterative bronchiolitis is an uncommon and severe form of obstructive airways disease involving the small airways . its diverse etiologies include smoking , collagen vascular diseases , inhalational injury , certain drugs , and organ transplantation . known inhalational exposures include chlorine , ammonia , oxides of nitrogen , sulfur dioxide , and other gases besides diacetyl , a chemical used to provide butter flavor in many foods . however , it has rarely been reported in association with organic dusts but never in relation to cotton - processing industry . while symptoms , plain radiological features , and the spirometric abnormality resemble those seen in copd , it is the radiological signs that differentiate the two . direct signs of bronchiolitis obliterans include ill - defined centrilobular nodules and tree - in - bud opacities whereas indirect signs of small airways disease include a mosaic pattern of attenuation ( on inspiratory ct scan ) and air trapping ( on expiratory ct scan ) . a fibreoptic transbronchial biopsy was not done in the present case because of resting hypoxemia and also because the yield in small airways disease is usually poor and would not have justified the risk . a histological confirmation by open or a video - assisted thoracoscopic lung biopsy was not considered necessary because the hrct picture was diagnostic . absence of a history of smoking ruled out respiratory bronchiolitis . in view of long occupational exposure to cotton dust , symptoms , characteristic picture on hrct scan , exclusion of other causes and supportive evidence on lung function testing the present case adds obliterative bronchiolitis to the spectrum of respiratory diseases known to occur with cotton dust exposure . an hrct of the chest is recommended in patients working in cotton - processing industry and reporting dyspnea .

the disease symptoms include increased bone density , short stature , hypoplasia of the mandible , an obtuse mandibular angle , dysplasia of the skull bones with unossified fontanels and separated cranial sutures , partial aplasia of the terminal phalanges , stubby hands and feet with dystrophic nails , spondylolysis of the lumbar vertebrae , clavicular dysplasia , and increased tendency towards pathological fractures . growth hormone ( gh ) secretion impairment and low insulin - like growth factor 1 ( igf-1 ) concentrations were also reported in these patients . in many other syndromes such as prader - willi and skeletal dysplasia , there is defect in growth hormone and igf-1 axis which hasnt been declared yet so as growth hormone deficiency in pycnodysotosis . the prevalence of pycnodysostosis has been reported to be approximately 1.7 per million individuals with equal sex distribution . mutations in the gene identified for coding of cathepsin k , a lysosomal cysteine protease localized exclusively in osteoclasts , were suggested to be responsible for this disease . considering the extremely short stature as well as other complications of these children in their adulthood and limited investigations on the treatment of this disorder , the present study aims to describe gh level and effect of this hormone on linear growth in eight children suffering from pycnodysostosis . the present study was conducted on 8 children with clinical and radiological features of pycnodysostosis in the age range of 3 - 6 years at endocrinology clinic , shiraz , southern iran , from may 2010 to november 2012 . before any interventions , all parents of the patients filled the consent forms for participating in this study . in the first visit , children with clinical and radiological symptoms of pycnodysostosis were included in this study and then all systemic diseases , adrenal insufficiency , hypothyroidism , and also the compliance of the patient s family were evaluated and in case of any dissatisfaction in this appraisal , the patient was excluded from the study . height standard deviation score ( htsds ) , body mass index ( bmi ) , and some other demographical characteristics of the patients were measured since 6 months prior to any treatment and continued during and after the end of the gh treatment . after obtaining a basal blood sample , a standard oral clonidine test ( 0.15 mg / m ) was performed twice ; in a way that serum samples were obtained every 30 minutes for two hours in order to measure the serum gh . with initiation of the trial , human gh was injected subcutaneously once a day 6 days a week for a period of 1.5 years ( 50g / kg / day ) . the patients were followed up for 3 months in order to document their height and bmi until 6 months after the end of the treatment . besides musculoskeletal examination ( like for scoliosis ) , serum glucose , thyroid hormone and cortisol level were attained in these follow ups in order to assess complications of gh administration ( every 6 months ) . igf-1 was measured every 6 months after beginning the treatment which was in the normal range based on age and sex and there was no need to adjust gh . one - way anova test was used for comparison of the study group in different courses of treatment . besides , p - value 0.05 was considered as statistically significant . participants of this study were 8 children ( 4 girls and 4 boys ) in the age range of 3 - 6 years . all of the subjects had gh deficiency and bone ages were lower than the chronological age . according to table 1 , the children s mean bmi at each visit had increased in comparison to the previous visit during gh therapy and in most of the cases it was decreased in the follow ups after discontinuing gh . in addition , table 2 shows that all the patients had a htsds below -3.5 at the first visit which continued to decrease during the 6 months before starting the treatment ; however , htsds started to increase after beginning of gh administration . 1 the mean value of growth was improved after gh therapy in the patients , in both males and females separately ( fig . 2 ) . patients data before receiving growth hormone ( gh ) treatment , at first visit , and body mass index ( bmi ) before , during , and after receiving gh treatment descriptive data of patients height sd score before , during , and after discontinuing gh treatment it was demonstrated that height velocity of patients during gh therapy was significantly different in comparison to growth rate before and after this time period . pycnodysostosis is an inherited disorder of the bone remodeling and dwarfism is one of its major features . this presentation is due to some reasons , such as involvement of vertebrae , chronic airway obstruction , hypoxemia , marked anemia , chromosomal anomalies associated with the disease , and malnutrition secondary to dental disorders . so far , no specific treatment has been validated in pycnodysostosis except for symptomatic therapy . up to now only a few studies have been conducted on growth hormone level and effect of growth hormone therapy on linear growth in the children with pycnodysostosis . the prevalence of growth hormone deficiency in pycnodysostosis is still unknown . in the present study which was conducted on 8 children suffering from this disease , all patients had growth hormone deficiency after 2 provocative growth hormone assay tests . in a case report , darcan et al described a patient with pycnodysostosis and growth hormone deficiency in whom the growth rate improved after growth hormone treatment . rothenbuhler et al demonstrated that gh therapy developed growth noticeably in the children with pycnodysostosis , reaching midparental height instead of the short 14010 cm height in adulthood resulting from the disease . in a study performed by soliman et al ( 1996 ) , defective gh secretion associated with hypoplasia of the pituitary gland was detected in 4 of the 8 children with pycnodysostosis which was explained later by increased intrasellar pressure due to the increased bone volume and leading to the pituitary hypoplasia and partial gh deficiency in some of these patients . soliman et al ( 2001 ) also showed that a lot of patients suffering from pycnodysostosis had gh secretion impairment and low igf - i concentrations . besides , they reported a dramatically increased igf - i secretion and improvement of linear growth after gh therapy . curved line : mean ( sd ) of the values in each visit ; htsds starts to increase after beginning of growth hormone ( gh ) treatment and decreases again after stopping gh administration mean ( sd ) of growth rates ( cm / month ) in patients suffering from pycnodysostosis . a indicates significant difference between the growth rate during growth hormone treatment versus before and after the treatment ( p<0.001 ) . the present clinical study revealed that gh administration had a positive impact on the linear growth of the children suffering from pycnodysostosis , which could be due to defective gh secretion and low igf - i concentrations in the setting of this disease . however , there are a few studies on pycnodysostosis which make this disease still a concern for researchers and clinicians . therefore , proposing agents affecting the final height of these patients could affect their future life and this study could be a step forward in this scenario ; nevertheless , other dosages , the exact needed period of therapy , and also complications are not investigated in this work ; therefore , this emphasizes the importance of continuance of this thesis via future studies . outcome of the present study implies that gh treatment is beneficial for linear growth of patients suffering from pycnodysostosis , particularly since they showed gh deficiency ; however , the effect of this hormone on the final height of the patients is not apparent and more researches are needed to determine all aspects and also complications of long term hormone therapy in these patients and prevent the progression of skeletal abnormalities and correct the abnormal bone metabolism in these cases . as this disease is so rare and all the children with pycnodysostosis participating in this study were suffering from growth hormone deficiency , it was impossible to investigate the effect of this agent in the patients with pycnodysotosis without gh deficiency . scarcity of the cases was also another limitation for this study and finding this amount of the patients was so unlikely . z. karamizadeh : concept , data interpretation , study supervision h. ilkhanipoor : acquisition of data , data analysis , drafting of the manuscript f. bagheri : data analysis , draft of manuscript all authors approved final version of the paper .

it is well known that the content of sea hare digestive glands is derived from the diet that this animal ingests [ 14 ] . a large number of cognate compounds have been isolated from both the red alga of the genus laurencia and the sea hare that preys on the red alga . metabolites isolated from sea hares exhibit various biological activities , for example , cytotoxic [ 57 ] , algicidal , ichthyotoxic , antifungal [ 6 , 8 ] , and neurotrophic activities . in the course of our search for new biologically active compounds from marine organisms [ 912 ] , we succeeded in isolating four compounds exhibiting cytotoxic and antibacterial activities from sea hare collected from toyama bay in the japan sea . here we report the isolation , structure determination , and the biological activities of the four compounds including two new derivatives . specimens of aplysia kurodai were collected by hand from toyama bay in the japan sea and kept frozen until extraction with meoh . the etoac layer , which showed cytotoxic and antibacterial activity against staphylococcus aureus , was fractionated by a combination of silica gel chromatography , silica gel hplc , and gel permeation on sephadex lh-20 to afford laurinterol acetate ( 1 ) , laurinterol ( 2 ) [ 13 , 14 ] , debromolaurinterol acetate ( 3 ) , and debromolaurinterol ( 4 ) [ 13 , 14 ] in yields of 2.3 10 , 3.8 10 , 2.6 10 , and 3.9 10% , respectively . the eims of 1 showed 1:1 doublet ion peaks at m / z 336 and 338 [ m ] , indicating the presence of a bromine atom in the molecule , and a molecular formula of c17h21o2br was established by hreims and c nmr data . the ir spectrum of 1 showed absorption at 1762 and 1600 cm , which indicated the presence of a conjugated ester group and an aromatic ring . the h nmr spectrum of 1 ( table 1 ) revealed two characteristic high - field signals assignable to a trisubstituted cyclopropane ring at 0.50 ( t , j = 4.6 hz ) and 0.55 ( dd , j = 4.6 , 8.8 hz ) , two singlet aromatic signals at 6.88 ( s ) and 7.82 ( s ) , four singlet methyls at 1.30 ( 3h , s ) , 1.34 ( 3h , s ) , 2.30 ( 3h , s ) , and 2.39 ( 3h , s ) . the analysis of cosy and hmbc spectra of 1 suggested the presence of a dimethylbicyclo[3.1.0]hexane group and a 1,2,4,5-tetrasubstituted aromatic ring ( fig . the hmbc cross - peak , h 1.34 ( h3 - 14)/c 139.7 ( c-6 ) , showed the connection between c-1 of the bicyclohexane ring and c-6 of the aromatic ring . the hmbc cross - peak , h 2.39 ( h3 - 15)/c 136.1 ( c-9 ) , revealed the methyl signal was attached to a carbon at c-9 , which was supported by the noe correlation between h3 - 15 and h-8 ( 6.88 , s ) . the magnitude of carbon chemical shifts of 148.3 ( c-7 ) and 121.2 ( c-10 ) indicated that an acetate and a bromine group were substituted at c-7 and c-10 , resepectively . these data suggested that 1 was an acetate of laurinterol ( 2 ) [ 13 , 14 ] . the acetylation of 2 afforded its monoacetate , of which the h nmr spectrum showed the appearance of an additional acetate signal at 2.30 ( 3h , s ) instead of the disappearence of a hydroxy signal at 5.39 ( s , 7-oh ) in 2 . the spectral data of the monoacetate were all identical with those of 1 including the specific rotation , which implied that 1 was laurinterol acetate . the ei mass spectrum of compound 3 exhibited an ion peak at m / z 258 [ m ] , which matched a formula of c17h22o2 . the h nmr spectrum of 3 was similar to that of 1 , except for the presence of two doublet aromatic protons at 6.98 ( 1h , d , j = 7.8 hz , h-10 ) and 7.57 ( 1h , d , j = 7.8 hz , h-11 ) and the absence of a singlet aromatic proton at 7.82 ( 1h , s , h-11 ) in 1 . these spectral features indicated that 3 was an acetate of debromolaurinterol ( 4 ) [ 13 , 14 ] . the acetylation of 4 afforded a monoacetate and its spectral data were identical with those of 3 . the antibacterial activity using staphylococcus aureus ( table 2 ) and cytotoxicity ( table 3 ) were tested for 14 . compared with acetates ( 1 and 3 ) and their original metabolites ( 2 and 4 ) , we have isolated laurinterol acetate ( 1 ) , laurinterol ( 2 ) [ 13 , 14 ] , debromolaurinterol acetate ( 3 ) , and debromolaurinterol ( 4 ) [ 13 , 14 ] from the sea hare , aplysia kurodai . isolation of 2 and 4 was reported from the red alga , laurencia intermedia yamada , and their acetates 1 and 3 were derived from 2 and 4 by acetylation , respectively [ 13 , 14 ] . however , this is the first isolation of 1 and 3 from natural sources . among the four metabolites , 1 , 2 , and 4 showed moderate cytotoxicity against tumor cells , and 2 and 4 exhibited more potent antibacterial activity against staphylococcus aureus than 1 and 3 . it is implied that the content of sea hare digestive glands is derived from the red alga of the genus laurencia that this animal ingests [ 14 ] and that the metabolites are used for the sake of their self - defense . although isolation of acetates 1 and 3 from red alga has not been reported so far , the acetates could be derived from the red alga ingested as diet or transformed from 2 and 4 in the digestive gland of the sea hare . all chemical shifts were reported with respect to the residual solvent peaks ( h 7.26 amd c 77.0 ) . the extract was concentrated under reduced pressure and extracted with etoac . the etoac layer ( 1.4 g ) was subjected to silica gel chromatography with a stepwise gradient of hexane / etoac to give two cytotoxic and antibacterial fractions , fr . 1 ( 642 mg ) eluted with hexane / etoac ( 19:1 ) and fr . 2 ( 354 mg ) eluted with hexane / etoac ( 9:1 ) . after purification by silica gel chromatography ( hexane / etoac ) followed by silica gel hplc ( hexane / etoac ) , fr . 1 gave laurinterol acetate ( 1 , 38.7 mg , 2.3 10% ) and debromolaurinterol acetate ( 3 , 4.5 mg , 2.6 10% ) . 2 was purified by silica gel chromatography ( hexane / etoac ) followed by sephadex lh-20 gel filtration ( hexane / etoac ) to give laurinterol ( 2 , 64.8 mg , 3.8 10% ) [ 13 , 14 ] and debromolaurinterol ( 4 , 6.8 mg , 3.9 10% ) [ 13 , 14 ] . laurinterol acetate ( 1 ) : [ ]d + 1.7 ( c 0.064 , chcl3 ) ; uv ( meoh ) max ( log ) 269 ( 3.5 ) and 279 nm ( 3.4 ) ; ir ( film ) max 3060 , 2930 , 1762 , 1600 , 1544 , 1482 , 1364 , 1203 cmh and c nmr ( cdcl3 ) , see table 1 ; eims m / z 336/338 ( intensity , 1:1 ) [ m ] ; hreims m / z 336.0707(calcd for c17h21o2br , 336.0725 ) . laurinterol ( 2 ) : h nmr ( cdcl3 ) 0.54 ( 1h , dd , j = 8.8 , 4.6 hz , h-12 ) , 0.57 ( 1h , t , j = 4.6 hz , h-12 ) , 1.13 ( 1h , dt , j = 8.8 , 4.6 hz , h-3 ) , 1.27 ( 1h , m , h-5 ) , 1.31 ( 3h , s , h3 - 13 ) , 1.40 ( 3h , s , h3 - 14 ) , 1.65 ( 1h , dd , j = 13.2 , 8.8 hz , h-4 ) , 1.94 ( 1h , m , h-4 ) , 2.10 ( 1h , dd , j = 13.2 , 8.8 hz , h-5 ) , 2.28 ( 3h , s , h3 - 15 ) , 5.39 ( 1h , br s , 7-oh ) , 6.61 ( 1h , s , h-8 ) , 7.60 ( 1h , s , h-11 ) ; eims m / z 294/296 ( intensity , 1:1 ) [ m ] . debromolaurinterol acetate ( 3 ) : [ ]d 4.7 ( c 0.092 , chcl3 ) ; uv ( meoh ) max ( log ) 265 ( 3.5 ) and 275 nm ( 3.4 ) ; ir ( film ) max 2920 , 2850 , 1730 , 1460 , 1177 , 1038 cmh nmr ( cdcl3 ) 0.50 ( 2h , h2 - 2 ) , 1.09 ( 1h , dt , j = 8.8 , 4.6 hz , h-3 ) , 1.28 ( 1h , m , h-5 ) , 1.30 ( 3h , s , h3 - 13 ) , 1.34 ( 3h , s , h3 - 14 ) , 1.65 ( 1h , dd , j = 13.2 , 8.8 hz , h-4 ) , 1.83 ( 1h , dd , j = 13.2 , 8.8 hz , h-5 ) , 1.94 ( 1h , m , h-4 ) , 2.31 ( 3h , s , 7-oac ) , 2.33 ( 3h , s , h3 - 15 ) , 6.81 ( 1h , s , h-8 ) , 6.98 ( 1h , d , j = 7.8 hz , h-10 ) , 7.57 ( 1h , d , j = 7.8 hz , h-11 ) ; eims m / z 258 [ m ] ; hreims m / z 258.1625 ( calcd for c17h22o2 , 258.1620 ) . debromolaurinterol ( 4 ) : h nmr ( cdcl3 ) 0.51 ( 1h , dd , j = 7.8 , 4.4 hz , h-12 ) , 0.58 ( 1h , t , j = 4.4 hz , h-12 ) , 1.13 ( 1h , dt , j = 7.8 , 4.4 hz , h-3 ) , 1.32 ( 1h , m , h-5 ) , 1.33 ( 3h , s , h3 - 13 ) , 1.43 ( 3h , s , h3 - 14 ) , 1.66 ( 1h , dd , j = 13.2 , 7.8 hz , h-4 ) , 1.95 ( 1h , m , h-4 ) , 2.11 ( 1h , dd , j = 13.2 , 7.8 hz , h-5 ) , 2.27 ( 3h , s , h3 - 15 ) , 5.05 ( 1h , br s , 7-oh ) , 6.55 ( 1h , s , h-8 ) , 6.68 ( 1h , d , j = 7.8 hz , h-10 ) , 7.39 ( 1h , d , j = 7.8 hz , h-11 ) ; c nmr ( cdcl3 ) 16.6 ( c-12 ) , 19.1 ( c-13 ) , 20.9 ( c-15 ) , 23.9 ( c-14 ) , 24.6 ( c-3 ) , 25.6 ( c-4 ) , 30.0 ( c-2 ) , 36.4 ( c-5 ) , 48.2 ( c-1 ) , 117.6 ( c-8 ) , 121.1 ( c-10 ) , 129.1 ( c-11 ) , 131.7 ( c-6 ) , 137.0 ( c-9 ) , 154.3 ( c-7 ) ; eims m / z 216 [ m ] . hela cells were grown in dulbecco s modified eagle s medium supplemented with 10% fetal calf serum , penicillin ( 50 units / ml ) , and streptomycin ( 50 g / ml ) under a humidified atmosphere of 5% co2 at 37 c . the cells were seeded into 96-well microplates ( 310 cells / well ) and pre - cultured for a day . the medium was replaced with that containing test compounds at various concentrations and the cells were further cultured at 37 c for 3 days . the medium was then replaced with 50 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) solution ( 0.2 mg / ml in medium ) and the cells were incubated under the same conditions for 4 h. after addition of 200 l of dmso , the optical density at 570 nm was measured with a microplate reader . paper disk ( 6 mm ) with sample ( 50 , 25 , or , 12.5 g ) was incubated on an agar plate containing bacterium at 37 c . all chemical shifts were reported with respect to the residual solvent peaks ( h 7.26 amd c 77.0 ) . the extract was concentrated under reduced pressure and extracted with etoac . the etoac layer ( 1.4 g ) was subjected to silica gel chromatography with a stepwise gradient of hexane / etoac to give two cytotoxic and antibacterial fractions , fr . 1 ( 642 mg ) eluted with hexane / etoac ( 19:1 ) and fr . 2 ( 354 mg ) eluted with hexane / etoac ( 9:1 ) . after purification by silica gel chromatography ( hexane / etoac ) followed by silica gel hplc ( hexane / etoac ) , fr . 1 gave laurinterol acetate ( 1 , 38.7 mg , 2.3 10% ) and debromolaurinterol acetate ( 3 , 4.5 mg , 2.6 10% ) . 2 was purified by silica gel chromatography ( hexane / etoac ) followed by sephadex lh-20 gel filtration ( hexane / etoac ) to give laurinterol ( 2 , 64.8 mg , 3.8 10% ) [ 13 , 14 ] and debromolaurinterol ( 4 , 6.8 mg , 3.9 10% ) [ 13 , 14 ] . laurinterol acetate ( 1 ) : [ ]d + 1.7 ( c 0.064 , chcl3 ) ; uv ( meoh ) max ( log ) 269 ( 3.5 ) and 279 nm ( 3.4 ) ; ir ( film ) max 3060 , 2930 , 1762 , 1600 , 1544 , 1482 , 1364 , 1203 cmh and c nmr ( cdcl3 ) , see table 1 ; eims m / z 336/338 ( intensity , 1:1 ) [ m ] ; hreims m / z 336.0707(calcd for c17h21o2br , 336.0725 ) . laurinterol ( 2 ) : h nmr ( cdcl3 ) 0.54 ( 1h , dd , j = 8.8 , 4.6 hz , h-12 ) , 0.57 ( 1h , t , j = 4.6 hz , h-12 ) , 1.13 ( 1h , dt , j = 8.8 , 4.6 hz , h-3 ) , 1.27 ( 1h , m , h-5 ) , 1.31 ( 3h , s , h3 - 13 ) , 1.40 ( 3h , s , h3 - 14 ) , 1.65 ( 1h , dd , j = 13.2 , 8.8 hz , h-4 ) , 1.94 ( 1h , m , h-4 ) , 2.10 ( 1h , dd , j = 13.2 , 8.8 hz , h-5 ) , 2.28 ( 3h , s , h3 - 15 ) , 5.39 ( 1h , br s , 7-oh ) , 6.61 ( 1h , s , h-8 ) , 7.60 ( 1h , s , h-11 ) ; eims m / z 294/296 ( intensity , 1:1 ) [ m ] . debromolaurinterol acetate ( 3 ) : [ ]d 4.7 ( c 0.092 , chcl3 ) ; uv ( meoh ) max ( log ) 265 ( 3.5 ) and 275 nm ( 3.4 ) ; ir ( film ) max 2920 , 2850 , 1730 , 1460 , 1177 , 1038 cmh nmr ( cdcl3 ) 0.50 ( 2h , h2 - 2 ) , 1.09 ( 1h , dt , j = 8.8 , 4.6 hz , h-3 ) , 1.28 ( 1h , m , h-5 ) , 1.30 ( 3h , s , h3 - 13 ) , 1.34 ( 3h , s , h3 - 14 ) , 1.65 ( 1h , dd , j = 13.2 , 8.8 hz , h-4 ) , 1.83 ( 1h , dd , j = 13.2 , 8.8 hz , h-5 ) , 1.94 ( 1h , m , h-4 ) , 2.31 ( 3h , s , 7-oac ) , 2.33 ( 3h , s , h3 - 15 ) , 6.81 ( 1h , s , h-8 ) , 6.98 ( 1h , d , j = 7.8 hz , h-10 ) , 7.57 ( 1h , d , j = 7.8 hz , h-11 ) ; eims m / z 258 [ m ] ; hreims m / z 258.1625 ( calcd for c17h22o2 , 258.1620 ) . debromolaurinterol ( 4 ) : h nmr ( cdcl3 ) 0.51 ( 1h , dd , j = 7.8 , 4.4 hz , h-12 ) , 0.58 ( 1h , t , j = 4.4 hz , h-12 ) , 1.13 ( 1h , dt , j = 7.8 , 4.4 hz , h-3 ) , 1.32 ( 1h , m , h-5 ) , 1.33 ( 3h , s , h3 - 13 ) , 1.43 ( 3h , s , h3 - 14 ) , 1.66 ( 1h , dd , j = 13.2 , 7.8 hz , h-4 ) , 1.95 ( 1h , m , h-4 ) , 2.11 ( 1h , dd , j = 13.2 , 7.8 hz , h-5 ) , 2.27 ( 3h , s , h3 - 15 ) , 5.05 ( 1h , br s , 7-oh ) , 6.55 ( 1h , s , h-8 ) , 6.68 ( 1h , d , j = 7.8 hz , h-10 ) , 7.39 ( 1h , d , j = 7.8 hz , h-11 ) ; c nmr ( cdcl3 ) 16.6 ( c-12 ) , 19.1 ( c-13 ) , 20.9 ( c-15 ) , 23.9 ( c-14 ) , 24.6 ( c-3 ) , 25.6 ( c-4 ) , 30.0 ( c-2 ) , 36.4 ( c-5 ) , 48.2 ( c-1 ) , 117.6 ( c-8 ) , 121.1 ( c-10 ) , 129.1 ( c-11 ) , 131.7 ( c-6 ) , 137.0 ( c-9 ) , 154.3 ( c-7 ) ; eims m / z 216 [ m ] . hela cells were grown in dulbecco s modified eagle s medium supplemented with 10% fetal calf serum , penicillin ( 50 units / ml ) , and streptomycin ( 50 g / ml ) under a humidified atmosphere of 5% co2 at 37 c . the cells were seeded into 96-well microplates ( 310 cells / well ) and pre - cultured for a day . the medium was replaced with that containing test compounds at various concentrations and the cells were further cultured at 37 c for 3 days . the medium was then replaced with 50 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) solution ( 0.2 mg / ml in medium ) and the cells were incubated under the same conditions for 4 h. after addition of 200 l of dmso , the optical density at 570 nm was measured with a microplate reader . growth - inhibitory activity was determined by the paper disk method . paper disk ( 6 mm ) with sample ( 50 , 25 , or , 12.5 g ) was incubated on an agar plate containing bacterium at 37 c .

hypertension has been acknowledged as one of the greatest and established risk factors for cardiovascular diseases ( cvd ) ( heart diseases and stroke ) [ 14 ] . particularly in east asian countries , where the mortality and morbidity of stroke have been higher than in western countries , measures against hypertension hypertension affects a majority of the elderly , and drug therapies for hypertension have greatly added to medical costs in most developed countries . on the other hand , japan is one of the countries with the longest longevity in the world , the major reason of which is regarded to be a great decline in stroke mortality due to a decrease in nationwide blood pressure ( bp ) levels . the author has been involved in epidemiological researches on the risk assessment and prevention of hypertension through several cohort studies in japan , their meta - analyses , and international cooperative studies . in this article , findings from these studies are reviewed from the viewpoints of population - wide and individual - based preventive approaches in japanese , and the reason of the longest longevity of japanese is discussed . the national integrated project for prospective observation of non - communicable disease and its trends in the aged ( nippon data ) consists of two cohort studies of representative japanese population samples who participated in the national surveys on circulatory disorders and the national nutrition surveys in 1980 ( nippon data80 ) and in 1990 ( nippon data90 ) . a total of approximately 10,000 ( nippon data80 ) and 8,000 ( nippon data90 ) participants were from 300 randomly selected areas from all over japan . this project has served a number of evidences on the relationships of major cvd risk factors on long - term cvd mortality risk , including a strong , continuous , graded , and independent relationship of bp levels to cvd risk [ 7 , 8 ] . in addition , follow - up surveys on activity of daily living ( adl ) in the nippon data80 participants reported a positive relation between high bp at middle age and future impaired adl , since a large part of impaired adl is caused by stroke occurrence in japan . we found that 45% of impaired adl could be explained by non - normal bp at baseline . the evidence for cardiovascular prevention from observational cohorts in japan ( epoch - japan ) is a meta - analysis of individual participants data from high - quality established 13 cohort studies in japan , including nippon data . the total number of epoch - japan participants was over 180 thousands , with approximately 10 years of follow - up , and sex- and age - specific risks ( relative and attributable ) of all - cause mortality with higher bp were estimated in this meta - analysis . adjusted all - cause mortality increased as bp increased in all age - groups ( fig . 1 ) , and this trend was more apparent in younger people than older people ; hazard ratio [ hr ] for 10 mm hg increase in systolic bp was 1.37 in men aged 4049 , whereas hr was 1.09 in men aged 8089 . younger people should pay more attention to bp increase to prevent future cardiovascular risk , and should modify their lifestyle to lower bp from their younger age . moreover , population - attributable fraction of deaths with higher bp above optimal ( < 120/80 mm hg ) was 23% in men and 18% in women . thus , a great impact of higher bp above optimal level on total deaths in japan was revealed . 1relation between blood pressure and multivariate - adjusted mortality rate by age range ( men and women combined ) . each dot represents a multivariate estimate of mortality rate after adjusting for sex , smoking , drinking , and body mass index , and each line shows the 95% ci relation between blood pressure and multivariate - adjusted mortality rate by age range ( men and women combined ) . each dot represents a multivariate estimate of mortality rate after adjusting for sex , smoking , drinking , and body mass index , and each line shows the 95% ci some epidemiologic studies reported that pulse pressure ( pp ) , the difference between systolic bp ( sbp ) and diastolic bp ( dbp ) , is a useful predictor for cvd especially in middle - aged or older people [ 11 , 12 ] ; however , it was uncertain whether pp is superior to sbp or dbp in predicting future cvd risk in various age - sex groups of general japanese population , where stroke is dominant among cvds . moreover , it is also important to assess whether dbp has any additional role in predicting risks independently from sbp . the author examined these questions in a population - based cohort study , the oyabe study , on long - term stroke incidence by four age - sex groups ( men and women aged 3564 and 6579 years ) . the study showed that relations of pp to stroke risk were less strong than those of sbp in all age - sex groups . in middle - aged men , the strongest relationship to stroke incidence was observed for dbp ( hr 1.79 for 1 standard deviation [ sd ] increase ) . in older men , the strongest relationship to stroke risk older women , both sbp ( hr 1.56 and 2.25 ) and dbp ( hr 1.63 and 2.46 ) showed similarly strong relationships to stroke risk . this issue was also examined by large - scale meta - analyses [ 3 , 14 ] . however , the majority of study participants in these studies were caucasians ( even in one study from the asia pacific region ) , and there have been few investigations including only asian people . moreover , it was still uncertain whether the importance of bp indices differs for predicting ischemic stroke and hemorrhagic stroke risks . the japan arteriosclerosis longitudinal study - existing cohorts combine ( jals - ecc ) is a pooling project based on individual participant data from existing high - quality prospective cohort studies on cvd in japan . from this project , the author reported the meta - analysis of 16 cohort studies which allowed detailed investigations with over 1,000 stroke events and 200 myocardial infarction ( mi ) events from over 400,000 person - years of follow - up in japanese . stroke risk was most strongly related to mean bp ( mbp ) and sbp in both sexes , and was most weakly related to pp ( fig . 2 ) . sbp and dbp were independently and positively related to stroke risk in both sexes , even after adjusted for each other . both stroke subtypes , ischemic and hemorrhagic , were most strongly related to mbp or sbp in both sexes . also in older men and women aged 7089 , mbp or sbp showed the strongest relation to all stroke risk . mi risk was most strongly related to sbp and mbp in both sexes . for any cvd endpoints in any age - sex groups , pp was not the strongest predictor . in this report , we concluded that the long - term incident risk of stroke and mi associated with high bp in east asian populations should be assessed mainly on the basis of sbp . hazard ratios are calculated for each bp index higher by 1standard deviation , and adjusted for age , body mass index , total cholesterol , and smoking using poisson regression . * sbp systolic blood pressure ; dbp diastolic blood pressure ; pp pulse pressure ; mbp mean blood pressure ; hr hazard ratio ; 95%ci 95% confidence interval adjusted hazard ratios for four blood pressure indexes for all stroke incidence by sex . hazard ratios are calculated for each bp index higher by 1standard deviation , and adjusted for age , body mass index , total cholesterol , and smoking using poisson regression . * two bp indices sbp systolic blood pressure ; dbp diastolic blood pressure ; pp pulse pressure ; mbp mean blood pressure ; hr hazard ratio ; 95%ci 95% confidence interval several factors have been reported to determine population - wide bp level and individual s bp in japan . in 1960 , dahl authored a famous study on the relationship between salt intake and bp in populations in various parts of the world , including japan . he reported that people living in northern japan consumed about 30 g salt per day and had an almost 40% prevalence of hypertension , whereas alaskan natives consumed less than 5 g of salt per day and had a prevalence of almost zero . the intersalt study was conducted , using 52 populations in 32 countries ( including three populations in japan ) ( a total of 10,079 participants ) , with strictly standardized bp measurement and evaluation of salt intake by 24-h urinary sodium excretion determination [ 1820 ] . the intersalt study found that urinary sodium excretion was significantly related to individual bp and to age - dependent bp elevation , after adjustment for age , gender , obesity and alcohol consumption . it was estimated that 100 mmol lower sodium excretion ( equivalent to about 6 g salt ) was related to 2.2 mmhg lower sbp , and to lower elevation in sbp and dbp by 1011 mmhg and 6 mmhg , respectively , between 25 and 55 years of age a period of 30 years . as also mentioned below the intersalt study also showed that sbp was significantly higher in men and women drinking 300 ml of ethanol / week ( approximately 238 g ) than in non - drinkers . other cross - sectional studies in japanese men showed that sbp was approximately 710 mmhg higher , and dbp was approximately 27 mmhg higher , in drinkers of three to six drinks / day ( approximately 210420 g of ethanol / week ) than in non - drinkers [ 22 , 23 ] , and that the relationship was not different among the types of beverage . moreover , a large - scale 7-year follow - up study of japanese male workers revealed that , when adjusted for age , weight change , and foods intake , average annual increases in sbp were 0.33 mm hg greater in drinkers of 300 g ethanol / week than in non - drinkers ; it means that sbp in drinkers will increase 3.3 mm hg greater in 10 years than in non - drinkers . a randomized controlled trial in japanese habitual male drinkers also showed that a reduction of ethanol consumption from 56 ml / day to 23 ml / day resulted in 3.6 mm hg decrease in sbp . the international study of macro - micro - nutrients and blood pressure ( intermap ) showed that mean ethanol intake in middle - aged men was the highest in japan ( 30 g / day ) among four countries , whereas it was 20 g / day in the uk , 17 g / day in china , and 11 g / day in the usa . therefore , higher alcohol consumption would contribute largely to increase bp level and the prevalence of hypertension in japanese men . the intermap is an international cross - sectional epidemiologic study of 4,680 men and women ages 4059 from 17 population samples in china ( three samples ) , japan ( four samples ) , uk ( two samples ) , and usa ( eight samples ) . nutrient intake data were based on four in - depth multi - pass 24-h dietary recalls / person and two timed 24-h urine collection / person , and bp was measured eight times at four visits [ 28 , 29 ] . the study has reported the relationships of several nutrients to bp , including the intakes of vegetable protein , omega-3 polyunsaturated fatty acids ( pfa ) , omega-6 pfa ( linoleic acid ) , dietary cholesterol , phosphorus , non - heme iron , and glutamic acid [ 3036 ] . among these nutrients , omega-3 pfa intake , which was inversely related to bp , was the highest in japan among four countries ( 3.4 g / day in men and 2.7 g / day in women ) due to higher fish intake , so that higher omega-3 pfa intake would affect favorably to bp level in japanese . on the other hand , dietary cholesterol intake , which was positively related to bp , was the highest in japan ( 446 mg / day in men and 359 mg / day in women ) largely due to higher egg intake , so that higher cholesterol intake would affect unfavorably to bp level in japanese . obesity is , of course , an important factor for high bp in japanese , although japanese people have been relatively lean compared with western people . the proportion of individuals with obesity - induced hypertension among total hypertensives was calculated using the baseline data of nippon data80 and nippon data90 . the estimated percentage whose hypertension was attributed by obesity ( bmi 25 kg / m ) in 1980 and 1990 was 11% and 15% for men and 19% and 22% for women , respectively . as the proportion of obesity has been increasing continuously in men and in older women in japan , these percentages would be higher now compared with those in 1980 and 1990 . during 1960 s , mortality due to stroke in the japanese population was markedly high by international standards , but has now been drastically reduced to approximately one - seventh of the previous level ( fig . this drastic change is the main cause of elongation in longevity of japanese during the past several decades , and it is presumably largely due to a steady decrease in the mean bp of the population . this decrease in bp was not only found in the elderly , but also in the middle - aged and younger generations . the national survey on circulatory disorders of japan showed that in the 30 years from 1971 to 2000 , the mean sbp for japanese men aged 40 to 49 decreased by 3.6 mmhg , from 134.8 mmhg to 131.2 mmhg , and for women aged 40 to 49 it decreased by 7.7 mmhg , from 132.6 mmhg to 124.8 mmhg ( fig . 3trend in age - adjusted mortality rate ( /100,000 ) of stroke in japan by sex and stroke subtypes , the vital statistics of japan ( 19502008 ) . . 4trends in mean systolic blood pressure in japanese men and women by age groups , the national survey on circulatory disorders of japan ( 19712000 ) trend in age - adjusted mortality rate ( /100,000 ) of stroke in japan by sex and stroke subtypes , the vital statistics of japan ( 19502008 ) . ci cerebral infarction ; ch cerebral hemorrhage ; sah subarachnoid hemorrhage trends in mean systolic blood pressure in japanese men and women by age groups , the national survey on circulatory disorders of japan ( 19712000 ) population - wide screening of high bp by law begun in 1960 s ; therefore , detection and pharmaceutical treatment of hypertension effectively decreased the prevalence of hypertension especially in elder population . however , as the mean bp decreased in the whole population including the younger generations , such a phenomenon can not be explained merely by the widespread use of anti - hypertensive agents ( e.g. 30% in 1986 to 40% in 2002 in hypertensive men ) , but should be largely due to a change in the japanese lifestyle and dietary habits . bp decreased despite unfavorable trends of other risk factors such as increased obesity ( excluding young women ) and an increase in alcohol consumption . several decades ago , people living in the northern part of japan consumed as much as 20 to 30 g of salt per day . the national nutrition survey reported that the daily salt intake of the japanese decreased by 3.8 g , from 14.5 g in 1973 to 10.7 g in 2009less than half of the estimated intake in the 1950s . the salt reduction in japan was accomplished in part by a campaign to reduce salt intake initiated by the japanese government in 1960 s , in addition to a big lifestyle change during the past several decades . people now do not need to preserve foods using salt through the spread of refrigerator and the development of distribution system , and the westernization of dietary habits reduced the use of soy sauce and miso ( fermented soybean paste ) which are major dietary sources of salt in japan . japan is one of the countries with the longest longevity in the world , and it was accomplished by a drastic decline in stroke mortality from 1960 s . the decline in stroke mortality would be largely explained by a population - wide decreasing trend in bp level both in men and women in all age - groups , which can not be fully explained by the widespread use of anti - hypertensive agents . the most probable reason for the population - wide bp decline in japan would be a large decline in dietary salt intake after 1950 s . however , recent increasing trend in obesity , together with high alcohol intake in men , physical inactivity , and unfavorable dietary habits ( high energy , high fat , and low vegetable ) in japan could increase bp level of japanese again . further continuous efforts for the primary prevention of hypertension are necessary in japan . from the experience and epidemiologic findings in japan , it can be concluded that bp management in individuals and in a whole population is very important to extend longevity . population - wide screening and early detection of high bp , using both systolic and diastolic bp , are essential , and detected high - risk individuals should modify their lifestyle and , if necessary , should be treated pharmaceutically . on the other hand , population - based approaches to change environments for bp reduction make the individual s effort easier [ 46 , 47 ] . such approaches for society , government , manufacturers , communities , worksites , schools , or mass - media should also be applied continuously in future .

technika przezcewnikowej wymiany zastawki aortalnej zostaa opracowana jako alternatywa dla pacjentw o wysokim ryzyku operacyjnym lub nieoperacyjnych z cik objawow stenoz aortaln . celem niniejszej pracy bya ocena wynikw pacjentw poddawanych przezkoniuszkowej wymianie zastawki aortalnej jako jedynej opcji terapeutycznej przez wielodyscyplinarny zesp sercowy w pojedynczym orodku . pomidzy czerwcem 2009 r. a grudniem 2014 r. 41 pacjentw poddano przezkoniuszkowej implantacji zastawki aortalnej ( ta - tavi ) w orodku autorw . wszyscy chorzy otrzymali balonowe zastawki osierdziowe edwards sapien ( edwards lifesciences , irvine , ca , usa ) . redni wiek pacjentw wynis 79,6 roku , redni wynik logistic euroscore wynosi 21,06 12,82% . wystpiy nastpujce komplikacje : udar mzgu ( n = 1 ) , reeksploracja ze wzgldu na krwawienie lub tamponad serca ( n = 4 ) , niewydolno nerek wymagajca czasowej hemodializy ( n = 4 ) oraz stae wszczepienie stymulatora ( n = 3 ) . czas pooperacyjnego pobytu na oddziale intensywnej terapii wynis 4,6 5,7 dnia , a redni czas hospitalizacji : 11,6 7,2 dnia . surgical aortic valve replacement ( avr ) still represents the gold standard among the therapeutic options in patients with severe symptomatic aortic valve stenosis . with aging of the population , the number of older and sicker patients has increased , and a significant portion of them are not indicated for surgical intervention because of advanced age and its associated comorbidities . transcatheter aortic valve implantation ( tavi ) represents an attractive approach to treat high - risk or inoperable patients , with promising results . transcatheter aortic valve implantation can be performed through several access approaches : transfemoral , transaortic , trans - subclavian and transapical . in most centers performing tavi , a transfemoral first approach policy is applied . however , unsuitable peripheral arterial anatomy , bleeding and vascular complications frequently occur and are associated with increased risk of perioperative morbidity and long - term mortality . the transapical approach ( ta - tavi ) is considered a more invasive and complex procedure requiring general anesthesia , but provides superior control of valve positioning due to shorter distance , potential reduction of stroke due to absence of retrograde crossing of the aortic valve , and fewer access site complications . the aim of this study was to evaluate the short - term and mid - term outcomes of patients undergoing ta - tavi as a single - strategy option by a single - center multidisciplinary heart team . all patients with severe symptomatic aortic stenosis and excessive risk related to open surgery were discussed by an institutional multidisciplinary heart team . patients were considered as suitable candidates for transcatheter aortic valve implantation for technical surgical reasons ( e.g. , previous cardiac surgery , calcified ascending aorta not accessible for surgery ) or because of a very high risk for a conventional operation ( all reasons preventing the insertion of a catheter for external blood circulation , all conditions preventing from aortic clamping , reasons preventing surgical access of the mediastinum the internal mammary artery or another critical conduit crossing the midline and/or adherent to the posterior table of the sternum , past history of mediastinal irradiation , burns , active mediastinitis ) . the absolute contraindications for tavi were active or recent endocarditis or an annulus size that exceeded the recommendations of the valve manufacturers . from june 2009 to december 2014 , 41 patients with severe symptomatic aortic stenosis and excessive risk related to surgery were considered as suitable candidates for transcatheter aortic valve implantation , and all of them underwent transapical tavi at our institution . the evaluation of early operative results was performed according to the valve academic research consortium ( varc)-2 consensus criteria . these include device success endpoints and safety endpoints ( all - cause mortality , major stroke , peri - procedural myocardial infarction , life - threatening bleeding , major vascular complications , and acute kidney injury ) , valve performance , and complications during hospitalization and at 30 days . the impact of the learning curve on patient outcomes was analyzed by comparing the overall survival of the first 30% of patients operated on during 2009 - 2012 versus the next 70% of patients operated on during 2013 - 2014 . also we evaluated the impact of previous cardiac operations and euroscore i on patients undergoing ta - tavi . in all patients , multimodality imaging ( transesophageal echocardiography , coronary angiography and multislice computed tomography ) was performed to assess anatomic suitability , diameters of the aortic annulus , distribution and amount of calcification and aortic root geometry . all ta - tavi procedures were performed in a cath lab suitable for the use of a cardiopulmonary bypass machine by a multidisciplinary team of dedicated cardiac surgeons , interventional cardiologists and cardiac anesthesiologists . the procedures were performed under general anesthesia , and the only implanted devices were the sapien pericardial transcatheter balloon expandable bioprosthesis ( edwards lifesciences , irvine , ca , usa ) . rapid ventricular pacing at 150 - 180 beats / min was performed to reduce cardiac motion , blood pressure and transvalvular flow during balloon dilatation . positioning and deployment of the valve was performed under fluoroscopic and transesophageal guidance . immediately after valve deployment patients underwent clinical and echocardiographic evaluation at our institute before the procedure , at discharge , at 1 , 6 and 12 months after tavi , and on a yearly basis thereafter . continuous variables were expressed as means ( m ) with standard deviation ( sd ) or medians ( md ) with interquartile range ( iqr ) and minimum - maximum range . normality of the distributions was assessed using the d'agostino - pearson omnibus k2 tests and with the shapiro - wilk normality test . non - parametric mann - whitney test was used to compare and study the relationships between the continuous variables . categorical variables were described as numbers and percentages ( % ) and analyzed using the test or fisher 's exact test , as appropriate . a confidence level of 95% was accepted as significant . survival curves for time - to - event variables were constructed on the basis of all available follow - up data using kaplan - meier estimates and were compared with the log rank test . a two - sided level of 0.05 was used for all superiority testing . a p - value < 0.05 was considered to be significant . the statistical analysis was performed with ms excel 2003 for windows xp and the statistical analysis system graphpad prism version 5.01 ( graph - pad software , inc . , san diego , california , usa ) . continuous variables were expressed as means ( m ) with standard deviation ( sd ) or medians ( md ) with interquartile range ( iqr ) and minimum - maximum range . normality of the distributions was assessed using the d'agostino - pearson omnibus k2 tests and with the shapiro - wilk normality test . non - parametric mann - whitney test was used to compare and study the relationships between the continuous variables . categorical variables were described as numbers and percentages ( % ) and analyzed using the test or fisher 's exact test , as appropriate . survival curves for time - to - event variables were constructed on the basis of all available follow - up data using kaplan - meier estimates and were compared with the log rank test . a two - sided level of 0.05 was used for all superiority testing . a p - value < 0.05 was considered to be significant . the statistical analysis was performed with ms excel 2003 for windows xp and the statistical analysis system graphpad prism version 5.01 ( graph - pad software , inc . between june 2009 and december 2014 , 41 patients with severe symptomatic aortic stenosis with excessive risk related to surgery underwent ta - tavi at our institution . preoperative clinical variables of patients are listed in table i. mean age was 79.6 6.3 years , and mean logistic euroscore i was 20.1 12.8% . the previous cardiac operation was cabg in 12 patients ( 29.3% ) , mitral valve replacement with bioprosthesis in 2 patients ( 4.8% ) and a combined procedure ( cabg + mitral valve replacement ) in 1 patient ( 2.4% ) . in 17 patients ( 41.5% ) with concomitant coronary artery disease , elective coronary artery stent implantation was performed , but not as a part of the tavi procedure . incidence of chronic renal insufficiency was 36.5% ( 15 patients ) preoperatively ; 1 patient was on chronic renal replacement therapy . preoperative characteristics lv left ventricle , cad coronary artery disease , copd chronic obstructive pulmonary disease , tia transient ischemic attack , ta - tavi transapical transcatheter aortic valve implantation the procedure was performed using edwards sapien valves . a 23 mm sapien valve was implanted in 9 patients ( 21.9% ) , a 26 mm prosthesis in 20 patients ( 48.8% ) and a 29 mm prosthesis in 13 patients ( 31.7% ) . the postoperative peak gradient over the prosthesis valve was 15.1 mmhg and the mean gradient was 7.7 mmhg as measured by transesophageal echocardiography . mild - to - moderate post - implant paravalvular regurgitation was found in 8 ( 19.5% ) patients . in 2 patients , post - dilatation of the valve was performed with reduction of the leakage to a mild degree . in this group of 8 patients , there were 5 patients with the position of the prosthesis deeper into the left ventricle outflow tract . clinical operative results ta - tavi transapical transcatheter aortic valve implantation clinical endpoints according to varc-2 definitions are reported in table iii . the need for re - exploration for bleeding or tamponade arose in 4 patients ( 9.7% ) . one patient experienced a major stroke ( 2.4% ) , and 4 patients ( 9.7% ) had acute kidney injury requiring renal replacement therapy . clinical endpoints according to varc-2 criteria ( n = 41 ) cva cerebrovascular accident , tia transient ischemic attack device success as defined by varc-2 was achieved in 75.6% of cases ( table iv ) . device success criteria were not met in 10 patients for the following reasons : procedural mortality in 1 patient ( 2.4% ) , suboptimal performance of the prosthetic heart valve , mild - to - moderate prosthetic paravalvular regurgitation in 8 patients ( 19.5% ) , and 1 ( 2.4% ) rescue valve - in - valve composite endpoints , device success according to varc-2 criteria ( n = 41 ) early safety at 30 days as defined by varc-2 was achieved in 24 patients ( 58.8% ) ( table v ) . all - cause mortality at 30 days or during procedure hospitalization was 17.1% ( 7 patients ) , and was classified as cardiovascular mortality . intraprocedural mortality occurred in 1 patient ( 2.4% ) , death of multiorgan failure in 4 patients ( 9.7% ) , severe hemorrhage in 1 patient ( 2.4% ) , and 1 patient died in a local hospital ( 2.4% ) . in 4 patients ( 9.7% ) after full sternotomy in 2 patients and thoracotomy in 2 patients , we found diffuse bleeding in 3 patients . in the last patient we found a hematoma around the aortic root and massive untreatable bleeding from the free wall of the left ventricle and base of the heart . in 1 patient ( 2.4% ) , conversion to sternotomy and surgical aortic valve replacement after deployment of the balloon - expandable prosthesis was necessary due to annular rupture . one patient experienced a major stroke ( 2.4% ) and 4 patients ( 9.7% ) had acute kidney injury requiring renal replacement therapy . composite endpoints , early safety ( at 30 days ) according to varc-2 criteria ( n = 41 ) aki acute kidney injury clinical efficacy after 30 days according to varc-2 clinical guidelines is reported in table vi and was reached in 61.7% of patients . incidence of permanent pacemaker implantation for complete atrioventricular conduction block was 7.3% ( 3 patients ) . two patients required hospitalization for worsening of congestive heart failure and thoracotomy wound healing complication during the follow - up . in 5 patients moderate paravalvular leakage without significant clinical symptoms persists . composite endpoints , clinical efficacy ( after 30 days ) according to varc-2 criteria ( n = 34 ) varc-2 included a new composite endpoint , time - related valve safety , which combines valve dysfunction , endocarditis , and thrombotic complications of the prosthesis . the specific composite endpoint time - related valve safety was achieved in 79.4% of surviving patients during the follow - up . in our group of patients we observed 1 infective endocarditis of the prosthesis , and we did not find structural deterioration of the prosthetic valve , but 1 calcified leaflet of the native valve caused the obstruction of the normal leaflet motion of the transcatheter valve prosthesis . median stay in the intensive care unit was 1 day ( interquartile range iqr , 1 - 6.5 days , range 0 - 24 days ) , and median hospital stay was 9 days ( iqr , 8 - 14.5 days , range 1 - 39 days ) . the median follow - up was 12.7 months ( iqr , 4.1 - 20.75 months , range 0.1 - 55.6 months ) . overall kaplan - meier 1-year survival was 77.3% 6.8% , 2-year survival was 55% 12.5% , and 4-year survival was 37% 17% ( fig . kaplan meier analysis of overall survival of all patients underwent ta - tavi we did not observe any statistical significant differences in 1-year survival at follow - up related to reoperation . kaplan - meier survival was similar for the patients who underwent ta - tavi as a first procedure ( 80.2% 8.1% ) and patients who underwent ta - tavi as a second operation ( 72% 12% , p = 0.87 ) ( fig . a ) all - cause kaplan - meier survival of group primo ( first operation ) ( black line ) and group redo ( reoperation ) ( grey line ) . b ) kaplan - meier analysis of overall mortality of patients operated between years 2009 - 2012 ( 30% ) ( grey line ) , and the patients operated between years 2013 - 2014 ( 70% ) ( black line ) . c ) kaplan - meier survival for patients with different risk profile according to classification of euroscore i : euroscore 30% ( grey line ) , euroscore > 30 ( black line ) kaplan - meier analysis of 1-year survival related to the learning curve was 62% 13.5% for the first 30% of patients and 82% 7.3% for the second 70% of patients ( p = 0.21 , statistically not significant ) . up to 14 months the differences between curves increased , but they were still not statistically significant in this small group of patients . there was a trend to worse survival at the beginning of the learning curve ( fig . i , in the group with the euroscore 30% , the survival rate at 1 year was 74.5% 9.9% , and in the group with the euroscore > 30% , the 1-year survival rate was 87.5% 11.7% ( statistically insignificant , p = 0.52 ) ( fig . the median follow - up was 12.7 months ( iqr , 4.1 - 20.75 months , range 0.1 - 55.6 months ) . overall kaplan - meier 1-year survival was 77.3% 6.8% , 2-year survival was 55% 12.5% , and 4-year survival was 37% 17% ( fig . kaplan meier analysis of overall survival of all patients underwent ta - tavi we did not observe any statistical significant differences in 1-year survival at follow - up related to reoperation . kaplan - meier survival was similar for the patients who underwent ta - tavi as a first procedure ( 80.2% 8.1% ) and patients who underwent ta - tavi as a second operation ( 72% 12% , p = 0.87 ) ( fig . a ) all - cause kaplan - meier survival of group primo ( first operation ) ( black line ) and group redo ( reoperation ) ( grey line ) . b ) kaplan - meier analysis of overall mortality of patients operated between years 2009 - 2012 ( 30% ) ( grey line ) , and the patients operated between years 2013 - 2014 ( 70% ) ( black line ) . c ) kaplan - meier survival for patients with different risk profile according to classification of euroscore i : euroscore 30% ( grey line ) , euroscore > 30 ( black line ) kaplan - meier analysis of 1-year survival related to the learning curve was 62% 13.5% for the first 30% of patients and 82% 7.3% for the second 70% of patients ( p = 0.21 , statistically not significant ) . up to 14 months the differences between curves increased , but they were still not statistically significant in this small group of patients . there was a trend to worse survival at the beginning of the learning curve ( fig . 2b ) . based on euroscore i , in the group with the euroscore 30% , the survival rate at 1 year was 74.5% 9.9% , and in the group with the euroscore > 30% , the 1-year survival rate was 87.5% 11.7% ( statistically insignificant , p = 0.52 ) ( fig . 2c ) . the prognosis in symptomatic patients with severe aortic valve stenosis is poor if treated medically . conventional open surgical treatment of severe aortic valve stenosis in high - risk patients can be effectively performed with excellent outcomes and an observed hospital mortality rate ranging from 0 to 8% and a 1-year survival rate of around 90% of patients in the majority of recently published series [ 9 , 10 ] . however , the risk of surgical mortality increases significantly with age . surgeons sometimes hesitate to perform surgical valve replacement in this group of elderly patients , or patients themselves or their practitioners are reluctant to undergo surgical intervention . transcatheter aortic valve implantation ( tavi ) has been developed as a minimally invasive alternative to open surgery , especially in patients with unacceptably high perioperative risk or patients who are not suitable for conventional surgery . tavi became a very attractive , safe and effective therapeutic option which can be performed with acceptable mortality rate . the salient findings from 2-year analysis of the randomized partner trial are as follows : mortality after transcatheter aortic valve replacement ( tavr ) remained similar to that after surgical replacement ; stroke frequency was similar in the surgery and tavr groups after 30 days ; periprocedural complications ( strokes , major bleeding , and major vascular events ) affected mortality after tavi or surgical replacement ; aortic regurgitation ( even mild ) after tavi was associated with increased long - term mortality ; and valve performance in the tavi group was maintained during the follow - up and was similar to that in the surgery group . in the literature there is limited available information which directly compares transfemoral transcatheter aortic valve implantation ( tf - tavi ) and transapical aortic valve implantation ( ta - tavi ) . van der boon and colleagues reported the results of a multicenter collaborative study . in that study a total of 882 patients underwent tavi , of whom only 10.1% ( 89 patients ) underwent ta - tavi . the authors found that in institutions with predominant tf - tavi practice , ta - tavi is associated with an increased risk of 30-day mortality and all - cause mortality during the follow - up . moreover , patients undergoing ta - tavi had a longer hospital stay but fewer vascular complications in comparison with tf - tavi . the transfemoral approach is preferably the first choice access because it can be carried out completely percutaneously and without any general anesthesia . transapical patients usually have a higher estimated preoperative risk prediction and higher incidence of comorbidities . our reasons for the strategy of a candidates for tavi are more precise control and deployment of the valve in the correct position , and better and more intuitive manipulation with the device at a shorter distance . in the last patients we decided to perform angiography during the valve deployment while inflating the balloon , as described by pasic et al . as part of the berlin addition . angiography enabled perfect visualization of the position of the prosthetic valve and its relationship to the coronary arteries , aortic valve annulus and valve cusps throughout the valve deployment . angiography during deployment of the valve presents the coronary arteries immediately at the end of the valve deployment . angiographic visualization improves the safety of transapical aortic valve implantation and simplifies valve positioning and the valve - deploying technique . the inflation of the balloon during valve deployment can be performed slowly , allowing easy correction of the position of the valve if necessary . advancing the wire in an antegrade direction through the valve is very easy , rapid , and smooth in comparison to the retrograde approach used with transfemoral implantation . also the transapical approach is independent of the degree of the patient 's peripheral artery disease , calcification , diameter and other anatomical variables of the inguinal and iliac vessels . the reported rates of moderate or severe regurgitation during the tavi procedure vary between 10% and up to 20% or more in larger series , regardless of the type of prosthesis . paravalvular regurgitation ( pvr ) of some degree is observed and accepted in the majority of transcatheter aortic valve implantation patients . regurgitation after tavi is known to have a negative impact on mid - term and long - term survival . unbehaun and colleagues reported mild to moderate regurgitation in 20% of patients [ 17 , 18 ] . results from the italian registry of ta - tavi showed that 45% of patients had mild or moderate aortic regurgitation at hospital discharge . summarized in their work that there are two anatomical , non - modifiable factors which have an impact on the likelihood of pvr : aortic valve calcification and lvot - ao angle . there are two other valve - related , modifiable factors that determine the incidence of pvr : prosthesis - annulus discongruence and valve position . in our series we observed mild - to - moderate regurgitation in 19.5% of patients . we observed only 1 post - procedural stroke ( 2.4% ) and no other stroke during the follow - up . our result is comparable to a recent meta - analysis of 10 037 patients from 53 studies showing the incidence rate of major stroke at 30 days being 2.9 1.8% . the absence of retrograde crossing of the aortic valve and lack of manipulation in the aortic arch may reduce or eliminate cerebral embolization during this phase of the procedure and decreased the rate of neurological complications . we observed 1 conversion to sternotomy ( 2.4% ) due to annular rupture and surgical replacement of the transcatheter valve with a bioprosthesis , as well as 4 re - explorations ( 9.7% ) because of bleeding or tamponade . our observed 30-day mortality rate was 17.1% , reflecting the high - risk profile of these patients and the early experience and learning curve with these methods . at the beginning of the tavi program in our institution , when all high - risk patients were considered as candidates for tavi regardless of comorbidities and clinical status , we did not follow the no exclusion policy as reported by pasic and his berlin colleagues . we tried to select the most high - risk , extremely fragile patients and did not schedule them for the tavi procedure . d'onofrio and colleagues reported a 30-day mortality of 9.9% in 774 patients undergoing ta - tavi . van der boone et al . observed 15.7% in - hospital mortality among 89 patients undergoing ta - tavi . johansson et al . reported a late survival after tavi of 77% both at 6 months and 1 year . published the results of 339 patients , including 177 patients after ta - tavi with a 4-year survival rate of 43% , and summarized that tavi in patients considered to be inoperable or at very high surgical risk was associated with good hemodynamic results translating into a significant functional improvement in most patients . the mortality rate at a mean follow - up of 3.5 years after the procedure remained relatively high ( > 50% ) . patients with copd , chronic kidney disease , frailty , and chronic atrial fibrillation were at higher risk of death within the few years after tavi , suggesting that a more careful evaluation and follow - up of patients with these comorbidities might translate into better mid- to long - term outcomes . published a single - center experience of 5-year outcomes in 24 transapical tavi patients with a survival rate of 35% . the early mortality is increased if an intraprocedural complication such as resuscitation , conversion to conventional surgery or reexploration because of bleeding occurs . with the tavi program confirmed that a small , but certain , number of patients would have a possibly detrimental intraprocedural complication ( e.g. , annular rupture ) that is difficult to manage . therefore , the main problem of tavi remains the uncertainty about the definitive result at the end of the procedure . unbehaun et al . found , most interestingly , that age itself was not predictive of survival in their patients . they observed that later survival is negatively influenced by various noncardiac comorbidities , such as reduced pulmonary function , chronic renal failure , or advanced stages of peripheral arterial diseases . the combination of several comorbidities , as reflected by higher values of the applied risk scores ( the logistic euroscore and the sts score ) , was not different between 30-day survivors and nonsurvivors but was found to be a very sensitive predictor for survival during follow - up . the study is limited by the small number of patients and the lack of a control group . transapical transcatheter aortic valve implantation should be considered as a feasible therapeutic option in a selected patient population . the transapical approach is a complex procedure that requires training , and the learning curve may affect the outcomes . the learning curve is crucial not only for the technique of implantation but also for patient selection , valve sizing , positioning and postdilatation , which can affect early mortality and morbidity of the patients . with increasing experience , the transapical approach is not associated with a higher incidence of apex - related complications . transcatheter aortic valve implantation can be performed with an acceptable early and mid - term survival rate . very low rates of strokes ( 2.4% ) and vascular complications ( % ) in our patients are further benefits of the short and antegrade approach .

sedentary behavior , particularly television ( tv ) watching , presents unique risks for obesity and related negative health outcomes in addition to risks from inactivity [ 1 , 2 ] ; even individuals who are very active show increased metabolic risk with higher amounts of tv watching . sedentary video gaming has also been implicated as a predictor of obesity [ 4 , 5 ] . video gaming using motion - based controllers has been proposed as a less sedentary alternative to tv watching or traditional video gaming , but these games vary widely in physical activity level produced during play . one factor that appears to have a large influence on energy expenditure is the type of controller used : for instance , dance and fitness - themed games using dance mat and camera - based controllers consistently show higher levels of activity during play when compared to games that use more traditional gamepad controllers [ 611 ] . this difference is to be expected , as gamepads rely on small finger movements , whereas other control schemes engage large muscles in the arms and legs . though dance and fitness - themed games are popular , action games remain the most widely played genre . action games typically emphasize skill , reflexes , and speed , and many of the most enduring video game franchises fall under this category ; examples include games that require jumping across platforms ( e.g. , super mario bros . and its many sequels ) or precisely shooting or punching enemies ( e.g. , games in the call of duty or street fighter series ) . integration of greater bodily motion into these traditional action video games may be a way to increase energy expended during play while retaining the features that make these games popular . supplanting button presses for more realistic mimicking of , for example , punching or sword - fighting would not necessarily change engaging aspects of these games ( storyline , skill - testing gameplay , graphics , etc . ) . the nintendo wii console comes packaged with a wiimote controller , which includes both buttons and motion - sensing functionality . many action games can be played on the wii console , but wii versions of the games generally change the control scheme to include motion control for such actions as aiming a weapon or throwing grenades . it is as yet unclear whether these motions produce greater energy expenditure than typical button presses in traditional games . if motion controllers produce greater energy expenditure than traditional controllers , they could produce positive health impacts at both the individual and public health level . the purpose of this study was to investigate the effects of motion - sensing controllers on energy expenditure during play of games from the action genre . the two games in each pair were different versions of the same game to minimize the presence of potential confounding variables . it was hypothesized that motion - controlled games would produce greater energy expenditure than the traditionally controlled games . participants were 100 1835-year olds , equal numbers of men and women , recruited primarily through a university online mailing list . the recruitment e - mail specifically solicited those interested in a video game research study . participants were also recruited via a general weight - related study advertisement for 1835-year olds shown on a local news network that directed interested viewers to a website with study descriptions and contact information . to be included , participants were required to weigh < 300 pounds ( necessary for the use of other game controllers in a larger study discussed elsewhere ) , have played video games at least 3 times over the past year , be willing to fast 2.5 hours ( necessary for indirect calorimetry ) , be videotaped during the study protocol , and have transportation to the study location . of 757 individuals who requested information and eligibility criteria , 325 completed eligibility information ; of those 325 , 169 potential participants were scheduled , and 100 completed the protocol . eligible participants who did not attend their appointments ( n = 49 ) were considered dropouts , 156 eligible participants were wait - listed , and 20 participated in a sub - study not reported here . all data were collected between april and august of 2009 . after providing informed consent , anthropometric measures ( height , weight ) participants were next led to a darkened video gaming laboratory and fitted with measurement equipment , then rested for 20 minutes . controller type ( traditional gamepad or motion wiimote motion controller ) was assigned randomly using a random number generator , and games were played in random order . all participants were provided with a visual aid to assist in learning controls , and study staff gave a brief introduction to the basic story and mechanics of the games . additionally , participants were randomized to play several other types of games either before or after these games as part of a larger study . tests of the effects of game order and play of other games prior to this study on energy expenditure did not show significant effects ( data not reported ) . games were played on a 58 high - definition television with optimized settings for each console . participants sat in a gaming chair with speakers in the headrest that provided surround sound . snack foods and drinks were available immediately after data collection concluded and water was available during rest periods . this protocol was approved by the university of north carolina public health - nursing institutional review board . participants were randomized to play three games using either a button - based traditional gamepad controller or a motion - based wiimote controller . all games were part of the broad genre of action games , rated m for mature , and released between the years of 2002 and 2007 . game pairs were chosen to represent different player perspectives , to investigate the effects of perspective and controller on psychological reactions not reported here . medal of honor : heroes 2 and medal of honor : airborne used a first - person perspective ( i.e. , players see through the eyes of their character ) , while resident evil 4 : wii edition and resident evil 4 used an over - the - shoulder perspective ( players view the game environment from over the shoulder of their character ) . the final pair was chosen to vary both controller as well as perspective , and thus these two games were not as similar than the games in the other pairs . resident evil used a third - person perspective and a traditional controller , and resident evil : the umbrella chronicles used a first - person perspective and a motion controller . the controllers used for the traditional play condition were the playstation 3 ( ps3 ) dualshock controller and the gamecube controller . the wiimote , shaped like a remote control , was held in the right hand and used for both button and motion - based inputs . the nunchuk was held in the left hand and used only for its button - based inputs . the indirect calorimeter was calibrated daily using a 3-liter syringe as well as prior to each test using certified gases . oxygen consumption ( vo2 ) and carbon dioxide expiration ( vco2 ) were measured on a breath - by - breath basis and converted to energy expenditure . enjoyment was measured using the interest / enjoyment subscale of the intrinsic motivation inventory , which is a well - validated measure that has been used in previous virtual reality physical activity studies [ 15 , 16 ] . participants were also asked about their general previous experience playing video games ( how much experience do you have playing video games ? with responses ranging from 1 , not a lot , to 7 , a lot ) and whether they had played each of the games previously ( dichotomous yes / no response ) . weight and height were measured using a calibrated scale ( tanita , arlington heights , il ) and wall - mounted stadiometer ( perspective enterprises , inc . , energy expenditure data were averaged over the ten - minute play period and corrected for body mass ( kcalkghr , equal to metabolic equivalents or mets ) . cut - points for sedentary behavior and light and moderate physical activity were taken from pate et al . . two mixed models ( dependent variables energy expenditure and enjoyment ) were created , with game pair as a repeated measure and controller condition as an independent variable . degrees of freedom were calculated using the kenward - roger method , and all tests of mean differences took into account row - wise degrees of freedom . because several studies of energy expenditure during video game play have found effects of bmi and gender [ 11 , 18 ] , these variables were included in the model . interaction terms for game pair controller condition were also included . to investigate interactions , post hoc contrasts with tukey - kramer corrections were used . games were contrasted within pairs and/or with the other games in their controller group ( motion or traditional ) . independent sample t - tests were also used for simple comparisons between groups , analyses of variance for tests of the impact of playing the game previously , and correlation analysis was used to investigate bivariate associations . the sas software package ( cary , nc ) version 9.2 was used for all analyses . table 1 displays participant characteristics . the sample was 73% white , 15% black , 8% asian , and 4% other race . most participants were college graduates ( 49% ) , followed by those with some college education ( 32% ) . most participants had not played the games previously : 18 of the 100 had played resident evil , 25 had played resident evil 4 , and 18 had played medal of honor . no association was found between energy expenditure and video game playing experience or previous play of each game ( p > .05 ) . video game playing experience was positively associated with enjoyment ( resident evil , r = .255 , p = .011 ; resident evil 4 , r = .307 , p = .002 ; medal of honor , r = .264 , p = .008 ) . having ever played the game before predicted enjoyment in resident evil ( b = 0.91 , se = .63 , p = .006 ) and medal of honor ( b = 1.62 , se = .63 , p < .001 ) but not resident evil 4 ( b = 0.54 , mean energy expenditure corrected for body mass for each game is shown in table 1 . no main effect was found for controller ( p = .121 ) or game pair ( p = .510 ) . however , an interaction between controller and game pair was found ( p = .004 ) . the motion - controlled resident evil game ( mean [ sd ] 0.96 [ 0.20 ] kcalkghr ) produced 0.10 ( 95% confidence interval [ ci ] 0.030.17 , p = .048 ) kcalkghr greater energy expenditure than its comparison , the traditionally controlled resident evil game ( 0.86 [ 0.17 ] kcalkghr ) . within the motion - controlled condition , the resident evil game produced 0.06 ( 95% ci 0.020.11 , p = .030 ) kcalkghr greater energy expenditure than resident evil 4 ( 0.90 [ 0.18 ] kcalkghr ) . no other energy expenditure differences between games in a pair or within controller condition were found ( p > .30 ) . participants with a higher bmi expended less energy during play than those with a lower bmi , b ( se ) = 0.01 ( 0.00 ) , p < .001 . all six games produced energy expenditure estimates below 1.5 kcalkghr ( equal to mets ) , indicating that all were sedentary . analyses of enjoyment ratings found significant differences by game ( p < .001 ) as well as an interaction between game and console ( p < .001 ) . the motion - controlled resident evil game produced a 1.39 ( 95% ci 0.841.95 ) point higher enjoyment rating ( on a scale of 17 ) than the traditionally controlled resident evil game . the other two traditionally controlled games were also rated as more enjoyable than the traditionally controlled resident evil game ( resident evil 4 , mean difference 1.32 , 95% ci 0.841.80 ; medal of honor , mean difference 1.51 , 95% ci 1.031.99 ) . males ( b = 1.19 , se = 0.20 , p < .001 ) and those with higher bmi ( b = 0.03 , se = 0.01 , p = .028 ) rated the games as more enjoyable than females and those with lower bmi . inclusion of enjoyment as a covariate in the energy expenditure model slightly attenuated but did not alter the significance of the interaction between game pair and controller type ( p = .026 ) . enjoyment was not associated with energy expenditure in any of the game pairs ( p > contrary to our hypothesis , play of action games with motion - sensing controllers did not produce greater energy expenditure than play of similar games with traditional gamepad controllers . the traditional and motion - controlled games studied here produced average energy expenditures of 0.89 and 0.93 kcalkghr , respectively , representing increases of 22 and 25 percent over rest measurements . in only one of three game pairs did the motion - controlled version produce significantly greater energy expenditure than a traditionally controlled version , a difference of 0.10 kcalkghr . however , that game also produced greater energy expenditure than another of the motion - controlled games , suggesting that how motion controls are integrated into gameplay likely influences the intensity of motions used and thus energy expenditure . some motion - controlled games hold potential to significantly increase energy expenditure during screen time as compared to similar traditionally controlled games . however , results of this study , showing only one of three games increasing expenditure , suggest that many action games likely do not . the results further suggest that the simple addition of motion control to traditional games , such as action games , is not sufficient to produce light or moderate intensity physical activity and will not consistently produce an improvement in activity intensity as compared to traditional controllers . replacement of traditional controllers with motion controllers during play of action games could positively affect health in several ways . even very low intensity muscle contractions , such as those involved in standing , can prevent or negate metabolic changes that result from sedentary behavior [ 20 , 21 ] . additionally , the muscle contractions necessary to play motion - controlled action games may interrupt longer stretches of sedentary gaming . breaks in sedentary time , regardless of their length or intensity , are associated with lower waist circumference , bmi , triglycerides , and 2-hour plasma glucose independent of total sedentary time and moderate - vigorous activity . in other words , even the smallest periods of very light intensity activity can be beneficial , particularly if they interrupt what would otherwise be a long session of sedentary behavior . thus , the motions required for play of motion - based action video games , though not strenuous enough to produce light intensity activity , may be sufficient to reduce risk associated with sedentary screen time . previous studies have found that use of the wiimote motion - sensing controller can produce light ( 1.53 mets ) to moderate ( > 3 mets ) intensity physical activity in several specific fitness- and sports - themed games ( e.g. , wii sports and wii fit ) , increasing energy expenditure over rest up to 328% [ 18 , 23 ] . though the wii games studied here used the same controller as the games previously studied , due to our study design , they more closely resembled traditional sedentary games in both content and energy expenditure [ 6 , 24 ] . the motions encouraged by games like wii sports differ greatly from those encouraged by other wii action games . whereas wii sports encourages large , exaggerated movements such as swinging a tennis racket or punching an opponent , action games require greater precision of movement to ensure success . the game that showed the greatest energy expenditure in this study ( resident evil : the umbrella chronicles ) encouraged frequent shooting by offering unlimited ammunition for the default weapon . it may be that the abundance of ammunition ( a rarity in action games , which often require management of scarce resources to increase suspense ) allowed participants to shoot more often than they did in the other games and , thus , expend more energy . though resident evil 4 : wii edition , which produced significantly lower energy expenditure than the umbrella chronicles , also used motion controls , it required more precise shooting less often and placed a greater emphasis on exploration of the game environment , which did not require player motion . even if motion controls are added to this genre of game , it may be that the characteristics of the genre itself generally discourage unnecessary movements . the game that produced the lowest energy expenditure ( traditionally controlled resident evil ) was also rated less enjoyable than its comparison motion - controlled game as well as the other two traditionally controlled games . this finding was surprising , as resident evil is considered a classic game , and the version used ( the 2002 gamecube remake ) received extremely high review scores . enjoyment was also predicted by overall gaming experience and having played the game before in resident evil and medal of honor games . it is unclear whether previous experience leads to increased enjoyment due to familiarity or if previous play and enjoyment both reflect an underlying variable such as a preference for specific types or series of games . a strength of this study was the experimental study design and inclusion of three different pairs of games . to our knowledge , this study was the largest one of motion - controlled gaming yet conducted , with a much larger proportion of female participants ( 50% ) than in past studies . as women represent approximately 40% of video game players , their inclusion in active gaming studies is important . participants were randomized to play iterations of the same game using different controllers , eliminating many potential confounding variables related to game content that may have biased previous studies . a limitation of the study was that only one of the three pairs included identical games . the other two pairs included games that were chosen to minimize potential confounding variables ; however , it was impossible to eliminate them . the pair that produced significant differences in energy expenditure , resident evil and resident evil the games shared characters , storyline , and setting , but differences in implementation of control scheme may have led to differences in gameplay . differences in this pair ( e.g. , more frequent shooting , greater enjoyment ) , offer insight into possible variables to manipulate in future studies that may produce increases in energy expenditure . other limitations include the cross - sectional nature of the data and measurement of only one side of energy balance . it is impossible based on this study design to determine potential effects of game play on subsequent energy intake and expenditure . play of video games with a traditional controller has been found to increase food consumption after a play session ; thus , it is possible that later increases in energy intake could erase the expenditure benefits of motion - controlled action gaming and result in overall positive energy balance . results should also be generalized with caution due to possible differences between eligible participants who completed the study and those who did not attend their appointment or who were eligible but not asked to participate because recruitment goals ( i.e. , 100 participants , 50 per gender ) were reached . action video games are extremely popular and are the most widely played genre of video game . addition of motion controls to this genre of game can produce small increases in energy expended during play . however , only some motion - controlled action games produce greater expenditure than equivalent traditionally controlled games ; these games also produce greater energy expenditure than some other motion - controlled games . the manner in which motions are integrated into gameplay likely has a large effect on the amount of energy expenditure than can be expected during play . though motion - controlled action games may not lead to changes in the intensity of physical activity , their potential for reducing risk associated with sedentary behavior should not be overlooked . little is currently known about the impact of motion - controlled games on sedentary behavior and physical activity over time . even less is known about the impact of these games on other behaviors that may be associated with obesity and other negative health outcomes , such as snacking during screen time . there is a particular need for investigation of game characteristics that encourage or discourage movement even in highly similar games , such as the two motion - controlled resident evil games played in this study that produced significantly different energy expenditure levels . randomized controlled trials are necessary to investigate the potential of games to contribute to obesity prevention and treatment ; even games that do not increase moderate - vigorous intensity physical activity may improve health by decreasing prolonged bouts of sedentary behavior . e. j. lyons , d. f. tate , and j. m. bowling planned and performed statistical analyses , and all authors participated in interpretation of analyses . d. f. tate , d. s. ward , k. m. ribisl , j. m. bowling , and s. kalyanaraman critically revised the paper .

regulated spatial and temporal control of gene transcription is a fundamental process for all metazoans . critical to this process is the interaction of transcription factors ( tfs ) with specific cis - regulatory dna sequences . these regulatory sequences for instance , enhancers and promoters are organized in a modular fashion , with each module containing one or more binding sites for a specific combination of tfs ( 1 ) . we use the term cis - regulatory module ( crm ) as a generic term to refer to all enhancers and similar regulatory elements that are located outside of the core promoter region and which function to regulate transcription in a spatio temporal - specific manner . we use the more general term cis - regulatory element to refer to either a crm or a tf binding site ( tfbs ) . despite the clear importance of cis - regulatory elements for many areas of biology for instance , crms and tfbss act as major control nodes in embryonic development , and variation in cis - regulatory elements plays an important role in both evolutionary change and normal phenotypic variation ( 2,3)our knowledge of these sequences is surprisingly limited . the vast majority of crms are not known and , of those that are , relatively few have been characterized in detail . even drosophila melanogaster , which is a well - studied organism with a richly annotated genome , only has identified crms associated with fewer than 2% of its 14 000 genes ( 4 ) . likewise , fewer than 1% of drosophila genes currently have annotated tfbs data ( 5 ) . a well - annotated collection of known crms and their constituent tfbss would be of significant use in many important areas of biological research , including studies of transcriptional regulation , genome structure and organization and the evolution of gene regulation . such a resource would have considerable value in aiding subsequent crm and tfbs discovery , for example , by providing training data for supervised learning or other bioinformatics approaches . currently , two databases play an important role in the study of cis - regulation in the model organism d. melanogaster : the flyreg dnase i footprint database ( 5 ) , a database of empirically defined tfbss ; and the redfly ( regulatory element database for fly ) database ( 4 ) , a highly annotated source of information on experimentally proven crms . these resources have served as the basis of a number of large - scale studies of cis - regulation and have allowed statistical , computational and comparative genomics methods to be brought to bear on its study ( 620 ) . in this report , we describe the merger of these two databases into redfly v2.0 . with this release , redfly has become a unified source of drosophila cis - regulatory element annotation with one - stop access to both crm and tfbs data for one of the best - studied model organisms , and the most comprehensive open - access resource for curated regulatory data in any metazoan species . redfly v2.0 ( august 2007 ) contains records for 665 crms ( up from 544 in the initial release ) and 1341 tfbss ( down from 1367 in the initial flyreg release due to removal of tfbs not attributed to target genes ) . the goal of redfly is to include all experimentally verified fly crms and tfbss along with their dna sequence , their associated genes , and the expression patterns they direct . at present , curation has focused on literature reports of sequences that have been unambiguously demonstrated to be sufficient to regulate gene expression , primarily through reporter gene assays in transgenic animals and on tfbss discovered by dnase i footprinting assays . for the most part , crms are included directly as reported in the literature . where multiple nested sequences with identical activity sequences with identical activity that are distinct but minimally overlapping are mostly reported separately , although in some instances of more substantial overlap , one or more sequences were omitted . tfbs records include primarily dnase i ( but not hydroxy - radical or copper nuclease ) footprinting experiments that used protein obtained from nuclear extract ( either crude or purified ) or recombinant expression ( either partial or full - length ) . when a binding factor purified from nuclear extract has been shown to be the derivative of a specific gene , footprints were attributed to the gene encoding that factor ; otherwise the binding factor for nuclear extract footprints has been left as unspecified. where possible , we followed the rule of precedence in attributing footprint data to a particular reference , unless members of the same research group reported refined coordinates in a subsequent publication . when two or more overlapping motifs for the same tf were reported for a single footprinted region , they were merged and annotated as one footprint . all redfly sequence features are mapped to the most current release ( release 5 ; http://www.fruitfly.org/sequence/release5genomic.shtml ) of the d. melanogaster genome sequence . coordinates are also provided for the two previous sequence releases for maximum convenience and back - compatibility with other sequence resources . we store the actual dna sequences as well as the coordinates so that sequences can be downloaded without ambiguity . because tfbs sequences are often short and therefore can not be uniquely mapped to the genome tfbs with flank option that provides 25 bp of additional sequence both 5 and 3 to the tfbs . all records contain hyperlinks to the flybase ( 21 ) and flymine ( 22 ) entries for the target gene whose expression is regulated by the crm or tfbs , and all features can be displayed on gbrowse or ucsc genome browsers ( 23,24 ) . for tfbs records , hyperlinks to flybase , flymine and flytf ( 16 ) are also available for the tf that binds the site , when known . for crm records , controlled vocabulary descriptions of the expression pattern mediated by the crm this is a key feature of redfly and allows users to search for expression patterns using a tree - based browser interface ( figure 1 ) . selecting a term from the tree will query redfly for any crms annotated with that term or any of its descendant terms . alternatively , users can search for only a single term . because expression patterns are described using the anatomy ontology , users can link from a crm record to any other redfly crms that are annotated as mediating the same gene expression pattern , or to records in flybase or the berkeley drosophila genome project 's in situ expression pattern database ( 26,27 ) for genes expressed in that pattern . these features promise to be highly useful for investigating properties of tissue - specific crms . for example , we recently made use of the expression pattern annotations to demonstrate that a certain class of crms those that drive gene expression in the drosophila early embryonic blastoderm have characteristics that distinguish them from other crms ( 6 ) . detailed instructions on using the ontology to facilitate searching for crms that regulate specific expression patterns are provided in redfly 's online help . figure 1.searching for regulatory elements based on the expression patterns they regulate using the ontology search function . browse ontology button ( circled ) will open a pop - up window with the ontology tree ( inset ) that can then be navigated until the desired anatomical feature at the preferred level of granularity is reached . all redfly records annotated with the selected term or any of its descendant terms will be returned in the database search . in the pictured example , the chosen term is somatic muscle primordium ; any records containing annotations for somatic muscle primordium or its two descendant terms embryonic somatic muscle and larval somatic muscle will be returned . alternatively , a user can type a term into the expression term or id field , in which case only records annotated with that term ( e.g. , somatic muscle primordium ) will be returned . searching for regulatory elements based on the expression patterns they regulate using the ontology search function . clicking on the browse ontology button ( circled ) will open a pop - up window with the ontology tree ( inset ) that can then be navigated until the desired anatomical feature at the preferred level of granularity is reached . all redfly records annotated with the selected term or any of its descendant terms will be returned in the database search . in the pictured example , the chosen term is somatic muscle primordium ; any records containing annotations for somatic muscle primordium or its two descendant terms embryonic somatic muscle and larval somatic muscle will be returned . alternatively , a user can type a term into the expression term or id field , in which case only records annotated with that term ( e.g. , somatic muscle primordium ) will be returned . a major advantage of integrating the redfly and flyreg databases is the unprecedented level of detailed information that can now be obtained by mapping tfbss directly to the crms of which they are a part . upon entry of a new crm or tfbs , the sequence coordinates of the new element are checked against the coordinates of all of the stored tfbss or crms , respectively . if a tfbs falls within a known crm , the name of the crm and a link to its redfly record is provided . similarly , all crm records are linked to the redfly annotations of any tfbss that fall within them ( figure 2 ) . searches of redfly can be restricted to just those tfbss that map to known crms , and vice - versa . currently , 70% of tfbss in redfly map to a known crm , while 26% of crms contain annotated tfbss . using these new redfly features , it is now possible , for example , to investigate the association of tfbs sequences with expression patterns via their corresponding crms . redfly is the only resource for regulatory bioinformatics that provides such a highly integrated annotation of crms and their constituent tfbss . an exciting new feature in redfly is its cross - referencing of crms with any tfbss that fall within them . panel a shows the detailed view page for the crm dll_208 , which contains three footprinted tfbss with records in redfly . the arrow indicates the link to the record for one of these three tfbss , ubx_dll : redfly : tf000513 . panel b shows the record obtained through the link indicated in panel a. note that the lookup is functional in both directions ; from this tfbs record page , there is a link to the crm in which the binding site is located ( arrow ) . an exciting new feature in redfly is its cross - referencing of crms with any tfbss that fall within them . panel a shows the detailed view page for the crm dll_208 , which contains three footprinted tfbss with records in redfly . the arrow indicates the link to the record for one of these three tfbss , ubx_dll : redfly : tf000513 . panel b shows the record obtained through the link indicated in panel a. note that the lookup is functional in both directions ; from this tfbs record page , there is a link to the crm in which the binding site is located ( arrow ) . the database schema has been designed to be both fast and extensible so that additional species can be added to the existing database structure at a later date and utilize the same search capabilities that have been developed for redfly 's drosophila data . the tables in the database are grouped into four categories as diagrammed at the redfly site at http://redfly.ccr.buffalo.edu/?content=/database.php : species - specific fixed terms ( outlined in red)the crm definition ( yellow)the binding site definition ( blue)external reference information ( green ) . species - specific fixed terms ( outlined in red ) the crm definition ( yellow ) the binding site definition ( blue ) external reference information ( green ) . the fixed - term tables equivalent to the dimension tables of a star database schema contain information that change infrequently , including anatomy terms ( using the controlled vocabulary ) , evidence terms ( also using a controlled vocabulary ) , chromosome numbers , sequences and gene names and ids . fixed - term information can be associated with a particular species , or can be common across all species . by utilizing tables of fixed terms we can load information for multiple species into the database and then reference this information from crms or binding sites without duplicating the information . fixed terms also allow us to reduce query times and prevent the introduction of typographical errors when entering data . the crm definition tables consist of the basic information describing each crm , such as the crm name , species to which the crm belongs , free text notes , references to information stored in the fixed - term tables , citation data and references to external websites . the binding site tables describe the tfbss and provide information similar to that found in the crm definition tables . a mapping also exists from crms to binding sites that are associated with that crm , and vice versa . any reference that a crm or tfbs record makes to an external site such as flybase ( 21)is considered an external reference . the external reference tables contain information on how to construct references to external sites such as a template for the url , required parameters , etc . snapshots of the mysql schema ( i.e. a database dump ) are recorded daily and are available for download . this provides extra backup and versioning protection , as well as an alternative method of access to the data for interested users . in addition to the inclusion of tfbss and their association with corresponding crms , we have implemented a number of other key improvements to redfly . in particular , we have developed extensible markup language ( xml ) representations for both crm and tfbs records and have enabled xml formatted data as one of the download options . the xml format is the most comprehensive available for redfly and allows for a complete dump of the database contents . development of the xml format has also helped us to automate our input procedures and should help to increase the pace of updates and additions to the database . we have also established data - sharing standards with the oreganno community regulatory annotation database ( 28 ) and implemented a two - way exchange of data . all redfly data are automatically shared with oreganno , where they represent 33 and 27% of the total number of curated crm and tfbs records in oreganno , respectively . although oreganno lacks drosophila - specific functionality and several of the detailed annotation fields contained in redfly , the inclusion of our data within oreganno allows for alternative access through the oreganno web - services and the newly implemented oreganno tracks in the ucsc browser ( 23 ) . redfly data that do not correspond to core oreganno fields are stored as metadata within the oreganno record . therefore , community users can annotate fly crms and tfbss via the easy - to - use oreganno interface that includes automatic mappings to the current genome build . redfly can then automatically retrieve these data from oreganno and map them to the appropriate redfly fields using the xml representations . during the synchronization process , redfly also performs real - time queries to ncbi and ucsc to augment the oreganno data with related information such as literature citations and mapping of feature locations to multiple genome sequence releases . the records are then passed to the redfly curators for validation , for the addition of any further annotation not provided in the oreganno metadata , and for connection to external drosophila - specific resources not supported by oreganno . over time , we anticipate that this will be the primary route of entry for redfly data , either from the community or by our own curators . in this way , we are able to take advantage of oreganno 's general database cross - referencing functions , community - based annotation model and ucsc browser tracks while still maintaining redfly 's ability to provide drosophila - specific information such as expression pattern data , links to external drosophila resources and cross - references between crms and tfbss . in addition to the inclusion of tfbss and their association with corresponding crms , we have implemented a number of other key improvements to redfly . in particular , we have developed extensible markup language ( xml ) representations for both crm and tfbs records and have enabled xml formatted data as one of the download options . the xml format is the most comprehensive available for redfly and allows for a complete dump of the database contents . development of the xml format has also helped us to automate our input procedures and should help to increase the pace of updates and additions to the database . we have also established data - sharing standards with the oreganno community regulatory annotation database ( 28 ) and implemented a two - way exchange of data . all redfly data are automatically shared with oreganno , where they represent 33 and 27% of the total number of curated crm and tfbs records in oreganno , respectively . although oreganno lacks drosophila - specific functionality and several of the detailed annotation fields contained in redfly , the inclusion of our data within oreganno allows for alternative access through the oreganno web - services and the newly implemented oreganno tracks in the ucsc browser ( 23 ) . therefore , community users can annotate fly crms and tfbss via the easy - to - use oreganno interface that includes automatic mappings to the current genome build . redfly can then automatically retrieve these data from oreganno and map them to the appropriate redfly fields using the xml representations . during the synchronization process , redfly also performs real - time queries to ncbi and ucsc to augment the oreganno data with related information such as literature citations and mapping of feature locations to multiple genome sequence releases . the records are then passed to the redfly curators for validation , for the addition of any further annotation not provided in the oreganno metadata , and for connection to external drosophila - specific resources not supported by oreganno . over time , we anticipate that this will be the primary route of entry for redfly data , either from the community or by our own curators . in this way , we are able to take advantage of oreganno 's general database cross - referencing functions , community - based annotation model and ucsc browser tracks while still maintaining redfly 's ability to provide drosophila - specific information such as expression pattern data , links to external drosophila resources and cross - references between crms and tfbss . in addition to continued curation of redfly , we have targeted several areas for development within the near future . important among these is to expand our curation to include tfbs data from sources other than footprint experiments , e.g from electrophoretic mobility shift assay ( emsa ) and chromatin immunoprecipitation experiments . we also plan to annotate a broader range of crms , including negative regulatory elements ( silencers ) , and to increase the amount of annotation for each crm to include features such as the flybase transgenic transposon i d for the reporter gene construct used in the assay that defined the crm , and the position of the crm with respect to the organization of the gene it regulates ( e.g. transcription start site , exon boundaries ) . these additions will increase the comprehensiveness of redfly as a source of drosophila cis - regulatory data and facilitate the mining of these data in more diverse and sophisticated ways . a further planned development is the addition of images of reporter gene expression driven by each crm in order to associate cis - regulatory sequences directly with embryonic expression patterns ; this work is being conducted in collaboration with the flyexpress project ( 29 ) . over the longer term , we plan to incorporate a more extensive use of formal ontologies to describe not only expression pattern data but also experimental evidence , assay types and sequence features in order to maximize opportunities for data mining and for interoperability with other databases . toward this goal , we have been working with the sequence ontology ( so ) developers ( 30 ) to expand and refine so 's treatment of cis - regulatory sequences . we note that redfly is easily adaptable to curation of cis - regulatory elements from species other than d. melanogaster with only minor modifications to the current schema that raises the possibility of incorporating multi - species regulatory data either by direct curation or via our links with oreganno into the database . this , along with our use of ontologies to allow interspecies mapping of genes and tissues , has the potential to make redfly an unparalleled platform for comparing regulatory strategies and studying the organization of regulatory elements throughout evolution .

diabetes , osteoporosis , chronic liver disease , and coronary artery disease are just some of the common metabolic conditions that affect millions of people , especially later in life . unfortunately , for those infected with hiv , these conditions tend to occur more frequently and at an earlier age compared with the general population . one theory is that the activation of the immune system and subsequent inflammatory reactions to hiv infection might cause premature aging , accelerating the onset of these metabolic complications , despite the use of effective antiretroviral therapy . alternatively , antiretroviral drugs themselves have been linked to metabolic complications , particularly the class of drugs called protease inhibitors . however , these metabolic complications are at least partly genetically determined ; in fact , disease heritability rates range from approximately 30 to 90% , depending on the condition . this highlights why it is important to gain a deeper understanding of how genetic factors contribute to metabolic complications in hiv - infected people . figure 1 summarizes genetic studies of common metabolic complications in the general population and in the setting of hiv - infection . summary of the genome - wide association studies ( gwas ) performed in the general population to discover genetic variations associated with various metabolic disorders , including the number of single nucleotide polymorphisms ( snps ) identified and whether there have been genetic studies conducted in hiv - infected individuals . the genetic factors contributing to these major metabolic disorders are typically complex and no single gene mutation or variant is likely to explain a large proportion of the differences in clinical presentation between individuals . rather , genome - wide association studies ( gwas ) , which look for genetic variations associated with a disease across the entire human genome in the general population , have identified dozens of common ( i.e. , present in at least 5% of the general population ) single nucleotide polymorphisms ( snps ) single base substitutions of one nucleotide for another associated with specific metabolic disorders . identification of these genetic differences may help to predict an individual 's likelihood of developing a disease as well as their response to treatment . a catalogue of published gwas is available online . in the case of type 2 diabetes , gwas of the general population this is significant because insulin resistance and type 2 diabetes are considered serious complications in hiv - infected patients , given the increased risk for premature cardiovascular disease . the importance of identifying these snps was confirmed very recently in a long - term study of 644 white hiv - infected patients . in the study , 20% of the patients had an unfavorable genetic risk score ( i.e. , those patients who carried multiple diabetes - associated snps ) , associated with a threefold increase in the risk of developing diabetes compared with patients who had a favorable genetic score . for comparison , this increased risk is similar to the effect of established risk factors such as advancing age but greater than the effect of antiretroviral therapy . interestingly , the effect of the snps was still less than the effect of having an increased body mass index . the situation is similar for people affected by dyslipidemia , a condition where patients suffer from abnormal levels of lipids ( such as cholesterol and triglycerides ) in the blood , predisposing them to heart disease . all snps consistently associated with serum lipid levels were validated in a recent paper analyzing 745 hiv - infected patients . a patient 's genetic background and use of antiretroviral therapy contributed to similar proportions of lipid variation . a favorable genetic score ( based on the patient 's number of dyslipidemia - associated snps ) bad cholesterol in 32% of patients taking antiretroviral drugs ; in patients with an unfavorable genetic score , this figure rose to 53% . good hdl cholesterol during protease inhibitor - based antiretroviral therapy were seen in 17% of patients with a favorable genetic score versus 42% with an unfavorable genetic score . there are also some preliminary studies suggesting that there is a genetic component to the mitochondrial dysfunction widely implicated in lipoatrophy , a physically disfiguring condition that results in localized loss of fat tissue and that has been linked to the use of certain antiretroviral drugs in hiv - infected patients . some evidence for this comes from a recent report involving 536 white multicenter aids cohort study participants , which demonstrated that having one of a group of snps in mitochondrial dna ( haplogroup h ) , is a risk factor for lipoatrophy . additionally , a recent study of 103 thai patients showed that the human leukocyte antigen gene hla - b*4001 was associated with lipotrophy , implicating the immune system in this disorder . however , these findings await confirmation in other populations and , as yet , no unifying hypothesis of the genetic predisposition to lipoatrophy has emerged . even less is understood about a direct genetic contribution to coronary artery disease risk in hiv - infected individuals , but the international magnificent consortium is intending to report the results of a study on this by summer 2011 . it features more than 600 hiv - infected patients with coronary artery disease and more than 1200 hiv - infected patients without this complication . the snps evaluated in magnificent will include snps associated with coronary artery disease that have already been identified in gwas of the general population . recently , a step towards identifying a genetic predisposition to cardiovascular problems in hiv - infected persons was made through a small gwas ( 177 white male subjects genotyped ) in which a snp involved in the development of atherosclerosis of the carotid arteries was reported . chronic liver disease , often following infection with hepatitis c virus , is another significant cause of death in hiv - infected patients . considerable enthusiasm was generated by the description of variation in il-28b ( the gene for interleukin 28b , which is responsible for the production of the antiviral cytokine known as interferon-3 ) that accounts for a large proportion of the variation in the spontaneous clearance of hepatitis c , as well as the response to treatment with pegylated interferon and ribavirin [ 12 - 15 ] . in addition , higher frequencies of the unfavorable il-28b gene variants appear to be responsible for the lower response rate to hepatitis c treatment seen in individuals of african or hispanic origin . il-28b genetic screening is already being advocated and is becoming a key element in the stratification of patients enrolled in clinical trials of new anti - hepatitis c compounds . il-28b plays a similarly important role in people infected with both hiv and hepatitis c , as having the favorable il-28b gene variants improved spontaneous hepatitis c viral clearance rates and treatment response compared with patients carrying the unfavorable form of the gene . on a similar note , a genetic analysis of osteoporosis risk in hiv - infected individuals is overdue , given the aging hiv - infected population , the remarkably increased rates of this complication in the setting of hiv - infection , and the large degree of heritability of osteoporosis ( estimated at 60 - 90% ) . several gwas in the general population ( reviewed in ) have identified around 30 osteoporosis - associated snps . unfortunately , there are also several chronic complications of hiv infection where there are no clear insights into the genetics involved in pathogenesis , such as kidney disease and hiv - associated neurocognitive disorders , although a gwas to evaluate a potential genetic predisposition in the latter is ongoing . in addition , no new reports have been published since the identification of a possible genetic association of a snp in resistin ( a gene previously implicated in obesity and insulin resistance ) with certain features of the metabolic syndrome in a study of 135 candidate genes in 189 genotyped participants on antiretroviral therapy in 2008 . despite the current gaps in our knowledge , genetic screening for hiv - related metabolic complications is emerging as a clinically relevant prediction tool . in fact , genetic prediction is already used in routine hiv care to identify individuals at risk of developing a hypersensitivity reaction to the antiretroviral drug abacavir . however , the nearly 100% accuracy of this test is because only a single locus ( hla*b5701 ) is involved . thus , the best guess based on current knowledge is that a panel of snps will need to be screened to produce a risk score for metabolic complications in hiv - infected individuals , and that this will need to be combined with clinical variables to give an accurate prediction . an additional , consistent lesson learned from gwas is that the known common snps for a disorder account for less than 10% of the variability in disease seen between individuals . this applies to all metabolic and non - metabolic disorders assessed in gwas . in the next few years , the genetic basis of an increased fraction of the variability between individuals is likely to be uncovered . this will be achieved by re - sequencing the risk genes documented in gwas ( in order to identify rare variants causally implicated in the phenotype ) , large scale genome sequencing projects ( see ) and whole genome sequencing of people with extreme versions of the disorders costs per patient are , as of june 2010 , 400 us dollars ( usd ) for a single gwas and around 10,000 usd for whole genome sequencing . until these tests become cheaper , re - assessing the snps associated with metabolic disorders in the general population and screening hiv - infected patients for them ( at a cost of 0.5 - 1 usd per snp , per patient )

in adults , routine injections of botulinum toxin are the treatment of choice for the management of essential blepharospasm [ 1 , 2 ] . the clinical picture is characterized by recurrent involuntary spasms in the region of the orbicularis oculi muscle . typically , at the time of the first manifestation , the patients are in their 5th to 7th decade of life . until today , > 2,500 patients presenting with this clinical picture have been treated with botulinum toxin in the department of ophthalmology of the university of bonn , bonn , germany . among these patients , blepharospasm mostly occurs as reflex phenomenon in conditions of the anterior eye segment such as , for instance , corneal affections or trichiasis , in the sense of the defense triad consisting of blepharospasm , photophobia and epiphora . so far , congenital blepharospasm devoid of conditions of the anterior eye segment has not been described in the literature . the treatment of children affected by cerebral palsy with botulinum toxin is considered safe and effective , but is only approved for children 2 years of age . other authors described off - label - use in younger children , the youngest being 11 months old . a 4-week - old girl was referred to our department because of absence of eye opening . since her birth , more in the evening , slit - like eye opening occurred for a maximum of 1 h ( fig . the child reacted with eyes closed to direct flashlight in the dark , e.g. , by turning away or grimacing . immediate massive blepharospasm was present even when lid opening was attempted in her sleep or following administration of local anesthetic eyedrops . pregnancy and delivery had been normal . a neuropediatric examination including ultrasound study of the brain was unremarkable . at the age of 2 months , an examination under general anesthesia was performed which revealed regular organ findings devoid of ocular irritation . during anesthesia , marked regression of spasms was observed . hence , periorbital injection of botulinum toxin ( 6.25 iu xeomin in each eye at 6 injection sites each equivalent to half of the starting dose of adults with essential blepharospasm ) was performed to prevent deprivation amblyopia . for the first time , the child started eye contact and briefly followed with her eyes . four weeks later , the mother reported that both eyes continued to be opened and closed smoothly . the deep - seated eyes were opened slit - like with free pupils , pronounced inward squinting and reduced abduction capacity ( abduction only to midline ) . at the age of 7 months , alternating esotropia and only slightly impaired abduction with persistent lid opening was observed . meanwhile , the child had established a good eye contact ability and was able to follow objects . further ophthalmologic follow - up examinations conducted to the current age of 3 years revealed regular eye opening and complete lid closure without recurrence of blepharospasm ( fig . no side effects of botulinum toxin therapy have occurred . a signed patient consent - to - disclose the child presented in this case report is the youngest patient ever treated with botulinum toxin . typical indications for botulinum toxin in young children are spasticity of the upper or lower limbs due to infantile cerebral palsy . botulinum toxin reduces spasticity , avoids contractures and delays surgery , but is not able to improve the achievement of motor milestones . the safety profile of botulinum toxin was similar to older children except for two cases of acute respiratory events in children with severe cerebral palsy , mask anesthesia and high - dose botulinum neurotoxin therapy . in our case , a single periorbital injection of botulinum toxin induced lid opening already persisting for nearly 3 years . in typical essential blepharospasm in adults ( focal dystonia ) , botulinum toxin injections are only effective for about 3 months blepharospasm may occur in children with schwartz - jampel syndrome , a rare neuromuscular disorder with autosomal recessive inheritance characterized by myotonia , distinctive facial features including blepharospasm and a puckered chin , short stature as well as skeletal dysplasia . a case of congenital blepharospasm without other neurologic signs has never been described in the literature before . it may be due to some delayed midbrain maturation . in older children , blepharospasm devoid of ocular irritation or lid abnormalities ( reflex blepharospasm ) a direct influence of the periorbital botulinum toxin injection on the abduction deficit seems virtually impossible . diffusion from the region of the orbicularis oculi muscle into both extraocular muscles seems pathophysiologically impossible . hence , one may assume that the abduction deficit had already preexisted and could hardly be examined before the botulinum toxin injection due to blepharospasm . the botulinum toxin injection resulted in a normal visual development of the child ; otherwise , sensory deprivation would have occurred . botulinum toxin can therefore be helpful to treat special conditions even in very young children without relevant side effects of the neurotoxin . dr . wabbels received research grants from allergan , ipsen and merz for medical writing and statistics .

conventional phenolics are cross - linked products of low molecular weight precursors , typically formed through a condensation reaction . the versatility of their structures and the fact that they display desirable properties such as good heat resistance , flame - retardant properties , low dielectric constants , and the production of these materials is relatively inexpensive . however , there are some shortcomings associated with these materials in that they have poor toughness properties , they have a poor shelf life , and the process of polymerization of these materials produces byproducts and requires , in many cases , the use of a strong acid or base catalyst to effect cure . in the search for higher performance replacements for phenolic resins many materials have received attention , and published reports of the preparation of aromatic oxazines , or benzoxazines , date back some 60 years ; although commercial exploitation of the corresponding polymers has only come about relatively recently , they are now receiving a great deal of academic and industrial interest . poly(bis - benzoxazine)s ( sometimes simply referred to as polybenzoxazines ) are a family of thermosetting polymers that are made up through step growth ring - opening polyaddition from bis - benzoxazine monomers ( figure 1 ) , which are in turn the products of the mannich reaction between a bis - phenol , formaldehyde , and a primary amine . the monomer oligomer ratio in the yield can also be influenced by using an excess of formaldehyde and amine during the synthesis , causing the products to form via a different mechanism and resulting in a greater proportion of monomer in the product . this , in turn , affects the properties of the resin before , during , and after cure ( the presence of oligomers bearing hydroxyl groups in the chain is known to enhance the reactivity of the benzoxazine ) . polymerization of bisbenzoxazines through ring - opening and cross - linking and representative network . commercial benzoxazines are currently being evaluated for use in the aerospace industry ( in which they would replace or augment phenolic polymers in secondary applications such as interior paneling ) . polybenzoxazines appear to incorporate the best properties from conventional phenolics and may find application in a number of their traditional niches , while improving on shelf life and offering the potential for greater toughness properties through their greater molecular flexibility ; the relative cheapness of the monomer is also an important factor influencing their adoption . however , while polybenzoxazines display many benefits over conventional phenolics , the relatively low fracture toughness that is achieved by cured polymers ( a kic value of ca . 0.510.54 mpam is typical , although later linear polymers achieve higher kic values ) and the comparatively poor reactivity araldite mt35600 commences its reaction at 180200 c ( dsc , 10 k / min ) in the absence of additive ( and 160 c with the addition of 5 wt % 3,3-thiodiphenol)are still problems in advanced aerospace applications when compared with competitor resins . unlike many other commercial thermosetting resins , which evolve condensation products such as water or ammonia , benzoxazine monomers react relatively cleanly to form a polymer with few reaction byproducts , and although a number of studies have been published , the exact manner of the polymerization reaction to form a network has not been fully elucidated . in our ongoing research programmes , the ultimate aim is to examine and increase our understanding of the influence of the benzoxazine monomer structure on reactivity , fracture toughness , and thermal stability . in this paper we present the results of a study into the influence of the nature of the additive on the reaction mechanism . significant differences in the mechanism of the network growth are observed via raman spectroscopy when different initiators are employed , leading to significant differences in the thermal and mechanical properties that are observed . the monomers were characterized fully using both spectroscopic and chromatographic techniques , as it is well - known that monomer purity ( in particular , the oligomer content ) can have a significant effect on both cure mechanism and kinetics . analysis using high performance liquid chromatography ( hplc ) suggested that ba - a was markedly more pure ( ca . 95% ) than bf - a ( ca . 80% and a mixture of isomers ) , but this was misleading . subsequent discussion with the materials supplier confirmed that bis - benzoxazine monomers are prone to undergo degradation via ring - opening on the surface of the column during reverse - phase analysis , and so these data can not be used to determine absolute purity . h and c nmr spectroscopic analyses were performed in parallel on the monomers ( e.g. , using dept-135 , hsqc , and hmbc pulse sequences ) , and these supported the observation that ba - a was the more pure , containing a lower concentration of unreacted starting materials , but not disproportionately so . the dsc data for the uninitiated polymerizations are displayed in figure 2 and given in table 1 , from which it can be seen that ba - a offers a potentially relatively wide processing window ( i.e. , the gap between tm = 43 c and the onset of polymerization ca . scanning dsc data for the thermally initiated polymerization of the benzoxazine monomers ( 10 k / min , nitrogen ) : ( a ) ba - a recrystallized , ( b ) ba - a as received , ( c ) bf - a as received , and ( d ) bt - a as received . tg1 = possible glass transition or melting transition of monomer determined using a heating rate of 10 k min ; top = observed onset of polymerization ; tmax = temperature of exothermic peak maximum ; tep = observed end of polymerization ; hp = enthalpy of exothermic peak ( both as j / g of monomer and kj / mol of benzoxazine ring ) ; tg2 = glass transition temperature of polymer determined using a heating rate of 10 k min ; tdp = 3,3-thiodiphenol ; tda = 3,3-thiodipropionic acid . the thermal data obtained in a previous paper showed that the onset of the thermal polymerization was lowest for the monomers formed from aniline and incorporating tdp and 4,4-biphenol ( with relatively broad exotherms spanning 160270 c , tmax 180 and 200 c , respectively ) , with more flexible bridging groups or conjugated ring moieties . in the current work , the monomer based on 3,3-thiodiphenol ( bt - a ) is also the most reactive of those studied . narrower , more symmetrical exotherms were previously recorded at higher temperatures ( tmax = 215 and 225 c ) for the monomers based on dihydroxyphenyl ether and bisphenol a , respectively , with more flexible bridging groups and/or electron donating moieties . conversely , the highest onset temperature ( 180 c ) was recorded for the monomer based on bisphenol af6 , with a highly electronegative bridge ( i.e. , a hexafluoroisopropyl moiety ) . it was notable that the enthalpies recorded for the polymerizations of the majority of the monomers were practically the same ( 147.9 7.4 or 74 kj / mol bz ring ) and compared favorably with literature values ( of 144.7 or 72.4 kj / mol bz ring ) . the generation of kinetic information from thermal data in thermosetting polymers was reviewed extensively in the context of epoxy resins by barton . of these , the kissinger method relates the peak maximum temperature , tmax , obtained using dynamic dsc and the activation energy ( ea ) for data obtained at different heating rates ( figure 3).1where rmax is the rate of reaction at the peak maximum derived from dsc and max is the conversion at the peak maximum . scanning dsc data under nitrogen for ba - a at a variety of heating rates . using this method , the kinetics of the polymerizations were modeled using dynamic dsc at a variety of heating rates , from which an activation energy of 81 kj mol was calculated for the monomer ba - a ( as received ) , which correlated well with literature values ( table 2 ) . the data are presented for the as - received ba - a sample ( figure 4a ) and following recrystallization ( figure 4b ) . kissinger ( ) and ozawa ( ) plots for the polymerization of ( a ) ba - a and ( b ) recrystallized ba - a . however , the dsc data show the reaction mechanism is more complex with the reaction evolving from being chemically controlled to physically controlled as the viscosity increases throughout the reaction . consequently , the kinetics were then determined using the following equation , which is better suited to an autocatalytic system than an nth - order model:2where is the degree of conversion , the heating rate , a the pre - exponential factor , r the gas constant , 8.314 j/(mol k ) , n and m the orders of reaction in the initial and later stages of reaction , respectively . this equation was solved by multiple linear regression to yield values of a , m , and n using the activation energy previously determined ; these values were found to differ significantly from those found in the literature . for example , jubsilp et al . achieved average values of 1.7 for n and 0.8 for m for a ba - a benzoxazine and ishida and rodriguez achieved average values of 1.4 for n and 1.0 for m. following recrystallization from ethanol , ba - a was reanalyzed using dsc , from which it was observed that the melting temperature , polymerization onset temperature , and peak maximum were all raised by some 15 k , suggesting the removal of one or more impurities ( figure 2a , b ) . when the same kinetic analysis was repeated on the recrystallized monomer ba - a , average values of 1.89 and 0.89 for n and m , respectively , were obtained which were more in line with the literature values for a 10 k / min heating rate . therefore , commercial ba - a contains components that are affecting the cure kinetics of the benzoxazine , in the absence of the additive . this is supported by the value of ea for recrystallized ba - a ( 94 kj mol ) , which compares favorably with reported values ( 102116 kj mol ) for an analogous material prepared in the laboratory . it is known that commercial benzoxazine monomers may be held at elevated temperature to yield soluble , low molecular weight oligomers , in which the presence of hydroxyl groups increase the reactivity . an autocatalytic model was used to calculate values for n and m. although bis - benzoxazines will polymerize through opening of the heterocyclic ring when subjected to heat , the use of initiators or catalysts is preferred since highly pure monomers are comparatively unreactive ( onset temperature of 225 c , at 10 k / min dsc scan , in the absence of deliberate catalysis / initiation ) . samples were also analyzed using dsc in order to establish the relative effects of the additives , and the thermal data for the uninitiated polymerization of ba - a are displayed in figure 5 along with the corresponding mixtures containing each of the additives ( 10 mol % ) . the dsc thermograms for the acid - initiated reactions show several almost sequential thermal events , unlike the uninitiated system ( which yields a near gaussian exotherm with a high temperature tail covering several almost simultaneous events ) ; this becomes more apparent when the heating rate is further reduced to 5 k / min ( figure 3 ) . dsc data ( 10 k / min , under nitrogen ) for ba - a ( a ) in the absence of catalyst , with 10 mol % tdp and with 10 mol % tda . 3545 c along with a more diffuse endotherm associated with the fusion of the additive in the mixture containing tdp . the monomer is most sensitive to the addition of the diacid ( tda ) , but although the onset of the polymerization and the peak maximum are moved significantly to a lower temperature , the end point of the exotherm does not move along with this , leading to a broad exotherm . the additive has the greatest impact on the initial stages of reaction ( ring opening ) , but the later processes associated with cross - linking are not affected to the same degree . speed of cure is a concern with polybenzoxazines , which are sluggish to react when highly pure , and this may hinder the more widespread use of these materials despite the interest in their thermomechanical and physical properties ( especially fst ) . using modelmaker ( version 3.0.4 , cherwell scientific ltd . ) , peaks were fitted to the plots and activation energy determined to compare to those obtained using traditional methods . the curves chosen for peak fitting were 15 mol % tda and 12 mol % tdp , as they gave the best improvement in temperature of onset , while still being able to discern the different reaction peaks . the best fit was obtained for the initiated systems when three peaks were used , especially in the tdp samples where the reactions are much more drawn out , but a fourth peak needed to be included in the first peak to prevent the fit distorting in the tdp plots , suggesting there may be more reactions occurring than are being observed . the 5 k / min experiments show that the initiated systems give very different models . in the uninitiated system the best peak fit was found using two peaks , which is consistent with two processes : ring - opening and bridge forming reactions . in the tdp plot ( figure 6 ) the initial stages of reaction are being drawn out , so that several consecutive reactions occur : i.e. , the first ( and smaller second ) peak is associated with ring - opening ; the third peak , which accounts for the bulk of the reaction , is associated with bridge forming , and the last peak is structural rearrangement or possible beginnings of degradation . conversely , the tda plot shows a more simultaneous reaction occurring where several reactions are occurring at once . the results of peak deconvoluting are debatable , but the models presented here represent the best fitting models ( using regression coefficient ) with the smallest number of peaks . peak fitting for dsc data ( 5 k / min , under nitrogen ) for ba - a ( a ) in the absence of catalyst and ( b ) with 12 mol % tdp and ( c ) with 10 mol % tda . using the kissinger method ( 1 ) , the activation energy of for each peak was calculated ( table 3 ) , and the activation energies for the second and third peaks in tdp are similar to those calculated for uninitiated ba - a . the activation energy for the second peak of tda is slightly lower but of a similar magnitude , suggesting a similar reaction . the activation energy of the first peak is some 20 kj mol lower than the subsequent peak in both instances ; this is due to the initiator instigating the ring - opening reaction . it is apparent that the degree of catalysis and the nature of the catalyst have an influence on the progress of the reaction , although this is more marked in the case of the diacid ( e.g. , the reduction is visible in both the onset temperature and the peak maximum of the polymerization exotherm when compared with the uncatalyzed monomer and the formulations containing the diphenol ) ( figure 7 ) . furthermore , the glass transition temperatures of the tda - cured polybenzoxazines ( tg2 , recorded during the dsc rescan experiment ) are significantly higher ( and proportionate to additive concentration ) , where the values are unchanged for the tdp - cured analogues . this supports the suggestion that the additives are giving rise to different polymerization mechanisms , since the cross - link density does not alter significantly for the tdp - initiated blends . plots of glass transition temperature , tmax , and h ( j / g ) for ba - a initiated with ( a ) tda and ( b ) tdp as a function of additive concentration . data from dynamic mechanical thermal analysis were acquired to explore the cross - link densities generated in the different network structures . representative storage modulus curves for pba - a when initiated with tdp and tda are overlaid for comparison in figure 8 ; similar trends in the data were observed for the other polybenzoxazines . the glass transition ( -transition ) is clearly visible in the loss modulus data at ca . 125225 c ( tmax = 175 c at 2 k / min ) with a drop in the storage modulus of around 3500 mpa ; a -transition is visible between 50 and 100 c . the polybenzoxazine that had been cured using a 5 wt % addition of tdp differs in several important respects : ( i ) the tg occurs at slightly lower temperatures as the tdp content increases , ( ii ) the magnitude of the tan peak increases relative to the loss modulus peak as tdp ( 5 wt % ) is added , but decreases slightly at 10 wt % ; ( iii ) the magnitude of the -transition increases as with tdp concentration . these observations ( which are similar for the other polybenzoxazines studied ) indicate the emergence of a different cured network with the use of the additive ; the effect on modulus and damping behavior ( figure 9 ) is clearly demonstrated . dynamic viscoelasticity data for thermally cured polybenzoxazines : ( a ) pba - a , ( b ) pba - a with 5 wt % tdp , ( c ) pba - a with 10 wt % tdp , and ( d ) pba - a with 10 wt % ( tda ) . n.b . : scale relates to plot ( a ) other data offset progressively downward by 1000 mpa for clarity . dynamic viscoelasticity data ( tan ) for thermally cured polybenzoxazines : ( a ) pba - a ) , ( b ) pba - a with 5 wt % tdp , ( c ) pba - a with 10 wt % tdp , and ( d ) pba - a with 10 wt % ( tda ) . n.b . : scale relates to plot ( a ) other data offset progressively downward by 1.0 for clarity . the breadth of the tan peaks show differences in the damping behavior of the polybenzoxazines , and the peak widths represent the temperature ranges over which the glass transition temperatures occur . thus , the broadest tan peak ( ba - a ) can be attributed to more heterogeneous networks containing both highly and less densely cross - linked regions . this , in turn , results in a broad distribution of molecular mobilities or relaxation times ( this is seen to be broadest for the uninitiated pba - a ) . the cross - link density ( ) for each polybenzoxazine was calculated from the dmta data using eq 3:3where is taken as unity and ge is the storage modulus strictly from a sample at equilibrium , but is taken at te , where te = tg + 50 k. this equation is technically most appropriate for lightly cross - linked materials so it should only be used as a comparison between similar materials . the cross - link density values are shown for the benzoxazines following polymerization in both the presence and absence of the additive ( table 4 ) . examination of the data for the neat polybenzoxazines indicates that the three commercial monomers develop very similar cross - link densities upon thermal cure , which is consistent with the structures differing only by the nature of the central bridge : isopropyl and methylene ; the sulfur - bridged monomer bt - a develops a higher cross - link density . for comparison , values of cross - link density of ( 1.110.5 ) 10 mol cm have been reported depending on the type of benzoxazine and the functionality . typically , ba - a is quoted as having a cross - link density of 1.1 10 mol dm , and the higher value found for the ba - a may be due to the higher oligomers or impurities as evidenced by the discrepancies in the reaction kinetics . however , it was not possible to obtain enough material to carry out dmta on the polymer arising from the recrystallized monomer . allen and ishida obtained a cross - link density of 47 mol m for polybenzoxazines based on long chain aliphatic diamines . of the two additives employed in this work , tda has the greatest impact on the cross - link density according to the dmta data ; e.g. , it leads to an increase in the tg of some 20 k ( pba - a ) , 30 k ( pbf - a ) , and 20 k ( pbt - a ) . polybenzoxazine degradation can be broken down into three events , although the processes are not entirely discrete as evidenced by the breadth of the peaks . there is a lower temperature event ( 200220 c ) for the initiated pba - a systems that is much higher for the pba - a alone ( around 290 c ) . this first degradation stage is associated with the breakdown of the mannich bridge and loss of the pendant amino groups and is most prominent for the formulations containing the lowest concentrations of initiators , suggesting there are more pendant moieties in these systems . this might be due to the initiator preventing reaction at the arylamine ring or affecting the chain growth , although this growth in weight loss may also be due to loss of initiator from the polymer matrix . the second degradation peak is associated with the breakdown of the polymer chain , leading to release of substituted phenols , and is the largest contributor to the thermal degradation process . the observed rise in the cross - link density and tg is also reflected in the measurement of initial thermal stability ( table 5 ) of the cured ( but uninitiated ) polybenzoxazines ; e.g. , the t5% rises from 258 c ( uninitiated pba - a ) to 294 c ( pba - a + 5% tda ) and from 263 c ( uninitiated pbf - a ) to 290 c ( pbf - a + 5% tda ) . although pbt - a is initially the more stable polymer , the mass loss tends to reverse beyond ca . 30% , suggesting a different degradation mechanism . work is currently underway within our group to examine this aspect and will be reported in a future paper . the presence of 10 wt % tda reduces thermal stability and leads to a more rapid loss in mass but provides an increase in tg . the char yields are ordered in the following way : pbt - a > pbf - a pba - a with the polymers containing the highest alkyl component providing the lowest char . this order of stability was confirmed by visual observations made on thermally cured and deliberately charred samples . all samples analyzed under nitrogen at a heating rate of 10 k / min . the introduction of tdp is beneficial at the 5 wt % level of incorporation : the initial stability ( t5% ) is raised for both pba - a and pbf - a but has no apparent effect on pbt - a . at higher mass losses ( e.g. , 30% ) , the stability of both pbf - a and pbt - a is elevated , but the effect on pba - a is to reduce stability . tdp is less influential at 10 wt % ( where it leads to a reduction in both cross - link density and thermal stability for all the polymers , accompanied by the release of volatiles during cure ) . tda can be incorporated at 5 and 10 wt % and leads to enhanced thermal stability in pba - a , but a reduction in the char yield of pbf - a and also pbt - a . the differences in cross - link density observed , coupled with the thermal behavior , may reflect a different initiation mechanism as the pka = 6.6 for tdp is greater than for tda ( pka = 4.11 ) . wang and ishida also saw improved char yield and tg when lewis acids were used to initiate the ring - opening reaction of ba - a and , when considering the first - derivative plot of the weight change from tga data , suggested that a different degradation mechanism occurred in the polybenzoxazines that arose from initiated and uninitiated mechanisms . this was attributed by the authors to the formation of a different polymer architecture caused by the presence of the initiator . dunkers and ishida discuss the use of both strong and weak protonic catalysts and found that a slightly different polymerization mechanism occurs depending on the pka of the acid . for acids with pka in the range 0.704.43 , this was explained by the formation of a stable iminium ion at early conversions , but generally the stronger the acid , the more rapidly the monomer was consumed and more rapidly converted to the tetra - substituted ring of the product , which is consistent with our findings . thermal analysis provides information about physical changes brought about during the thermal treatment of the monomer , but ( without hyphenation ) no chemical information . consequently , the cure of monomer ba - a was monitored initially in situ using raman spectroscopy using a heated cell ( ramped rapidly to 180 c and held isothermally for 120 min ; spectra being taken at intervals of 7.5 min ) , but while a stacked spectral plot could be produced as a function of cure time ( figure 10 ) the fluorescence background produced by the thermal degradation at an impurity level caused the signal to be increasingly swamped between 1800 and 3400 cm so that any data in this region could not be used quantitatively . it is apparent that the vibrational spectra of the monomers and propagating species are quite complex and assignments in the literature are occasionally contradictory , but arguably , the most diagnostically useful bands in the spectra are those associated with the constitution of the benzoxazine ring , given that the ring opens to form an oligomer during polymerization . the band corresponding to the presence of the c c asymmetric stretch will be present only in the benzoxazine and can be found at 10701200 cm and the symmetric stretch at 800860 cm . similarly , the bands for c o c stretching can be found at ca . stacked raman spectra for the polymerization of ba - a acquired in situ ( comprising 16 spectra , sampling rate 1 every 7.5 min ) . the peaks can not be assigned unequivocally to pure monomer or dimer / oligomer as the same functional groups appear in both species ( and some studies have reached tentative assignments ) , but while the basic bands can be assigned with confidence the data from several published studies ( table 6 ) are sometimes contradictory on key points . consequently , it was decided to employ chemometrics techniques ( particularly principal components analysis , pca ) as our group has already demonstrated the benefits of this approach in unrelated areas of polymer chemistry . pca is a multivariate analysis technique that utilizes all the data and uses a holistic approach to determine similar and dissimilar spectra . the extraction of the principal components ( pcs ) , which account for the variance in data , enables the particular analysis to be simplified but makes no suppositions about the chemical meaning of the data . this technique has been shown to yield useful information in the correlation of infrared spectra of cyanate ester / bismaleimide blends with thermomechanical properties and has also been employed to probe the mechanism by which our previous complexes have undergone dissociation . figure 11 shows the pca data arising from the raw raman spectra for the time course of uncatalyzed ba - a in a series of in situ measurements . the data show that two principal components ( pcs ) account for the variation in the data . a plot of pc1 ( accounting for 97% ) vs pc2 ( accounting for 3% ) shows regular incremental changes in the data until spectrum 7 ( i.e. , after 52 min at 180 c ) , at which point there is a large change in the plot , indicating that a significant change in the curing polymer is occurring at this point . most of the spectral changes observed during the experiment occur in regions associated with the aromatic rings and the phenolic moiety . the main band changes are reductions at 700800 cm ( c h out - of - plane bending vibrations ) , 9901050 cm ( c h in - plane vibrations ) , and 11701250 cm ( c n c stretch ) and increases at 12601300 cm ( c o stretch ) , 14001500 cm ( c h deformations ) , and 15301630 cm ( skeletal vibrations such as quadrant stretching bands ) . these changes in the bands associated with aryl moieties upon heating is consistent with the monomer undergoing ring - opening leading to changes in the rotational freedom of the phenyl ring . the scores plot of pc-2 versus pc-1 indicates that the spectra are very similar and show very little difference during the first five acquisitions . spectrum 6 begins to differ , and then there is a progressive change in the spectral characteristics , with pc-1 becoming increasingly influential , up to spectrum 9 . from spectrum 10 onward until the end of the experiment there is a change in the trend , perhaps related to the change from chemical to physical control ( and from chain extension to cross - linking ) . similar plots were obtained for the range of bis - benzoxazine monomers and support the observation that similar changes are observed in key peaks in the spectra during cure ( this , in turn , suggests that the uninitiated thermal cure mechanism is largely independent of the nature of the bis - phenol of the benzoxazines studied here ) . unfortunately , the samples could only be cured up to 180 c for 2 h in the in situ raman experiment , due to instrumental limitations making it impossible to monitor reactions cured / postcured at 200 c . consequently , in order to validate the two experimental methods , it was decided to perform an ex situ analysis ( i.e. , curing individual samples in a fan - assisted oven to differing degrees of cure following the cure schedule2 h at 180 c and analyzing these samples individually with respect to cure time ) before comparing the results of the two methods . the data may be compared directly since the sampling intervals are the same and it is clear that there is no significant effect on the pathway of the reaction by changing the sampling method . there is inevitably a slight delay / variation in this method associated with removal of the samples from the oven , quenching them in liquid nitrogen before introducing them into the raman spectrometer . for comparison , the data for the uninitiated ba - a ( as received ) and following recrystallization ( figure 11 , bottom ) are presented , showing graphically the differences in the scores plot ( marked by a reduction in the significance of pc-2 is evident and a more symmetrical presentation of the data for the latter ) . pca data from cure of ba - a as received ( top ) and recrystallized ( below ) . raman data from in situ experiment showing ( left ) scores for pc2 versus pc1 and ( right ) loadings for pc1 . n.b . : the first data point ( monomer ) has been omitted from this treatment . the bis - benzoxazine monomers were each formulated with tdp or tda at 10 mol % addition , and ex situ analysis was performed using the same conditions ( heated at 2 k / min ) . using the same data treatment , the pca results arising from these spectra are presented for the polymerization reaction initiated with tdp ( figure 12 ) and tda ( figure 13 ) . these treatments indicate that the initiated systems obey very different kinetics during the early stages of the reaction ; e.g. , the changes in the scores plots occur over a markedly shorter time scale during both the early and later stages of reaction when compared with the uninitiated blend ( confirming that the effect of the additives is to induce a faster rate of cure ) . furthermore , in the case of tdp only one pc is required to describe the data ( whereas in the case of tda two pcs are needed : pc-1 covers > 98% and pc-2 > however , it is possible to state that the final networks yielded different spectra in the presence of the additives , possibly due to a different termination mechanism , increased cross - link density , and consequent differences in thermo - oxidative stability ( discussed below in the context of the tga data ) . pca data from in situ cure of ba - a ( as received ) with 10 mol % tdp . raman data showing ( top left ) scores for pc2 versus pc1 and ( top right ) loadings for pc1 . n.b . : pca data from in situ cure of ba - a ( as received ) with 10 mol % tda . raman data showing ( top left ) scores for pc2 versus pc1 and ( top right ) loadings for pc1 . n.b . : the first , second , and third data points were omitted from this treatment . the difference in the scores plots suggests differences in the mechanisms . from an overlay plot of pc1 for the different samples ( figure 14 ) , there are spectral regions ( 15501700 cm ) that overlay well in terms of intensity , suggesting that the observed differences in intensity are real . this would indicate that during the initial stages of the polymerization reaction the greatest reductions are in the bands due to aromatic substitution . for instance , reductions are observed in the c h out - of - plane deformation at 750 and 793 cm attributed by dunkers and ishida to 1,2,4-trisubstituted rings and 999 cm ( mono- , 1,3- , or 1,3,5-trisubstitution perhaps in the arylamine ) . overlay of plots of pc1 for ba - a , ba - a in the presence of tda ( 10 mol % ) , and ba - a in the presence of tdp ( 10 mol % ) . this is accompanied by reductions in the bands at 1031 cm ( from c c ring stretch or c o c stretch ) and 1102 ( possibly c n c stretch ) and 1208 cm ( c n stretch ) , 13001310 cm ( c o stretch ) , and 14501500 cm ( ch2 scissoring ) . over the same period , increases in band intensity are observed for 1050 ( c o stretch ) and 1114 cm ( c n stretch ) and 1518 and 1619 cm ( aromatic ring breathing ) , while bands appear to shift between 1150 and 1250 cm . to explore the mechanisms further , selected individual spectral bands within pc1 for the thermal polymerization of ba - a in the absence of additive and with both tdp and tda were monitored as a function of reaction time ( figure 15 ) over a total period of 185 min . variation in intensity of selected spectral bands ( cm ) as a function of reaction time for ba - a ( a ) in the absence of additive , ( b ) in the presence of tdp ( 5 mol % ) , and ( c ) in the presence of tda ( 5 mol % ) . the profiles for ba - a when thermally initiated or in the presence of tdp are quite similar , albeit with the initiated polymerization occurring earlier . at the point at which crossover occurs the sample appears to be undergoing some molecular change , e.g. , gelation causing a change in the kinetics , as this is the same point at which the parabola changes gradient in the scores plot ( 6080 min for the tdp - initiated reaction ; 100120 min for the thermally initiated reaction ) . this demonstrates the sluggish nature of the reaction in the absence of initiators or catalysts ; the observations would have been retarded still further on the purified ba - a monomer . there are reductions in the bands at 793 , 999 , 1032 , and 1104 cm ( associated with the ring - closed benzoxazine structure ) from about 85 min in the tdp - initiated ba - a and from 35 to 115 min in the ba - a alone . all the aforementioned bands appear to drop at a similar rate , while the intensity of the band at 751 cm decreases more rapidly and earlier in the polymerization reaction . conversely , the bands at 1052 , 1312 , and 1487 cm , which are attributed to aromatic ring functions , increase at approximately the same rate , and bands at 1619 and 1581 cm initially increase at the same rate , before 1581 cm increases its growth . these observations imply that the bands associated with 1581 and 751 cm are related , as are those at 999 and 1619 cm . when compared with ba - a alone the differences in the growth of the band at 1581 and 1619 cm are much more marked for the tdp initiated system , with 1581 cm increasing more rapidly and to a greater intensity , whereas in ba - a alone the increase in 1619 cm is greater than tdp , relative to many of the other bands . a similar analysis of the spectral data relating to the ba - a initiated with tda yields quite different results . for instance , the bands at 999 and 1619 cm are negatively correlated , as are 1581 and 751 cm . this observation is confirmed by the pc1 loadings plots as these are the most intense peaks ( either positive or negative ) and have similar loadings although 1581 cm is slightly greater . the increase in the degree of substitution of the newly formed phenolic ring can be monitored by observing the reduction in the intensities of the characteristic phenolic bands at 999 and 1032 cm ; the latter is assigned to the c o c stretching mode of the benzoxazine ring . thus , these may relate to the changing nature of the benzene ring substitution through benzoxazine ring - opening and bridge - forming . dunkers and ishida also observe a band at 1581 cm in a monofunctional benzoxazine with alkylamine groups , but this apparently shifts to 1600 cm in the corresponding dimer , suggesting that this band is related to the closed ring structure alone ; the band at 1620 cm is reported in both instances . the same authors note that the oxazine ring typically gives rise to a band around 960920 cm due to an out - of - plane b2 mode with medium to strong intensity in the ir spectrum . the changes in peak intensities show a different pattern for the tda initiated monomer ( figure 15c ) : the bands at 751 , 793 , 1032 , and 1104 cm increase at a similar rate , but 999 cm appears to increase more rapidly and over a longer period of time . the bands at 1312 , 1487 , and 1052 cm increase at the same rate , but 1052 cm reaches a much lower ultimate intensity . in fact , in ba - a , tdp , and tda , the difference in intensity between 1312 and 1487 cm and 1052 cm increase from nearly 0 to 1 arbitrary unit . the bands at 1312 and 1487 cm are most likely to reflect aromatic functions or possibly the methylene groups in the bridges , but 1052 cm is probably a c o c stretching mode . examining figure 15c , the relationship between 1581 and 1619 cm differs from 751 and 999 cm ; 1581 and 751 cm appear to be associated , as are 1619 and 999 cm ( unlike both ba - a and tdp ) . the thermal initiation and the tdp initiation of ba - a conform to a similar polymerization mechanism , but initiation shifts the reaction to a lower temperature regime and the resulting polymer networks display similar cross - link density and glass transition temperatures ( spanning a wider range implying a more heterogeneous network ) . the initiation of ba - a with tda progresses via a different mechanism involving the paired bands at 999 and 1581 cm and 751 and 1619 cm , which are all aromatic vibration modes . the tda works more efficiently , leading to the opening of more benzoxazine rings , and propagation is less linear ( more cluster - like ) prior to cross - linking . the result is that there are more points of reaction leading to a higher cross - link density and consequent higher tg . the effect of additive concentration [ tda ] is initially really marked but reaches an apparent plateau at 1015 mol % . a combination of thermal and spectroscopic analysis has proven successful in offering support to the proposed ring - opening mechanisms associated with benzoxazine monomers . the presence of the additive improves the temperature of polymerization , by encouraging the ring - opening reaction at a lower temperature , and its presence also appears to improve the thermal stability of the final polymer . spectral and thermal data suggest both initiated and uninitiated blends undergo the same mechanism : suggesting a step growth mechanism whereby the rings open and an active center reacts so there may be several centers active at one time . the selection of an initiator based on its pka appears to be able to influence both the nature of the network architecture and potentially the final properties of the polybenzoxazine as the tg ( determined from dmta ) is elevated when 3,3-thiodipropionic acid ( tda ) is used in preference to 3,3-thiodiphenol . tda is found to be the superior initiator , offering a more rapid polymerization mechanism and a higher glass transition temperature in the cured polybenzoxazines . the monomers based on bisphenol a ( ba - a ) , a mixture of 4,4- , 2,4- , and 2,2-bisphenol f ( bf - a ) , 3,3-thiodiphenol ( bt - a ) , and additives 3,3-thiodiphenol ( tdp ) and 3,3-thiodipropionic acid ( tda ) were all supplied by huntsman advanced materials ( basel , switzerland ) and having been characterized using h nmr , raman spectroscopy , and elemental analysis were generally used as received without further purification . monomer ( ba - a ) was recrystallized from ethanol ( 96% ) prior to dsc analysis when examining the kinetics of reaction . the structures and designations for the monomers studied are given in table 7 ; the corresponding polybenzoxazine of ba - a is designated pba - a . where additives were used , these were first ground ( together with the monomer ) and then heated to 90100 c ( to melt the additive ) and degassed for 23 h under vacuum depending on the level of foaming . where additives were not used , monomers were first degassed at 100120 c ( 23 h depending on the level of foaming ) and cured in aluminum dishes ( 55 mm diameter , depth 10 mm ) in a fan - assisted oven : heating at 2 k / min to 180 c ( 2 h isothermal ) + heating at 2 k / min to 200 c ( 2 h isothermal ) followed by a gradual cool ( 3 k / min ) to room temperature . cured samples were cut to the correct token size for analysis using a diamond saw . for the dynamic cure study 100 c ( 1 h ) and cured in aluminum dishes ( 55 mm diameter , depth 10 mm ) and transferred to a fan - assisted oven set at 90 c . the samples were allowed to equilibrate before being heated at 2 , 8 , or 15 k / min to 180 c ( 2 h isothermal ) + heating at 2 k / min to 200 c ( 2 h isothermal ) followed by a gradual cool ( 3 k / min ) to room temperature . cured samples were cut to the correct token size for analysis using a diamond saw . producing flat , void - free plaques for dmta provided a big challenge as the benzoxazines were difficult to degas , and the addition of the initiators compounded the problem as they seemed to undergo degradation during cure to produce gas bubbles ; this was particularly problematic at higher concentrations of tda . vibrational spectra were obtained using a perkinelmer system 2000 ft - nir - raman spectrometer operating at 250500 mw ( nd : yag laser ) and a perkinelmer ftir system 2000 spectrometer . samples that were analyzed in situ during the cure process using raman spectroscopy employed a heated cell that was ramped rapidly from 50 to 180 c and held isothermally for 120 min ; spectra being taken at intervals of 7.5 min . in contrast , samples analyzed ex situ were heated in a fan - assisted oven that was ramped rapidly to 180 c and held isothermally for 120 min ; samples being taken at intervals of 7.5 min and quenched in liquid nitrogen . samples for ir analysis were cast from chloroform ( gpr ) on to kbr disks and the solvent removed in a fan oven ( 50 c , 30 min ) . for each measurement , 10 spectra were obtained at a resolution of 4 cm and coadded to produce the final spectrum . chemometrics analysis ( pca ) was carried out on the spectral data using the unscrambler x , v10.1 software ( camo , oslo ) . nuclear magnetic resonance ( nmr ) spectra ( including dept-135 , hsqc and hmbc pulse sequences to assign individual environments ) were obtained for h ( 500 mhz ) and c ( 75 mhz ) using a bruker drx500 ft - nmr at 298 k in cdcl3 with tms . high performance liquid chromatography ( hplc ) was undertaken using a varian 920-lc using c18 column and uv detection ( = 254 nm ) . samples were dissolved in hplc grade chcl3 , and the mobile phase ( flow rate 0.7 ml / min , 40 c ) was 100% chcl3/100% methanol gradient ( both hplc grade ) . differential scanning calorimetry ( dsc ) was undertaken using a ta instruments q1000 running ta q series advantage software on samples ( 4.0 0.5 mg ) in hermetically sealed aluminum pans . experiments were conducted at a heating rate of 5 , 8 , 10 , 12 , and 15 k / min from room temperature to 300 c ( heat / cool / heat ) under flowing nitrogen ( 50 cm / min . ) . in order to gauge the reactivity of the monomer in the bulk thermogravimetric analysis ( tga ) was performed on a ta q500 on milled , cured resin samples ( 5.5 0.5 mg ) in a platinum crucible from 20 to 1000 c at 10 k / min in air and nitrogen ( 40 cm / min ) . dynamic mechanical thermal analysis ( dmta ) ( in single cantilever mode at a frequency of 1 hz ) was carried out on cured neat resin samples ( 2 mm 10 mm 17 mm ) using a ta q800 in static air .

the international diabetes federation estimated the prevalence of diabetes mellitus in spain to be approximately 10.4% , with around 3.6 million adults diagnosed with diabetes and 1.3 million undiagnosed in 2015 . the disease is a considerable burden on spanish patients and , in 2015 , diabetes mellitus was ranked eighth among causes for disability - adjusted life - years in spain . patients with diabetes are often affected by multiple morbidities , including foot ulcer , cardiovascular disease or renal failure [ 35 ] . diabetes also causes considerable costs to the spanish healthcare system and economy . in 2009 , diabetes - related costs accounted for 8% of all healthcare system expenditures , with eur 5.1 billion in direct costs and eur 2.8 billion in lost labor productivity . a study in the catalonia region estimated that , in 2011 , annual direct medical costs for a patient with type 2 diabetes were eur 3110 , compared with eur 1803 for a patient without diabetes . extrapolating these results to all of spain and assuming a prevalence of 7.8% , the authors estimated yearly direct costs of type 2 diabetes to be around eur 10 billion . the clinical and economic burden imposed by diabetes can be reduced if patients are treated effectively , i.e. if they meet glycemic targets to reduce the risk of micro- and macrovascular complications [ 810 ] . in line with other guidelines , spanish treatment guidelines recommend glycated hemoglobin ( hba1c ) target levels of < 6.5% ( 47.5 mmol / mol ) if patients are newly diagnosed with type 2 diabetes , younger than 70 years and without diabetes - related complications , and < 7.5% ( 58.5 mmol / mol ) otherwise . however , only 32 and 68% of spanish patients are reaching hba1c levels of < 6.5% ( 47.5 mmol / mol ) and < 7.5% ( 58.5 mmol / mol ) , respectively , and only 55% of patients are adequately controlled with respect to individualized glycemic targets . in catalonia , between 2007 and 2013 the percentages of patients who reached an hba1c value of 7% ( 53 mmol / mol ) ranged between 52 and 56% but did not notably increase over time . the spanish diabetes society recommends the use of metformin as the first - line pharmaceutical therapy for patients with type 2 diabetes and an hba1c level of 8.5% ( 69.4 mmol / mol ) . if treatment targets are not met within 3 months , second - line treatments should be added . glucagon - like peptide-1 ( glp-1 ) receptor agonists are attractive second - line treatments as they lead to improved glycemic control and weight loss and are associated with a low risk of hypoglycemia [ 1416 ] . several glp-1 receptor agonists , including liraglutide ( 1.2 mg or 1.8 mg ) , lixisenatide ( 10 g or 20 g once daily ) , exenatide ( 5 g or 10 g twice daily ) and exenatide once weekly , have already been prescribed to spanish patients , mostly in specialized diabetes care settings . both 1.2 mg and 1.8 mg doses of liraglutide have been shown to be cost - effective from a spanish healthcare payer perspective when compared with the dipeptidyl peptidase-4 ( dpp-4 ) inhibitor sitagliptin in patients who are unresponsive to metformin monotherapy [ 18 , 19 ] . in addition , the ability of liraglutide to reduce hba1c and weight was demonstrated in real - world clinical practice in spain . however , no studies comparing the cost - effectiveness of liraglutide versus lixisenatide in the spanish setting have been published to date . the recent publication of the lira - lixi trial has provided high - quality data which allows comparison of the long - term cost - effectiveness of liraglutide 1.8 mg with lixisenatide 20 g , both administered once daily , in the spanish setting for treatment of patients with type 2 diabetes who failed to achieve glycemic control on metformin monotherapy . the ims core diabetes model ( ims health , basel , switzerland ) was used to evaluate long - term outcomes . the model is a policy analysis tool that allows estimation of clinical and cost trajectories of patients with diabetes over longer time horizons than are feasible in clinical trials . it has been successfully validated against published data from clinical and epidemiological studies on initial publication in 2003 and following a series of updates in 2014 [ 2224 ] . the model contains several inter - dependent sub - models to simulate diabetes - related complications ( angina , cataract , congestive heart failure , diabetic retinopathy , foot ulcer and amputation , hypoglycemia , ketoacidosis , lactic acidosis , macular edema , myocardial infarction , nephropathy and end - stage renal disease , neuropathy , peripheral vascular disease , stroke and non - specific mortality ) . sub - models have a semi - markov structure and use time , time - in - state and diabetes type - dependent probabilities derived from published sources . while standard markov models are memory - less , the ims core model uses monte carlo simulation with tracker variables to model patient history and to allow for interactions between sub - models . in line with spanish and international recommendations on the economic evaluation of health technologies , long - term complications and costs as well as their impact on ( quality - adjusted ) life expectancy future costs and benefits were discounted at 3% annually , as per recommendations for spain . the lira - lixi trial ( nct01973231 ) provided the baseline cohort characteristics and treatment effects modeled in the analysis . the trial was a 26-week , open - label study in nine european countries that enrolled 404 adults with type 2 diabetes who had failed to meet glycemic targets on metformin monotherapy . study participants were randomly allocated in equal numbers to therapy with liraglutide 1.8 mg or therapy with lixisenatide 20 g , both to be administered once daily . at baseline , the cohort had a mean age of 56.2 [ standard deviation ( sd ) 10.3 ] years and a mean body mass index ( bmi ) of 34.7 kg / m ( sd 6.7 kg / m ) , with a mean glycated hemoglobin ( hba1c ) of 8.4% ( sd 0.8% ) and a mean diabetes duration of 6.4 ( sd 5.1 ) years . the lira - lixi trial data did not provide data on smoking and alcohol consumption so these were retrieved from external population data sources for spain [ 27 , 28 ] . after 26 weeks , liraglutide 1.8 mg was associated with a larger decrease in hba1c ( 1.83% ) than lixisenatide 20 g ( 1.21% ) , with an hba1c difference of 0.62% ( 95% confidence interval 0.80 to 0.44% , p < 0.0001 ) ( table 1 ) . glycemic targets of hba1c of < 7.0% and hba1c of 6.5% were achieved by a statistically significantly larger proportion ( p < 0.0001 for all targets ) of patients receiving liraglutide 1.8 mg compared with patients receiving lixisenatide 20 g , but no statistically significant differences between treatments were observed for changes in bmi , lipid profile , blood pressure or the number of hypoglycemic episodes ( table 1 ) . outcomes at the end of the trial were used in the analysis as first - year treatment effects of initiating glp-1 receptor agonists . after the first year , the analysis assumed that hba1c and systolic blood pressure followed natural progression algorithms based on the united kingdom prospective diabetes study ( ukpds ) and that serum lipids followed progression equations based on the framingham heart study . during glp-1 receptor agonist treatment , bmi was assumed to remain constant before returning to baseline level on treatment intensification.table 1treatment effects applied in the first year of the analysisliraglutide 1.8 mg ( mean)lixisenatide 20 g ( mean ) p value for differencehba1c ( % ) 1.831.210.0001systolic blood pressure ( mmhg)4.703.490.3722total cholesterol ( mg / dl)7.751.940.0755ldl cholesterol ( mg / dl)4.24 + 1.060.1140hdl cholesterol ( mg / dl)+0.93 + 1.860.4625triglycerides ( mg / dl)34.5620.740.1044body mass index ( kg / m)1.481.280.2255severe hypoglycemic event rate ( events per 100 patient years)0.000.00non - severe hypoglycemic event rate ( events per 100 patient glycated hemoglobin , hdl high density lipoprotein , ldl low density lipoprotein treatment effects applied in the first year of the analysis hba1c glycated hemoglobin , hdl high density lipoprotein , ldl low density lipoprotein the simulated cohort of patients received glp-1 receptor agonists for 3 years . a 3-year period was chosen based on the results of the leader trial which reported a mean time of exposure to liraglutide of 3.1 years . after 3 years and for the rest of the simulation , patients were treated with insulin glargine . both the timing of the switch and the type of insulin switched to were varied in sensitivity analyses . costs were accounted in 2015 euros ( eur ) from the spanish national health system payer perspective . annual treatment costs were based on published wholesale acquisition prices and included the costs of glp-1 receptor agonists and the needles needed for their injection , of concomitant metformin therapy and of self - monitoring of blood glucose testing , which was assumed to be three tests per week in both arms of the analysis . costs of treating diabetes - related complications were identified from literature reviews and spanish databases and were inflated to 2015 prices using the consumer price index for health [ 3139 ] . diabetes and its complications affect the quality of life , and this was captured by applying published event disutilities in the year when the complication occurred and published state utilities in all subsequent years [ 4043 ] . all values have been used in published cost - effectiveness analyses of liraglutide [ 19 , 44 , 45 ] . projection of quality - adjusted life expectancy [ measured in quality - adjusted life years ( qalys ) ] is used widely in cost - effectiveness analyses and is accepted as a useful , relevant metric for decision - makers and payers , although this approach is not without its limitations [ 46 , 47 ] . a number of sensitivity analyses were conducted as part of this study , in line with published recommendations . the time horizon was reduced to 10 and 20 years to assess the impact of varying the time horizon on health economic outcomes . the impact of discounting was evaluated by conducting analyses with discount rates that varied between 0 and 5% . key drivers of clinical outcomes were identified by individually setting differences in clinical parameters ( e.g. hba1c , bmi or hypoglycemic event rates ) between treatment arms to zero . an additional analysis set all clinical parameters equal to their values in the lixisenatide arm , except for the statistically significant difference in hba1c in the liraglutide arm . several analyses were performed to evaluate the impact of alternative treatment - switching options as patients treated with glp-1 receptor agonist therapy will eventually require insulin . first , in both arms treatment was switched to insulin glargine only after 5 years of glp-1 receptor agonist therapy , which approximated the upper observation time limit in the leader trial . third , patients switched to neutral protamine hagedorn ( nph ) insulin instead of insulin glargine after 3 years of glp-1 receptor agonist therapy . over- or underestimation of direct costs of diabetes - related complications might also influence results . therefore , the analysis was conducted with costs increased and decreased by 10% , respectively . a further sensitivity analysis was performed using an updated version of the ims core diabetes model , which was released in 2014 and incorporates data from the ukpds 82 study . the model proprietors recommend that this version is used only for sensitivity analyses and the previous version is used for the base case . probabilistic sensitivity analysis ( psa ) was performed using a second - order monte carlo approach . transition probabilities ( sampled based on regression co - efficients ) , utilities ( beta distributions ) direct costs ( log - normal distributions ) , treatment effects ( beta distributions ) and cohort characteristics ( normal distributions ) were sampled . one thousand cohorts of 1000 patients were simulated in the psa and used to generate cost - effectiveness scatterplots and acceptability curves which were used to analyze the probability that a treatment may be cost - effective over a range of willingness to pay thresholds . no fixed willingness to pay threshold exists in spain but earlier studies have used thresholds of between eur 20,000 and eur 30,000 per qaly gained [ 48 , 49 ] . this article does not contain any new studies of human or animal subjects performed by any of the authors . the ims core diabetes model ( ims health , basel , switzerland ) was used to evaluate long - term outcomes . the model is a policy analysis tool that allows estimation of clinical and cost trajectories of patients with diabetes over longer time horizons than are feasible in clinical trials . it has been successfully validated against published data from clinical and epidemiological studies on initial publication in 2003 and following a series of updates in 2014 [ 2224 ] . the model contains several inter - dependent sub - models to simulate diabetes - related complications ( angina , cataract , congestive heart failure , diabetic retinopathy , foot ulcer and amputation , hypoglycemia , ketoacidosis , lactic acidosis , macular edema , myocardial infarction , nephropathy and end - stage renal disease , neuropathy , peripheral vascular disease , stroke and non - specific mortality ) . sub - models have a semi - markov structure and use time , time - in - state and diabetes type - dependent probabilities derived from published sources . while standard markov models are memory - less , the ims core model uses monte carlo simulation with tracker variables to model patient history and to allow for interactions between sub - models . in line with spanish and international recommendations on the economic evaluation of health technologies , long - term complications and costs as well as their impact on ( quality - adjusted ) life expectancy future costs and benefits were discounted at 3% annually , as per recommendations for spain . the lira - lixi trial ( nct01973231 ) provided the baseline cohort characteristics and treatment effects modeled in the analysis . the trial was a 26-week , open - label study in nine european countries that enrolled 404 adults with type 2 diabetes who had failed to meet glycemic targets on metformin monotherapy . study participants were randomly allocated in equal numbers to therapy with liraglutide 1.8 mg or therapy with lixisenatide 20 g , both to be administered once daily . at baseline , the cohort had a mean age of 56.2 [ standard deviation ( sd ) 10.3 ] years and a mean body mass index ( bmi ) of 34.7 kg / m ( sd 6.7 kg / m ) , with a mean glycated hemoglobin ( hba1c ) of 8.4% ( sd 0.8% ) and a mean diabetes duration of 6.4 ( sd 5.1 ) years . the lira - lixi trial data did not provide data on smoking and alcohol consumption so these were retrieved from external population data sources for spain [ 27 , 28 ] . after 26 weeks , liraglutide 1.8 mg was associated with a larger decrease in hba1c ( 1.83% ) than lixisenatide 20 g ( 1.21% ) , with an hba1c difference of 0.62% ( 95% confidence interval 0.80 to 0.44% , p < 0.0001 ) ( table 1 ) . glycemic targets of hba1c of < 7.0% and hba1c of 6.5% were achieved by a statistically significantly larger proportion ( p < 0.0001 for all targets ) of patients receiving liraglutide 1.8 mg compared with patients receiving lixisenatide 20 g , but no statistically significant differences between treatments were observed for changes in bmi , lipid profile , blood pressure or the number of hypoglycemic episodes ( table 1 ) . outcomes at the end of the trial were used in the analysis as first - year treatment effects of initiating glp-1 receptor agonists . after the first year , the analysis assumed that hba1c and systolic blood pressure followed natural progression algorithms based on the united kingdom prospective diabetes study ( ukpds ) and that serum lipids followed progression equations based on the framingham heart study . during glp-1 receptor agonist treatment , bmi was assumed to remain constant before returning to baseline level on treatment intensification.table 1treatment effects applied in the first year of the analysisliraglutide 1.8 mg ( mean)lixisenatide 20 g ( mean ) p value for differencehba1c ( % ) 1.831.210.0001systolic blood pressure ( mmhg)4.703.490.3722total cholesterol ( mg / dl)7.751.940.0755ldl cholesterol ( mg / dl)4.24 + 1.060.1140hdl cholesterol ( mg / dl)+0.93 + 1.860.4625triglycerides ( mg / dl)34.5620.740.1044body mass index ( kg / m)1.481.280.2255severe hypoglycemic event rate ( events per 100 patient years)0.000.00non - severe hypoglycemic event rate ( events per 100 patient years)4.208.700.4980 hba1c glycated hemoglobin , hdl high density lipoprotein , ldl low density lipoprotein treatment effects applied in the first year of the analysis hba1c glycated hemoglobin , hdl high density lipoprotein , ldl low density lipoprotein the simulated cohort of patients received glp-1 receptor agonists for 3 years . a 3-year period was chosen based on the results of the leader trial which reported a mean time of exposure to liraglutide of 3.1 years . after 3 years and for the rest of the simulation , patients were treated with insulin glargine . both the timing of the switch and the type of insulin switched to were varied in sensitivity analyses . costs were accounted in 2015 euros ( eur ) from the spanish national health system payer perspective . annual treatment costs were based on published wholesale acquisition prices and included the costs of glp-1 receptor agonists and the needles needed for their injection , of concomitant metformin therapy and of self - monitoring of blood glucose testing , which was assumed to be three tests per week in both arms of the analysis . costs of treating diabetes - related complications were identified from literature reviews and spanish databases and were inflated to 2015 prices using the consumer price index for health [ 3139 ] . diabetes and its complications affect the quality of life , and this was captured by applying published event disutilities in the year when the complication occurred and published state utilities in all subsequent years [ 4043 ] . all values have been used in published cost - effectiveness analyses of liraglutide [ 19 , 44 , 45 ] . projection of quality - adjusted life expectancy [ measured in quality - adjusted life years ( qalys ) ] is used widely in cost - effectiveness analyses and is accepted as a useful , relevant metric for decision - makers and payers , although this approach is not without its limitations [ 46 , 47 ] . a number of sensitivity analyses were conducted as part of this study , in line with published recommendations . the time horizon was reduced to 10 and 20 years to assess the impact of varying the time horizon on health economic outcomes . the impact of discounting was evaluated by conducting analyses with discount rates that varied between 0 and 5% . key drivers of clinical outcomes were identified by individually setting differences in clinical parameters ( e.g. hba1c , bmi or hypoglycemic event rates ) between treatment arms to zero . an additional analysis set all clinical parameters equal to their values in the lixisenatide arm , except for the statistically significant difference in hba1c in the liraglutide arm . several analyses were performed to evaluate the impact of alternative treatment - switching options as patients treated with glp-1 receptor agonist therapy will eventually require insulin . first , in both arms treatment was switched to insulin glargine only after 5 years of glp-1 receptor agonist therapy , which approximated the upper observation time limit in the leader trial . third , patients switched to neutral protamine hagedorn ( nph ) insulin instead of insulin glargine after 3 years of glp-1 receptor agonist therapy . over- or underestimation of direct costs of diabetes - related complications might also influence results . therefore , the analysis was conducted with costs increased and decreased by 10% , respectively . a further sensitivity analysis was performed using an updated version of the ims core diabetes model , which was released in 2014 and incorporates data from the ukpds 82 study . the model proprietors recommend that this version is used only for sensitivity analyses and the previous version is used for the base case . probabilistic sensitivity analysis ( psa ) was performed using a second - order monte carlo approach . transition probabilities ( sampled based on regression co - efficients ) , utilities ( beta distributions ) direct costs ( log - normal distributions ) , treatment effects ( beta distributions ) and cohort characteristics ( normal distributions ) were sampled . one thousand cohorts of 1000 patients were simulated in the psa and used to generate cost - effectiveness scatterplots and acceptability curves which were used to analyze the probability that a treatment may be cost - effective over a range of willingness to pay thresholds . no fixed willingness to pay threshold exists in spain but earlier studies have used thresholds of between eur 20,000 and eur 30,000 per qaly gained [ 48 , 49 ] . this article does not contain any new studies of human or animal subjects performed by any of the authors . liraglutide 1.8 mg was associated with improved discounted life expectancy ( by 0.12 years ) and discounted quality - adjusted life expectancy ( by 0.13 qalys ) compared with lixisenatide 20 g in patients with type 2 diabetes failing metformin monotherapy ( table 1 ) . patients receiving liraglutide 1.8 mg benefitted from a reduced cumulative incidence of diabetes - related complications over their lifetimes ( fig . 1 ) . when diabetes - related complications occurred , they occurred , on average , 0.35 years later in patients treated with liraglutide 1.8 mg than in those treated with lixisenatide 20 g . a delayed onset was observed for all complications , most notably for neuropathy which occurred , on average , 0.45 years later in patients treated with liraglutide 1.8 mg.fig . 1comparison of treatment with liraglutide 1.8 mg vs. lixisenatide 20 g in terms of cumulative lifetime incidence of diabetes - related complications . all differences in incidences between liraglutide 1.8 mg and lixisenatide 20 g were statistically significant at the 5% level of significance comparison of treatment with liraglutide 1.8 mg vs. lixisenatide 20 g in terms of cumulative lifetime incidence of diabetes - related complications . all differences in incidences between liraglutide 1.8 mg and lixisenatide 20 g were statistically significant at the 5% level of significance treatment with liraglutide 1.8 mg was associated with higher direct costs over patient lifetimes , with a mean difference of eur 545 per patient ( fig . 2 ) . higher treatment costs over the first 3 years of the analysis were responsible for the higher overall costs of liraglutide 1.8 mg . lower costs of treatment of diabetes - related complications , however , partly offset the higher pharmacy costs . liraglutide 1.8 mg notably reduced costs of treating diabetic foot complications compared with lixisenatide 20 g , with mean cost savings of eur 641 per patient.fig . 2direct costs of treatment with liraglutide 1.8 mg versus lixisenatide 20 g over patient lifetimes . eur 2015 euros direct costs of treatment with liraglutide 1.8 mg versus lixisenatide 20 g over patient lifetimes . eur 2015 euros combining clinical and economic results generated incremental cost - effectiveness ratios ( icers ) of eur 4493 per life - year gained and eur 4113 per qaly gained for liraglutide 1.8 mg versus lixisenatide 20 g ( table 2).table 2long - term cost - effectiveness outcomes of treatment with liraglutide 1.8 mg versus lixisenatide 20 g liraglutide 1.8 mglixisenatide 20 gdifferencediscounted life expectancy ( years)14.42 ( 0.18)14.29 ( 0.19)+0.12discounted quality - adjusted life expectancy ( qalys)9.40 ( 0.12)9.26 ( 0.13)+0.13discounted direct costs ( eur)42,689 ( 1125)42,143 ( 1088)+545icer ( life expectancy)eur 4493 per life year gainedicer ( quality - adjusted life expectancy)eur 4113 per qaly gainedvalues in table are given as the mean with the standard deviation ( sd ) in parenthesis eur 2015 euros , icer incremental cost - effectiveness ratio , qaly quality - adjusted life year long - term cost - effectiveness outcomes of treatment with liraglutide 1.8 mg versus lixisenatide 20 g values in table are given as the mean with the standard deviation ( sd ) in parenthesis eur 2015 euros , icer incremental cost - effectiveness ratio , qaly quality - adjusted life year sensitivity analyses showed that variation in the time horizon , the timing of the switch from glp-1 receptor agonist treatment to insulin , the use of a 7.5% hba1c threshold to trigger the switch to insulin and the difference in hba1c between the treatment arms had the greatest impact on cost - effectiveness outcomes ( table 3).table 3results of sensitivity analysessensitivity analysisdiscounted quality - adjusted life expectancy ( qalys)discounted costs ( eur)icer ( eur per qaly gained)probability ( % ) that liraglutide 1.8 mg considered cost - effective at wtp threshold ( eur per qaly gained)liraglutide 1.8 mglixisenatide 20 gdifferenceliraglutide 1.8 mglixisenatide 20 gdifference20,00030,00050,000base case9.409.260.1342,68942,143545411374.275.578.220-year time horizon8.228.130.0831,95731,533424510467.470.271.810-year time horizon5.485.430.0517,35016,46388717,13052.662.069.00% discount rates13.1812.970.2167,07366,732340163576.577.077.55% discount rates7.777.660.1033,11732,479638622571.373.876.9hba1c difference abolished9.309.260.0443,46742,143132437,28243.149.154.0blood pressure difference abolished9.389.260.1242,76042,143616528369.171.072.1lipid difference abolished9.389.260.1242,72242,143579480372.574.476.1bmi difference abolished9.399.260.1242,62242,143478386273.274.175.2hypoglycemia difference abolished9.399.260.1342,68942,143545412673.975.278.1only statistically significant differences9.369.260.1042,71742,143573600964.266.067.9treatment switching at 5 years9.419.270.1444,41942,943147610,54966.072.075.9treatment switch at 7.5% hba1c threshold9.419.270.1343,58841,752183513,87259.465.970.7switch to neutral protamine hagedorn ( nph ) at 3 years9.409.260.1340,62440,098526396474.175.378.2costs of complications + 10%9.409.260.1345,64945,206443334274.476.278.5costs of complications 10%9.409.260.1339,72739,079648488573.575.477.9ukpds 82 and 68 equations applied9.679.560.1143,33042,695635571268.571.173.5psa9.139.020.1142,06341,170892826465.569.573.1 bmi body mass index , eur 2015 euros , hba1c glycated hemoglobin , icer incremental cost - effectiveness ratio , psa probabilistic sensitivity analysis , qaly quality - adjusted life year , ukpds united kingdom prospective diabetes study , wtp willingness to pay results of sensitivity analyses bmi body mass index , eur 2015 euros , hba1c glycated hemoglobin , icer incremental cost - effectiveness ratio , psa probabilistic sensitivity analysis , qaly quality - adjusted life year , ukpds united kingdom prospective diabetes study , wtp willingness to pay when shorter time horizons were used , icers increased to eur 17,130 and eur 5104 per qaly gained for 10- and 20-year horizons , respectively . shorter time horizons did not capture fully the long - term clinical benefits of liraglutide 1.8 mg , thereby resulting in increased icers . the icer decreased when current and future benefits and costs were discounted at 0% per annum . in contrast , the icer increased when future benefits and costs were discounted more heavily at a discount rate of 5% annually . improved hba1c in the liraglutide 1.8 mg arm compared with the lixisenatide 20 g arm was the main driver of improved clinical outcomes . abolishing the difference in hba1c reduced the difference in quality - adjusted life expectancy to only 0.04 qalys and increased the icer to eur 37,282 per qaly gained . when only the statistically significant difference in hba1c between treatment arms was retained , with all other between - treatment differences set to zero , the icer was eur 6009 per qaly gained . switching to insulin glargine after 5 years increased the icer to eur 10,549 per qaly gained . the icer also increased when treatment was switched to insulin glargine asymmetrically after an hba1c threshold of 7.5% was exceeded . the threshold was exceeded after 4 years of liraglutide 1.8 mg therapy and 2 years of lixisenatide 20 g therapy . when patients switched to nph insulin instead of insulin glargine after 3 years , treatment costs in both arms fell and the icer decreased slightly relative to the base case . increasing the costs of diabetes - related complications by 10% decreased the icer to eur 3342 per qaly gained , as the higher number of complications avoided resulted in larger avoided costs in the liraglutide 1.8 mg arm compared with the lixisenatide 20 g arm . conversely , a 10% decrease in diabetes - related costs increased the icer to eur 4885 per qaly gained . using the alternative ukpds equations decreased the difference in quality - adjusted life expectancy between treatments and reduced the cost offsets of complications avoided with liraglutide 1.8 mg more than those of lixisenatide 20 g , resulting in an icer of eur 5712 per qaly gained . the probabilistic sensitivity analysis indicated a 74.2% probability that liraglutide 1.8 mg would be considered cost - effective versus lixisenatide 20 g at a willingness - to - pay threshold of eur 20,000 per qaly gained . at a willingness - to - pay threshold of eur 30,000 per qaly gained , the probability increased to 75.5% ( fig . probabilities that liraglutide 1.8 mg was considered to be cost - effective were 74.2 , 75.5 and 78.2% at willingness - to - pay thresholds of eur 20,000 , eur 30,000 and eur 50,000 per quality - adjusted life year ( qaly ) gained , respectively . probabilities that liraglutide 1.8 mg was considered to be cost - effective were 74.2 , 75.5 and 78.2% at willingness - to - pay thresholds of eur 20,000 , eur 30,000 and eur 50,000 per quality - adjusted life year ( qaly ) gained , respectively . liraglutide 1.8 mg was associated with improved discounted life expectancy ( by 0.12 years ) and discounted quality - adjusted life expectancy ( by 0.13 qalys ) compared with lixisenatide 20 g in patients with type 2 diabetes failing metformin monotherapy ( table 1 ) . patients receiving liraglutide 1.8 mg benefitted from a reduced cumulative incidence of diabetes - related complications over their lifetimes ( fig . 1 ) . when diabetes - related complications occurred , they occurred , on average , 0.35 years later in patients treated with liraglutide 1.8 mg than in those treated with lixisenatide 20 g . a delayed onset was observed for all complications , most notably for neuropathy which occurred , on average , 0.45 years later in patients treated with liraglutide 1.8 mg.fig . 1comparison of treatment with liraglutide 1.8 mg vs. lixisenatide 20 g in terms of cumulative lifetime incidence of diabetes - related complications . all differences in incidences between liraglutide 1.8 mg and lixisenatide 20 g were statistically significant at the 5% level of significance comparison of treatment with liraglutide 1.8 mg vs. lixisenatide 20 g in terms of cumulative lifetime incidence of diabetes - related complications . all differences in incidences between liraglutide 1.8 mg and lixisenatide 20 g were statistically significant at the 5% level of significance treatment with liraglutide 1.8 mg was associated with higher direct costs over patient lifetimes , with a mean difference of eur 545 per patient ( fig . 2 ) . higher treatment costs over the first 3 years of the analysis were responsible for the higher overall costs of liraglutide 1.8 mg . lower costs of treatment of diabetes - related complications , however , partly offset the higher pharmacy costs . liraglutide 1.8 mg notably reduced costs of treating diabetic foot complications compared with lixisenatide 20 g , with mean cost savings of eur 641 per patient.fig . 2direct costs of treatment with liraglutide 1.8 mg versus lixisenatide 20 g over patient lifetimes . eur 2015 euros direct costs of treatment with liraglutide 1.8 mg versus lixisenatide 20 g over patient lifetimes . eur 2015 euros combining clinical and economic results generated incremental cost - effectiveness ratios ( icers ) of eur 4493 per life - year gained and eur 4113 per qaly gained for liraglutide 1.8 mg versus lixisenatide 20 g ( table 2).table 2long - term cost - effectiveness outcomes of treatment with liraglutide 1.8 mg versus lixisenatide 20 g liraglutide 1.8 mglixisenatide 20 gdifferencediscounted life expectancy ( years)14.42 ( 0.18)14.29 ( 0.19)+0.12discounted quality - adjusted life expectancy ( qalys)9.40 ( 0.12)9.26 ( 0.13)+0.13discounted direct costs ( eur)42,689 ( 1125)42,143 ( 1088)+545icer ( life expectancy)eur 4493 per life year gainedicer ( quality - adjusted life expectancy)eur 4113 per qaly gainedvalues in table are given as the mean with the standard deviation ( sd ) in parenthesis eur 2015 euros , icer incremental cost - effectiveness ratio , qaly quality - adjusted life year long - term cost - effectiveness outcomes of treatment with liraglutide 1.8 mg versus lixisenatide 20 g values in table are given as the mean with the standard deviation ( sd ) in parenthesis eur 2015 euros , icer incremental cost - effectiveness ratio , qaly quality - adjusted life year sensitivity analyses showed that variation in the time horizon , the timing of the switch from glp-1 receptor agonist treatment to insulin , the use of a 7.5% hba1c threshold to trigger the switch to insulin and the difference in hba1c between the treatment arms had the greatest impact on cost - effectiveness outcomes ( table 3).table 3results of sensitivity analysessensitivity analysisdiscounted quality - adjusted life expectancy ( qalys)discounted costs ( eur)icer ( eur per qaly gained)probability ( % ) that liraglutide 1.8 mg considered cost - effective at wtp threshold ( eur per qaly gained)liraglutide 1.8 mglixisenatide 20 gdifferenceliraglutide 1.8 mglixisenatide 20 gdifference20,00030,00050,000base case9.409.260.1342,68942,143545411374.275.578.220-year time horizon8.228.130.0831,95731,533424510467.470.271.810-year time horizon5.485.430.0517,35016,46388717,13052.662.069.00% discount rates13.1812.970.2167,07366,732340163576.577.077.55% discount rates7.777.660.1033,11732,479638622571.373.876.9hba1c difference abolished9.309.260.0443,46742,143132437,28243.149.154.0blood pressure difference abolished9.389.260.1242,76042,143616528369.171.072.1lipid difference abolished9.389.260.1242,72242,143579480372.574.476.1bmi difference abolished9.399.260.1242,62242,143478386273.274.175.2hypoglycemia difference abolished9.399.260.1342,68942,143545412673.975.278.1only statistically significant differences9.369.260.1042,71742,143573600964.266.067.9treatment switching at 5 years9.419.270.1444,41942,943147610,54966.072.075.9treatment switch at 7.5% hba1c threshold9.419.270.1343,58841,752183513,87259.465.970.7switch to neutral protamine hagedorn ( nph ) at 3 years9.409.260.1340,62440,098526396474.175.378.2costs of complications + 10%9.409.260.1345,64945,206443334274.476.278.5costs of complications 10%9.409.260.1339,72739,079648488573.575.477.9ukpds 82 and 68 equations applied9.679.560.1143,33042,695635571268.571.173.5psa9.139.020.1142,06341,170892826465.569.573.1 bmi body mass index , eur 2015 euros , hba1c glycated hemoglobin , icer incremental cost - effectiveness ratio , psa probabilistic sensitivity analysis , qaly quality - adjusted life year , ukpds united kingdom prospective diabetes study , wtp willingness to pay results of sensitivity analyses bmi body mass index , eur 2015 euros , hba1c glycated hemoglobin , icer incremental cost - effectiveness ratio , psa probabilistic sensitivity analysis , qaly quality - adjusted life year , ukpds united kingdom prospective diabetes study , wtp willingness to pay when shorter time horizons were used , icers increased to eur 17,130 and eur 5104 per qaly gained for 10- and 20-year horizons , respectively . shorter time horizons did not capture fully the long - term clinical benefits of liraglutide 1.8 mg , thereby resulting in increased icers . the icer decreased when current and future benefits and costs were discounted at 0% per annum . in contrast , the icer increased when future benefits and costs were discounted more heavily at a discount rate of 5% annually . improved hba1c in the liraglutide 1.8 mg arm compared with the lixisenatide 20 g arm was the main driver of improved clinical outcomes . abolishing the difference in hba1c reduced the difference in quality - adjusted life expectancy to only 0.04 qalys and increased the icer to eur 37,282 per qaly gained . when only the statistically significant difference in hba1c between treatment arms was retained , with all other between - treatment differences set to zero , the icer was eur 6009 per qaly gained . switching to insulin glargine after 5 years increased the icer to eur 10,549 per qaly gained . the icer also increased when treatment was switched to insulin glargine asymmetrically after an hba1c threshold of 7.5% was exceeded . the threshold was exceeded after 4 years of liraglutide 1.8 mg therapy and 2 years of lixisenatide 20 g therapy . when patients switched to nph insulin instead of insulin glargine after 3 years , treatment costs in both arms fell and the icer decreased slightly relative to the base case . increasing the costs of diabetes - related complications by 10% decreased the icer to eur 3342 per qaly gained , as the higher number of complications avoided resulted in larger avoided costs in the liraglutide 1.8 mg arm compared with the lixisenatide 20 g arm . conversely , a 10% decrease in diabetes - related costs increased the icer to eur 4885 per qaly gained . using the alternative ukpds equations decreased the difference in quality - adjusted life expectancy between treatments and reduced the cost offsets of complications avoided with liraglutide 1.8 mg more than those of lixisenatide 20 g , resulting in an icer of eur 5712 per qaly gained . the probabilistic sensitivity analysis indicated a 74.2% probability that liraglutide 1.8 mg would be considered cost - effective versus lixisenatide 20 g at a willingness - to - pay threshold of eur 20,000 per qaly gained . at a willingness - to - pay threshold of eur 30,000 per qaly gained , the probability increased to 75.5% ( fig . probabilities that liraglutide 1.8 mg was considered to be cost - effective were 74.2 , 75.5 and 78.2% at willingness - to - pay thresholds of eur 20,000 , eur 30,000 and eur 50,000 per quality - adjusted life year ( qaly ) gained , respectively . probabilities that liraglutide 1.8 mg was considered to be cost - effective were 74.2 , 75.5 and 78.2% at willingness - to - pay thresholds of eur 20,000 , eur 30,000 and eur 50,000 per quality - adjusted life year ( qaly ) gained , respectively . the present analysis showed that patients with type 2 diabetes failing metformin monotherapy were likely to benefit from improved long - term clinical outcomes when treated with liraglutide 1.8 mg compared with lixisenatide 20 g . improved glycemic control with liraglutide 1.8 mg resulted in fewer diabetes - related complications , and improved life expectancy and quality - adjusted life expectancy . while liraglutide 1.8 mg treatment was associated with increased direct costs compared with lixisenatide 20 g , lower costs of treating complications partly offset the higher acquisition costs . combining clinical and economic outcomes yielded an icer of eur 4113 per qaly gained for liraglutide 1.8 mg versus lixisenatide 20 g . this icer falls well below the willingness - to - pay threshold of eur 20,000 to eur 30,000 per qaly gained that is commonly referenced in the spanish setting . from the perspective of a spanish national health system payer , liraglutide 1.8 mg is likely to be considered a cost - effective add - on therapy to metformin for spanish patients with type 2 diabetes . the improved glycemic control associated with liraglutide 1.8 mg compared with lixisenatide 20 g in the lira - lixi trial was the key driver of the long - term benefit of liraglutide 1.8 mg . sensitivity analyses showed that the icer would increase to eur 37,282 per qaly gained if the hba1c difference between the two treatments were to be abolished . additional sensitivity analyses indicated that results were robust to changes in modeling assumptions and input parameters , with no icer higher than eur 17,130 per qaly gained reported . as the benefits of liraglutide 1.8 mg accrue over patient lifetimes , a long - term perspective was found to be important . mmol / mol ) decrease in hba1c observed with liraglutide 1.8 mg in the lira - lixi trial was higher than the average decrease of 1.15% calculated in a meta - analysis of seven clinical trials from the liraglutide clinical trial program . however , the effect of lixisenatide 20 g on hba1c was also greater than observed previously . the lira - lixi trial identified a reduction in hba1c of 1.21% with lixisenatide 20 g , while the lixisenatide trial program identified reductions of between 0.8 and 0.9% . it is currently unclear why glycemic control improved to a greater extent in both arms of the lira - lixi trial compared with earlier studies . multifactorial treatments of type 2 diabetes target both glycemic control and other risk factors for diabetes - related complications , including hypertension or dyslipidemia . several studies , particularly the steno-2 and addition trials , have compared the effects of multifactorial and conventional treatment approaches on risk factors and rates of diabetes - related complications [ 52 , 53 ] . multifactorial treatment was associated with reduced aortic stiffness over 6.2 years of follow - up , with a decreased risk of modeled cardiovascular disease , decreased risks of all - cause and cardiovascular mortality , autonomic neuropathy , nephropathy and retinopathy , and a median gain of 7.9 life - years over a follow - up of 21.2 years [ 10 , 5456 ] . as glp-1 receptors are present in a number of tissues throughout the body , glp-1 receptor agonists have numerous physiological effects , including inhibited glucagon release , glucose - dependent stimulation of insulin secretion and delayed gastric emptying , which makes them well suited as a multifactorial treatment for diabetes [ 14 , 15 ] . liraglutide was also associated with statistically significant reductions in nephropathy , cardiovascular disease risk and death from any cause compared with placebo treatment in the leader trial . a similar trial that compared lixisenatide 20 g with placebo [ evaluation of lixisenatide in acute coronary syndrome ( elixa ) trial ] did not find statistically significant differences between the treatment and control arms for the primary endpoint of death from cardiovascular causes , nonfatal stroke , nonfatal myocardial infarction or unstable angina over a mean follow - up of 25 months . while further trials on the long - term cardiovascular effects of glp-1 receptor agonists are necessary , the early evidence now available suggests that liraglutide has a protective cardiovascular effect while lixisenatide has a neutral cardiovascular risk profile . the effect of exclusion of this potential benefit in the present analysis was likely to be conservative . results from randomized controlled trials suggest that liraglutide 1.8 mg is more effective in reaching glycemic targets than other glp-1 receptor agonists or dpp-4 inhibitors , including exenatide once weekly , exenatide twice daily or sitagliptin [ 5860 ] . in addition , the clinical effectiveness and cost - effectiveness of both liraglutide 1.2 mg and liraglutide 1.8 mg , compared with sitagliptin , have been demonstrated in spain [ 1820 ] . the present analysis suggests that liraglutide 1.8 mg was also cost - effective compared with lixisenatide 20 g in the spanish setting . these data further suggest that liraglutide 1.8 mg , and glp-1 receptor agonists in general , may well represent clinically and economically valuable treatment options for many patients in spain [ 17 , 61 ] . the present cost - effectiveness study compared two of the treatment options available to spanish patients , based on recently available , high - quality trial data , to provide timely information for patients , physicians and decision - makers in the spanish setting . a full cost - effectiveness analysis comparing all treatment options for patients not achieving glycemic control on metformin monotherapy , which was beyond the scope of this study , the present analysis is possibly limited by the use of short - term clinical trial data to project long - term outcomes , a limitation faced by many cost - effectiveness analyses . using a published and validated diabetes model accepted by reimbursement authorities worldwide likely minimized the effect of using short - term data . however , modeling studies based on short - term data can be a valuable resource to decision - makers in the absence of long - term data . the open - label design of the lira - lixi trial may be a second limitation , which was unavoidable given the different titration protocols of liraglutide 1.8 mg and lixisenatide 20 g . it is conceivable that knowledge about which drug was assigned to a patient affected expectations with regard to medication results , adherence to medication or lifestyle recommendations , as well as reporting or assessment of adverse events . a further limitation may be that the lira - lixi trial did not include participants from spain , possibly limiting the generalizability of trial results to the spanish setting . while this possibility needs to be acknowledged , it was considered likely that the lira - lixi trial results are generalizable to spain and , therefore , that the conclusions of the present analysis are valid . the trial interventions were judged to be as feasible , acceptable and able to achieve broad coverage in spain as they were in the trial countries ( including italy , france and germany ) , thereby fulfilling an important set of generalizability criteria . in addition , in the absence of other efficacy data specific to spain , spanish health economic guidelines recommend the use of trials with high internal validity , to be combined with cost data specific to the spanish setting , as was done in this analysis . if efficacy data for spain become available in the future , the present analysis could be replicated with those new data . this cost - effectiveness analysis showed that the improved clinical outcomes associated with liraglutide 1.8 mg compared with lixisenatide 20 g are likely to result in improved life expectancy and quality - adjusted life expectancy . patients treated with liraglutide 1.8 mg benefitted in particular from reduced levels of hba1c compared with patients treated with lixisenatide 20 g . the clinical benefits associated with liraglutide 1.8 mg came at an increased cost for spanish healthcare payers , as acquisition costs of liraglutide 1.8 mg were higher than for lixisenatide 20 g , although the reduced incidence , and therefore treatment costs , of diabetes - related complications partially offset higher acquisition costs . liraglutide 1.8 mg was associated with an incremental cost - effectiveness ratio of eur 4113 per qaly gained versus lixisenatide 20 g . in the spanish setting , liraglutide 1.8 mg , compared with lixisenatide 20 g , is likely to be considered a cost - effective add - on to metformin in patients with type 2 diabetes who had not achieved glycemic control targets on metformin monotherapy . p. mezquita - raya is a scientific collaborator with novo nordisk and has participated in advisory boards and clinical trials . j. phlmann is an employee of ossian health economics and communications , which received a consulting fee from novo nordisk to support the study . w. valentine is an employee of ossian health economics and communications , which received a consulting fee from novo nordisk to support the study . b. hunt is an employee of ossian health economics and communications , which received a consulting fee from novo nordisk to support the study . this article does not contain any new studies of human or animal subjects performed by any of the authors . the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

non - communicable diseases ( ncd ) killed 38 million people globally in 2012 , accounting for 68% of total annual death ; and cardiovascular diseases alone were responsible for 17.5 million deaths in the year . the global prevalence of high blood pressure in adults is approximately 22% , and high blood pressure is estimated to cause 9.4 million deaths annually . recent studies have indicated that high blood pressure is also becoming an issue for children and adolescents , although it has historically been considered a disease of adults [ 46 ] . obesity is an important modifiable risk factor for prevention of a large number of ncds , including hypertension . globally , 39% of adults older than 18 years in 2014 were overweight , and 13% were obese . in recent decades , the prevalence of childhood obesity has become an important global health concern [ 810 ] . overweight status and low physical activity in adolescents are also associated with metabolic disorders , including insulin resistance and lipid profile alteration . in addition , childhood obesity and hypertension are associated with a number of adult non - communicable diseases and conditions , such as obesity , hypertension , cardiovascular diseases , diabetes , and premature death [ 7,1417 ] . cardiovascular risk factors , including hypertension , are highly prevalent in latin america . in peru , ncds are estimated to account for 66% of total deaths , and cardiovascular diseases alone cause 22% of all deaths . according to national estimates , 15.7% of peruvian adults in 2008 were obese , and 21.0% had high blood pressure . a study in peru found that 8% of boys and 3.3% of girls were obese . however , the burden of obesity and hypertension among peruvian school students is unknown , as only a small number of studies have examined these issues . moreover , schools are considered suitable places for the screening of obesity and hypertension and for promoting a healthy lifestyle to prevent and control obesity and hypertension among children and adolescents . to promote health through schools , the world health organization ( who ) has made recommendations to schools , including education regarding critical health and life skills . thus , the objective of this study was to determine the prevalence of overweight , obesity , and hypertension and to examine the relationship between obesity and hypertension among peruvian school - going adolescents . in addition , this study aims to assess the need for health - promoting school programs in the study area . a cross - sectional school - based survey was conducted among school - going adolescents . the field study was conducted from september 15 to october 31 , 2014 , in collaboration with korea international cooperation agency ( koica ) , peru office . the survey was conducted in one district of lima ( comas ) and two districts of callao ( bellavista and ventanilla ) . three areas from comas ( santa luzmila ii , laura rodriguez dulanto , and carlos philips ) , one area from bellavista ( bellavista ) , and two areas from ventanilla ( pachacutec and mi peru ) were selected for the survey . of 17 secondary schools in the study areas , 11 were randomly selected for the survey . total calculated sample size was 975 using the following formula : m = [ z v m]/[d ( m 1 ) + z v ] ( def ) ( tnr ) , where m is students to be estimated , z is value of the normal distribution ( z = 1.96 ) , p is prevalence is of smoking or alcohol among school students . ( p = 0.23 ) , v = p q ( q = 1 p = 0.77 ) , m is total number of students in the study area ( m = 14,787 ) , d is margin of error ( d = 0.0307 ) , def is clustering effect of the distribution of estimates ( def = 1.2 ) , and tnr is an adjustment factor due to non - response ( tnr = 1.18 ) . a total of 981 students were randomly selected and invited to participate in the study . of those invited to participate , 5 did not complete the questionnaire items about socio - demographics or were absent on the day of information collection . during analysis trained enumerators provided questionnaires to the randomly selected students in their classrooms during regular school hours . eight registered nurses were assigned to measure blood pressure ( bp ) , and 8 nutritionists collected the anthropometrics of all randomly selected students . weight and height were measured with a digital scale and extensometer , respectively . bp was measured in all subjects with a validated automatic blood pressure monitor ( omron hem-7113 , omron corporation , kyoto - shi , japan ) . for analysis , measured bp was categorized according to the standard for age , sex , and height . prehypertension was defined as systolic blood pressure ( sbp ) and/or diastolic blood pressure ( dbp ) greater than or equal to the 90 percentile but less than the 95 percentile ; hypertension was defined as sbp and/or dbp greater than or equal to the 95th percentile of bp for age , sex , and height . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . bmi was classified based on its distribution by sex and age according to the growth chart developed by the center for disease control and prevention into underweight ( less than the 5 percentile ) , normal ( greater than or equal to the 5 percentile and less than the 85 percentile ) , overweight ( greater than or equal to the 85 percentile and less than the 95 percentile ) , and obese ( the 95 percentile ) . ethical approval for this study was obtained from the institutional review board of wonju campus , yonsei university ( 1041849 - 201410-bm-048 - 02 ) and the diresa callao ( local government of peru ) . the chi - square test was conducted to examine the association between bmi and bp category . to determine the relationship between bmi and bp level , bp was categorized into two groups , normal and elevated ( those with prehypertension or hypertension ) . odds ratios were calculated to examine the relationship between elevated bp and bmi categories using univariate logistic regression analysis . bmi status was also categorized into two groups , normal bmi and elevated bmi ( overweight and obese students ) . prevalence of elevated bp and elevated bmi was calculated by sex , age group , and area , and associations were observed according to p - values at a 95% confidence interval ( ci ) . a cross - sectional school - based survey was conducted among school - going adolescents . the field study was conducted from september 15 to october 31 , 2014 , in collaboration with korea international cooperation agency ( koica ) , peru office . the survey was conducted in one district of lima ( comas ) and two districts of callao ( bellavista and ventanilla ) . three areas from comas ( santa luzmila ii , laura rodriguez dulanto , and carlos philips ) , one area from bellavista ( bellavista ) , and two areas from ventanilla ( pachacutec and mi peru ) were selected for the survey . of 17 secondary schools in the study areas , 11 were randomly selected for the survey . total calculated sample size was 975 using the following formula : m = [ z v m]/[d ( m 1 ) + z v ] ( def ) ( tnr ) , where m is students to be estimated , z is value of the normal distribution ( z = 1.96 ) , p is prevalence is of smoking or alcohol among school students . ( p = 0.23 ) , v = p q ( q = 1 p = 0.77 ) , m is total number of students in the study area ( m = 14,787 ) , d is margin of error ( d = 0.0307 ) , def is clustering effect of the distribution of estimates ( def = 1.2 ) , and tnr is an adjustment factor due to non - response ( tnr = 1.18 ) . a total of 981 students were randomly selected and invited to participate in the study . of those invited to participate , 5 did not complete the questionnaire items about socio - demographics or were absent on the day of information collection . during analysis socio - demographic characteristics were assessed using an anonymous self - administered questionnaire . trained enumerators provided questionnaires to the randomly selected students in their classrooms during regular school hours . eight registered nurses were assigned to measure blood pressure ( bp ) , and 8 nutritionists collected the anthropometrics of all randomly selected students . bp was measured in all subjects with a validated automatic blood pressure monitor ( omron hem-7113 , omron corporation , kyoto - shi , japan ) . for analysis , measured bp was categorized according to the standard for age , sex , and height . prehypertension was defined as systolic blood pressure ( sbp ) and/or diastolic blood pressure ( dbp ) greater than or equal to the 90 percentile but less than the 95 percentile ; hypertension was defined as sbp and/or dbp greater than or equal to the 95th percentile of bp for age , sex , and height . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . bmi was classified based on its distribution by sex and age according to the growth chart developed by the center for disease control and prevention into underweight ( less than the 5 percentile ) , normal ( greater than or equal to the 5 percentile and less than the 85 percentile ) , overweight ( greater than or equal to the 85 percentile and less than the 95 percentile ) , and obese ( the 95 percentile ) . ethical approval for this study was obtained from the institutional review board of wonju campus , yonsei university ( 1041849 - 201410-bm-048 - 02 ) and the diresa callao ( local government of peru ) . the chi - square test was conducted to examine the association between bmi and bp category . to determine the relationship between bmi and bp level , bp was categorized into two groups , normal and elevated ( those with prehypertension or hypertension ) . odds ratios were calculated to examine the relationship between elevated bp and bmi categories using univariate logistic regression analysis . bmi status was also categorized into two groups , normal bmi and elevated bmi ( overweight and obese students ) . prevalence of elevated bp and elevated bmi was calculated by sex , age group , and area , and associations were observed according to p - values at a 95% confidence interval ( ci ) . table 1 summarizes anthropometric outcomes and distribution of blood pressure and bmi status among survey respondents by sex . of the 952 subjects included in this study , 46.4% were boys and 53.6% were girls . participant age range was 1218 years ( mean sd , 14.6 1.6 ) . the mean weight in kg ( 56.53 11.31 vs. 52.60 9.55 ) , height in cm ( 160.57 8.18 vs. 153.34 5.87 ) , and sbp in mmhg ( 122.07 13.72 vs. 113.22 12.40 ) were higher in boys than in girls . the prevalence of overweight and obesity was 17.4% and 11.1% among boys and 22.5% and 8.0% among girls , respectively . table 2 shows the distribution and associations of blood pressure by sex and bmi status . the prevalence of hypertension in boys and girls was 29.5% and 16.1% for normal weight subjects , 45.5% and 25.2% for overweight subjects , and 55.1% and 36.6% for obese subjects , respectively . the relationship between bmi and bp was statistically significant in both boys and girls ( p < 0.01 ) . table 3 shows the prevalence of elevated bp by sex , age , bmi status , and study area . boys were more likely to have elevated blood pressure than girls [ ( 44.6% vs. 28.6% ) ; or , 2.00 ; ci 1.532.62 ] . overweight and obese students had a greater chance of having elevated bp [ ( or , 1.69 ; ci , 1.222.36 for overweight subjects ) and ( or , 2.98 ; ci , 1.904.68 for obese subjects ) ] . the prevalence of elevated bp was lowest in santa luzmilla ii ( 15.3% ) and highest in mi per ( 54.5% ) . table 4 shows the prevalence of elevated bmi by sex , age group , and study area . the prevalence of elevated bmi was slightly lower among boys ( 29% vs. 31% ) and was highest among participants aged 1213 years ( 39.7% ) and lowest among those aged 1618 years ( 24.1% ) . the prevalence of elevated bmi was highest in the bellavista area ( 35.8% ) and lowest in the pachacutec area ( 23.9% ) . table 1 summarizes anthropometric outcomes and distribution of blood pressure and bmi status among survey respondents by sex . of the 952 subjects included in this study , 46.4% were boys and 53.6% were girls . participant age range was 1218 years ( mean sd , 14.6 1.6 ) . the mean weight in kg ( 56.53 11.31 vs. 52.60 9.55 ) , height in cm ( 160.57 8.18 vs. 153.34 5.87 ) , and sbp in mmhg ( 122.07 13.72 vs. 113.22 12.40 ) were higher in boys than in girls . the prevalence of overweight and obesity was 17.4% and 11.1% among boys and 22.5% and 8.0% among girls , respectively . table 2 shows the distribution and associations of blood pressure by sex and bmi status . the prevalence of hypertension in boys and girls was 29.5% and 16.1% for normal weight subjects , 45.5% and 25.2% for overweight subjects , and 55.1% and 36.6% for obese subjects , respectively . the relationship between bmi and bp was statistically significant in both boys and girls ( p < 0.01 ) . table 3 shows the prevalence of elevated bp by sex , age , bmi status , and study area . boys were more likely to have elevated blood pressure than girls [ ( 44.6% vs. 28.6% ) ; or , 2.00 ; ci 1.532.62 ] . overweight and obese students had a greater chance of having elevated bp [ ( or , 1.69 ; ci , 1.222.36 for overweight subjects ) and ( or , 2.98 ; ci , 1.904.68 for obese subjects ) ] . the prevalence of elevated bp was lowest in santa luzmilla ii ( 15.3% ) and highest in mi per ( 54.5% ) . table 4 shows the prevalence of elevated bmi by sex , age group , and study area . the prevalence of elevated bmi was slightly lower among boys ( 29% vs. 31% ) and was highest among participants aged 1213 years ( 39.7% ) and lowest among those aged 1618 years ( 24.1% ) . the prevalence of elevated bmi was highest in the bellavista area ( 35.8% ) and lowest in the pachacutec area ( 23.9% ) . the present study found a higher prevalence of obesity ( 9.5% ) relative to the results of previous studies focused on different locations , such as south east nigeria ( 1.9% ) , portugal ( 5.3% ) , south iran ( 7% ) , india ( 4.8% ) , and china ( 4.1% and 8.7% ) . however , a similar prevalence of obesity was found in nigeria ( 9.4% ) , and a higher prevalence was observed in nepal ( 11.3% ) . in contrast , a study conducted in south china revealed that more than one - third of students were underweight . similar to our study , a higher prevalence of obesity was found among boys . the current study observed a higher prevalence of hypertension , 26.7% of the study population , which is an alarming situation , particularly when compared with the prevalence reported by previous studies in other locations . the rate of adolescent hypertension was nearly equal to the national estimate of adult hypertension rates in peru and far higher than the rate observed in a study of peruvian adolescents . in contrast to the present study , a prevalence ranging from 5.2% to 17.2% has been reported in other studies : 5.4% in southeast nigeria , 8.12% in brazil , 9.8% in portugal , 11.8% in southern iran , 12% in nepal , and 17.2% in israel . in the present study , in contrast , other studies have reported a higher prevalence of hypertension among girls relative to boys . in the present study , bmi level was significantly associated with hypertension in both boys and girls . overweight students were 1.6 times more likely and obese students were 2.9 times more likely to have elevated blood pressure . evidence of obesity as a risk factor of adolescent hypertension has been documented by numerous studies conducted in varied geographical and socioeconomic contexts [ 21,27,31,34,3641 ] . a similar prevalence of elevated bmi was observed in six different study areas , with the highest prevalence in the bellavista area . unlike elevated bmi , elevated bp varied greatly among the study areas , with the highest in the mi per area . the area - wide variation in prevalence of hypertension requires further study in order to determine the specific risk factors present in these areas . obesity and hypertension monitoring and other specific measures are needed to prevent and control disease among children and adolescents . however , in a comprehensive model , screening programs alone are not sufficient to prevent health problems ; many intervention studies of childhood obesity prevention have stressed the importance of holistic implementation of school health programs [ 22 , 4346 ] . the world health organization ( who ) , in its action plan for the prevention and control of non - communicable diseases , has recognized schools as an important sector for the reduction of modifiable and preventable risk factors of non - communicable diseases . physical inactivity , harmful use of alcohol , tobacco consumption , and unhealthy diet are preventable and modifiable risk factors of non - communicable diseases . school - based interventions such as nutrition , physical and lifestyle education , and physical activity promotion has the greatest impact on the prevention and reduction of childhood obesity - related problems . as childhood obesity is the result of multiple factors , including socio - economic , cultural , and environmental factors , multi - level approaches that address individual factors , community characteristics , and governmental influences school - based interventions that combine behavioral and environmental approaches to dietary intake and physical activity , as well as involvement of family and community , are necessary to promote and sustain healthy lifestyles . in addition , policy interventions are recommended to support healthy lifestyles for children and families [ 4548 ] . this study found relatively high prevalence of overweight , obesity , and hypertension among school - going adolescents in peru . obesity prevention activities may help to reduce the burden of hypertension and other related disorders . it is essential to have health - promoting school programs to address the issues of obesity and hypertension among school - going adolescents in peru . school administrations should consider the great burden of these problems and initiate coordinated efforts in all concerned sectors to conduct regular screening programs and other health - promoting school activities . in an effort to reduce the burdens of obesity and hypertension among children and adolescents , as well as adults , prevention and control activities schools , as principal institutions for the socialization of children , are ideal settings for these programs not only because children spend most of their time in school , but also because they are in a unique position to promote healthy lifestyles and prevent lifestyle - related problems . moreover , the ministry of health , the ministry of education , and other national and international organizations should initiate and support school development into health - promoting settings . a variety of evidence - based measures to reduce obesity and related problems among students can be successfully implemented in school settings . based on these findings , the koica and yonsei global health centre have developed health - promoting school programs for three years in the study areas . these programs include health screening and counseling on risk factors , nutrition education , physical education , and provision of mini gym facilities in schools to address these problems and promote better health among students and staff .

serum calcium concentrations are maintained in the normal range until very late in chronic kidney disease ( ckd ) when it decreases slightly ( 1,2 ) . gastrointestinal ( gi ) calcium absorption has been shown to be lower in ckd subjects on normal calcium intake than in controls yet remains dependent on calcium intake ( 3 ) . it is generally believed that decreased levels of circulating 1,25 dihydroxy - vitamin d ( 1,25-d ) , the principal hormone regulating transcellular calcium absorption in the gastrointestinal tract ( gi)(4 ) , results in decreased calcium absorption and net negative calcium balance , providing a stimulus for secondary hyperparathyroidism early in ckd . late in ckd serum calcium concentrations decrease , as circulating ( 1,25-d ) continues to decrease , lending support to this hypothesis . the decreasing 1,25-d concentrations coupled with a decrease in serum calcium concentrations late in ckd suggest that ckd patients may be in net negative calcium balance and that calcium supplementation may be needed to correct the calcium absorption defect . to date , there are no adequate calcium balance studies in ckd . in normal adults , calcium balance is relatively neutral when calcium intake ranges between 750 mg to 1740 mg per day , although significant individual variability exists ( 5 ) . twenty - four hour urinary calcium excretion generally ranges between 100 to 300 mg per day largely reflecting dietary intake with a fractional gi absorption between 10 - 60% depending on 1,25-d concentrations and calcium intake ( 6,7 ) . however , studies that have measured 24 hour urinary calcium excretion in patients with advanced ckd consistently show low 24 hour excretion , usually less than 80 mg / day ( 8,9 ) . even ckd patients on escalating doses of calcium based phosphate binders consistently had low urinary calcium excretion often in the range of 20 - 60 mg / day ( 10 ) . understanding calcium balance is particularly important in ckd as it is characterized by a high prevalence of vascular calcification , which is associated with cardiovascular and all - cause mortality ( 11 ) . while vascular calcification is thought to result from a complex disruption of normal vascular biology ( 12 ) , there is ongoing controversy whether supplemental calcium accelerates this process both in ckd and in dialysis patients ( 13 ) . furthermore , the increasing concern over phosphorus balance early in ckd and the observational finding that ckd non - dialysis patients treated with phosphate binder have improved survival ( 14 ) may increase the use of calcium containing phosphate binders in ckd despite the lack of long term safety data . therefore , the purpose of this study was to formally evaluate calcium balance in late stage 3 and stage 4 ckd compared to control subjects . we hypothesized that subjects with advanced ckd are in positive calcium balance on high dietary calcium intake and that this positive balance is not reflected in the serum calcium concentration which is tightly regulated . if true this hypothesis would suggest that exposure to high calcium intake results in positive calcium balance in ckd leading to calcium loading which may precipitate or worsen vascular and soft tissue calcification . six subjects with advanced stage 3 or stage 4 ckd and 6 control subjects completed both diet arms . one control subject was dropped from the study due to inability to eat the prescribed diet during the first diet period and was not included in the data analysis . two other control subjects were consented but failed vitamin d screening or withdrew consent and never started the active diet phase . one ckd subject had an inter - diet collection period that exceeded the study protocol by several months due to an inter - current illness . all subjects did a good job with the diets eating only , and most of the food prepared . the average calcium and phosphorus intake for subjects during their 48-hour in - patient diet periods were as follows : ckd- low calcium diet : ca 8124 , phos 157559 ; high calcium diet : ca 20099 , phos 167262 ; control - low calcium diet : ca 78742 , phos 1518136 ; high calcium diet : ca 1903160 , phos 1545125 mg / day . serum calcium concentrations were not different between ckd and control subjects on either diet and there was no intra - patient difference between the 800 and 2000 mg calcium diets ( figure 2a ) . serum phosphorus was significantly higher in ckd subjects on the 800 mg ( 4.150.20 vs. 3.560.09 mg / dl , p=0.02 ) and trended higher on the 2000 mg calcium diet ( 4.060.20 vs. 3.630.11 mg / dl , p=0.10 ) , but again , did not show an intra - patient difference between the low and high calcium diets ( figure 2b ) . however , in response to the increase calcium in the diet , both the serum 1,25-d and the ipth decreased significantly when subjects were on the 2000 mg calcium diet compared with the 800 mg calcium diet ( table 1 , figure 3 ) . 25-d levels were lower in ckd subjects than controls ( 33.41.9 vs. 43.21.8 ng / ml ; p=0.001 ) but did not change between diets in either group . total urinary phosphorus excretion was not different between ckd and controls and did not vary by calcium diet ( table 2 , figure 4a ) . however , the tubular reabsorption of phosphorus was significantly lower in ckd than controls ( 62.412.8 vs. 85.42.2 ; p=0.002 ) and fit closely with the logarithmic exponent as previously reported ( 15)(figure 4b ) . while some patients demonstrated an increase in urinary calcium on the higher calcium intake ( table 2 ) , the intra - patient urinary calcium excretion was not different for ckd or control groups . however , the urinary calcium excretion was significantly lower in ckd subjects compared to controls on both the 800 ( 83.047.6 vs. 272.1103.6 ; p<0.05 ) and 2000 mg ( 99.071.7 vs. 333.9166.3 ; p<0.05 ) calcium diets ( table 2 , figure 5a ) . the stool calcium / phosphorus ratio was slightly , although not significantly lower in controls vs. ckd subjects on the 800 mg calcium diet possibly suggesting greater calcium absorption in controls consistent with the higher 1,25-d concentrations ( figure 5b ) . on the 2000 mg calcium diet both control and ckd subjects demonstrated an increase in stool calcium / phosphorus compared with the 800 mg calcium diet as expected , although this was slightly greater and only statistically significant in the control group . there was no difference in the stool calcium phosphorus ratio between groups on either diet ( figure 5b ) . the fact that ckd subjects did not increase their stool calcium / phosphorus ratio more than controls , strongly suggests that marked down regulation or impairment of gi calcium absorption is not occurring in ckd and can not account for the low urinary calcium excretion in this group . the estimated percent of ingested dietary calcium absorbed increased from 521% to 4710% in ckd subjects and from 1626% to 4111% in controls on the 800 and 2000 mg calcium diets respectively . estimated calcium balance was slightly negative in ckd subjects on the 800 mg calcium intake ( although the 95% ci crossed the neutral line ) , but markedly positive on the 2000 mg calcium intake ( table 2 , figure 6 ) . control subjects were also in slightly negative calcium balance on the 800 mg calcium diet , but again this was not statistically significant . control subjects were also in positive calcium balance on the 2000 mg calcium diets , although significantly less positive than ckd subjects . we found that subjects with late stage 3 and stage 4 ckd were in slightly negative to neutral calcium balance on a daily intake of 800 mg , but were in marked positive balance on 2000 mg per day . this finding is in agreement with modeled balance data in dialysis patients predicting negative balance when intake is less than 800 and positive balance when calcium intake exceeds 1500 mg / day ( 16 ) . our finding of low urinary calcium excretion in ckd is in agreement with previously reported 24-hour urine calcium excretion in subjects with ckd ( 8,9 ) . the finding that urinary calcium did not increase in ckd subjects on the high calcium diet while the stool calcium to phosphorus ratio did not differ from controls supports our finding of marked positive calcium balance in the ckd subjects on the high calcium diet and suggests that significant down regulation or impairment of gi calcium absorption is not occurring in ckd . together , these findings demonstrate that ckd patients receiving 2000 mg of elemental calcium have a substantial increase in calcium absorption resulting in positive balance without escapeover the time period studied . the finding that 1,25-d and ipth concentrations decreased on the high calcium diet supports the finding that increasing calcium absorption is down regulating these hormones . however despite the decrease in 1,25-d , net calcium absorption increased on the high calcium diet possibly related to absorption via the less regulated , non - saturable paracellular pathway ( 4).the finding that the dietary calcium absorption was slightly lower in ckd subjects on the low calcium diet than controls is consistent with the lower 1,25-d levels , the principal hormone regulating transcellular calcium absorption in the gi tract when dietary calcium is low . on the high calcium intake , the percent calcium absorption in this study was quite variable between patients but consistent with early studies reporting absorption between 26 - 63% on 800 mg / day and between 18 - 30% on 2000 mg / day ( 17 ) . control subjects were in slightly negative to neutral calcium balance on the low calcium diet and in positive balance on the 2000 mg per day calcium diet , although not as positive as ckd subjects . the observation that the serum calcium concentrations did not change in either group is consistent with the knowledge that the serum calcium concentration is tightly regulated ( 17 ) and suggests that calcium loading occurs when ckd and possibly control subjects are exposed to high calcium intake . this is consistent with studies in pediatric patients with ckd showing vascular calcium loading in children with advanced ckd ( 18 ) . however , the study was tightly controlled and the findings are consistent across subjects . due to inaccuracies in the stool dye technique while a stable calcium isotope could be used to measure gi calcium absorption the method is expensive . we therefore chose to estimate the 24 hour stool production by assuming that patients were in neutral phosphorus balance , and normalizing the stool calcium to 24-hours based on these calculations . while this was an assumption we could not test furthermore , if incorrect and ckd subjects were in positive rather than neutral phosphorus balance this would only serve to increase their net calcium balance . if ckd subjects were in negative phosphorus balance ( due to continued urinary losses in the face of decreased absorption from calcium binding ) then our results would overestimate net calcium balance . however , even a 20 or 40% overestimation would still place the ckd subjects in net positive calcium balance . it is possible that both control and ckd subjects may have been accruing bone mass when on the high calcium diet . it is also possibly that the 7 days of diet equilibration was not long enough for subjects to return to a steady state , especially on the high calcium diet , and given more time control subjects may have been able to increase their urinary calcium excretion to achieve balance . there is currently very little literature to define a time course for return to steady state following changes in dietary calcium . however , it has been shown that following a decrease in dietary calcium intake from 2000 mg to 300 mg / day , urinary calcium nadirs by week one and the counter - regulatory hormones , pth and 1,25-d reach a maximum at 1 week ( 20 ) . in general , renal conservation of a filtered substance takes longer than decreased reabsorption suggesting that 7 days may be adequate , although we can not be certain that our control or ckd subjects were in a true steady - state . it is also possible that ckd subjects may require a longer time period than controls to reestablish balance . however , a long term study ( 6 weeks ) following urinary calcium excretion in ckd subjects treated with increasing dosing of calcium based binders failed to show a significant increase in urinary calcium excretion , suggesting that even after 6 weeks , ckd subjects fail to increase their urinary calcium excretion in response to increased calcium intake ( 10 ) . finally , although this study strongly suggests that ckd subjects on a high calcium diet are in positive calcium balance we can not be certain that this calcium is being deposited in soft tissue . however , it is unlikely that the bones could continually accumulate calcium in ckd subjects if positive calcium balance were maintained over weeks to months as might occur with long term high calcium intake . in summary , this study found that subjects with advance stage 3 or stage 4 ckd are in slightly negative to neutral calcium balance on 800 mg calcium intake but in marked positive balance when ingesting 2000 mg of elemental calcium . the increased calcium load when moving from an 800 to a 2000 mg calcium intake resulted in lower 1,25-vitamin d and ipth but did not result in overall net decreased calcium absorption or an increase in the serum calcium concentration suggesting that the calcium is being deposited in tissue . this finding is consistent with what is known regarding sodium and water balance in ckd where flexibility in urinary excretion is lost and dietary intake needs to be adjusted for patients to remain in balance . if confirmed in larger balance studies these findings suggest that elemental calcium intake should be limited to 800 - 1200 mg in individuals with late stage 3 or stage 4 ckd not on active vitamin d analogues and possibly to 800 to 1000 mg in subjects receiving active vitamin d ( although this was not studied in this trial ) . finally , this study underscores the fact that the serum calcium can not be used as a guide to evaluate calcium balance and normal serum calcium concentrations do not preclude calcium loading . this study suggests that knowledge of dietary calcium intake and the use of active vitamin d need to be considered when calcium is prescribed to patients with ckd . furthermore in adults with ckd , total elemental calcium intake should be within 800 - 1200 mg / day to prevent calcium deficiency and calcium loading . these values are close to the current estimated average requirement ( 800 - 1000 mg / day ) and the recommended dietary allowance 1000 - 1200 mg / day ( rda amount needed to meet or exceed the requirement of 97.5% of healthy adults ) in the recent institute of medicine report on dietary reference intakes for calcium ( 21 ) . this study used a cross - over designed to evaluate calcium balance on an 800 mg elemental calcium per day diet versus a 2000 mg per day diet in late stage 3 and stage 4 ckd subjects versus controls with normal kidney function . if < 30 ng / ml subjects were supplemented with vitamin d ( ergocalciferol 50,000 iu weekly ) and rescreened at 4 weeks . if the 25-d was still < 30 ng / ml subjects could undergo a second supplementation period followed by rescreening . no subjects were receiving any bone modifying medications and all subjects were instructed to discontinue all calcium supplements and any vitamins or antacids containing calcium at least 1 month prior to study entry . subject who passed the 25-d screening were assigned to the calcium diets in random order . diet 1 : 800 mg elemental calcium and 1600 mg phosphorus ; diet 2 : 2000 mg elemental calcium and 1600 mg phosphorus . each diet phase was a total of 9 days , the first 7 as an out - patient . for each active diet phase , subjects were supplied with all out - patient meals and snacks prepared by the clinical trials research center ( ctrc ) dietary facilities at the university of colorado . subjects were instructed to eat only food and non - water drinks provided but could drink water ad lib . subjects were instructed to return any uneaten food . following this outpatient phase , subjects were admitted to the ctrc for 48 hours . the experimental diet was continued during this stay and subjects were given de - ionized water ad lib . during the in - patient stay each subject had two 24-hour urine collections for calcium , phosphorus , and creatinine and fasting morning labs for creatinine , calcium , phosphorus , 25-d , 1,25-d , and ipth . during this time period all stool usa ) for measurement of calcium and phosphorus content for determination of 24-hour stool calcium excretion ( see below ) . any patient with only 1 stool during the 48 hour stay was asked to collect the next sample at home in an iced container . all but 1 control subject had at least 2 stool samples used in the analysis . following the first in - patient stay , subjects resumed their normal diet for a period of at least 1 week but up to 4 weeks before initiating the second diet phase . during the entire study period subjects continued to receive vitamin d supplementation , either their routine home vitamin or ergocalciferol 50,000 iu every month . serum and urine calcium , phosphorus , and urine creatinine were measured in the university of colorado clinical laboratory . serum creatinine , 25-d , 1,25-d , and ipth were measured in the denver children s hospital clinical laboratory . the tubular reabsorption of phosphorus ( trphos ) was calculated from the 24-hour urine collections and the fasting phosphorus by the equation : 1-[(uphos / serum phos ) x ( serum creat / ucreat ) x 100 ] . all stool collected for each subject was combined , weighed , dried , precharred , and ashed overnight in a muffle set to maintain 500c . the ashed sample was re - ashed with nitric acid , treated with hydrochloric acid , taken to dryness , and put into a solution of 5% hydrochloric acid . the amount of calcium and phosphorus was determined at appropriate wavelengths by comparing the emission of the unknown sample , measured on the inductively coupled plasma spectrometer , with the emission of the standard solutions . the stool calcium / phosphorus ratio was calculated as the total stool calcium divided by the total stool phosphorus . the initial plan for the calculation of the 24 hour stool calcium excretion was to perform a double labeled stool collection by administration of brilliant blue ( 100 mg ) 24 hours prior to ctrc admission and at the midpoint of the hospital stay . stool collected between the two blue - stained stool samples was to be collected and analyzed and would represent 48 hours of stool . however , due to marked inaccuracy and variation with gi transit time , dye streaking , and sometimes no clear dye free stool this method it was not utilized for the study . this assumption is in agreement with published findings ( 15,19 ) and supported by the finding that all subjects ( both ckd and control ) excreted equal amounts of phosphorus in the urine during both diet phases . although dietary calcium and to a lesser extent 1,25-d concentrations are known to exert modulating effects on gi phosphate absorption , based on the fixed and equal phosphorus content of both diets and equal urinary phosphorus excretion we felt the assumption that all patients were in phosphorus balance was reasonable . therefore , the phosphorus intake minus the urinary phosphorus excretion allowed for a close estimate of the expected 24-hour stool phosphorus . the total stool phosphorus content was then divided by the expected 24 hour stool phosphorus to determine the time period of the stool analyzed . the total stool calcium was then divided by this time factor to determine the 24-hour stool calcium content for each subject for each diet phase . for example : the expected 24 hour stool phosphorus for a subject with 900 mg of urinary phosphorus would be calculated as intake ( 1600 mg ) minus the 24 hour urine phosphorus ( 900 mg ) = 700 mg . the actual total stool phosphorus content measured ( ex : 1100 mg ) was divided by 700 mg to determine the number of days the total collected stool represented . in this example ; 1100/700 = 1.57 days . the total measured stool calcium was then divided by this number to estimate the 24 hour stool calcium excretion . although calcium is known to be secreted into the gi tract and small amounts are lost in sweat ( 22 ) this study used the simplified definition of balance as previous reported ( 5 ) as balance = oral intake - urine output - stool output . as the serum calcium concentrations did not change significantly on either diet it was not felt that the small changes observed needed to be included into the balance equation . differences between groups were analyzed by the non - paired t test for two groups or 1-way anova with tukey post - test for multiple comparisons . this study was conducted in accordance with the ethical standards of the responsible committee on human experimentation and was approved by the institutional review boards of the university of colorado # 070959 . all stool collected for each subject was combined , weighed , dried , precharred , and ashed overnight in a muffle set to maintain 500c . the ashed sample was re - ashed with nitric acid , treated with hydrochloric acid , taken to dryness , and put into a solution of 5% hydrochloric acid . the amount of calcium and phosphorus was determined at appropriate wavelengths by comparing the emission of the unknown sample , measured on the inductively coupled plasma spectrometer , with the emission of the standard solutions . the stool calcium / phosphorus ratio was calculated as the total stool calcium divided by the total stool phosphorus . the initial plan for the calculation of the 24 hour stool calcium excretion was to perform a double labeled stool collection by administration of brilliant blue ( 100 mg ) 24 hours prior to ctrc admission and at the midpoint of the hospital stay . stool collected between the two blue - stained stool samples was to be collected and analyzed and would represent 48 hours of stool . however , due to marked inaccuracy and variation with gi transit time , dye streaking , and sometimes no clear dye free stool this method it was not utilized for the study . instead 24 hour stool excretion was estimated as follows . this assumption is in agreement with published findings ( 15,19 ) and supported by the finding that all subjects ( both ckd and control ) excreted equal amounts of phosphorus in the urine during both diet phases . although dietary calcium and to a lesser extent 1,25-d concentrations are known to exert modulating effects on gi phosphate absorption , based on the fixed and equal phosphorus content of both diets and equal urinary phosphorus excretion we felt the assumption that all patients were in phosphorus balance was reasonable . therefore , the phosphorus intake minus the urinary phosphorus excretion allowed for a close estimate of the expected 24-hour stool phosphorus . the total stool phosphorus content was then divided by the expected 24 hour stool phosphorus to determine the time period of the stool analyzed . the total stool calcium was then divided by this time factor to determine the 24-hour stool calcium content for each subject for each diet phase . for example : the expected 24 hour stool phosphorus for a subject with 900 mg of urinary phosphorus would be calculated as intake ( 1600 mg ) minus the 24 hour urine phosphorus ( 900 mg ) = 700 mg . the actual total stool phosphorus content measured ( ex : 1100 mg ) was divided by 700 mg to determine the number of days the total collected stool represented . the total measured stool calcium was then divided by this number to estimate the 24 hour stool calcium excretion . although calcium is known to be secreted into the gi tract and small amounts are lost in sweat ( 22 ) this study used the simplified definition of balance as previous reported ( 5 ) as balance = oral intake - urine output - stool output . as the serum calcium concentrations did not change significantly on either diet it was not felt that the small changes observed needed to be included into the balance equation . differences between groups were analyzed by the non - paired t test for two groups or 1-way anova with tukey post - test for multiple comparisons . this study was conducted in accordance with the ethical standards of the responsible committee on human experimentation and was approved by the institutional review boards of the university of colorado # 070959 .

it poses an important public health challenge to both economically developing and developed countries , including asia . the prevalence and rate of diagnosis of hypertension in children and adolescents appears to be increasing . hypertension confers the highest attributable risk to deaths from cardiovascular disease and epidemiological data provide convincing evidence that the risk of cardiovascular disease related to blood pressure is graded and continuous . this risk is evident even in childhood ; with elevated blood pressure predicting hypertension in adulthood , and adverse effects of elevated blood pressure in childhood on vascular structure and function , specifically left ventricular hypertrophy , are already apparent in youth.[810 ] reduction of blood pressure reduces this risk in people with and without hypertension and is a desired goal in children and adults . even as most studies describe knowledge of hypertension and its risk factors in older adults and the elderly , there is a paucity of such data among teenagers and young adults , as they are considered to be at a lower risk of developing the disease . with a growing problem of hypertension worldwide , there is a concern that hypertension in young adults may also be on the rise and that cases are not detected because of inadequate screening in this age group . the epidemiology of demographic transition states that a long - term shift occurs in mortality and disease patterns , whereby infectious diseases are gradually displaced by degenerative and man - made diseases as the chief form of morbidity and death . furthermore , evidence shows that uae is a country in transition where people have adopted western living patterns ; risk factors such as sedentary lifestyle ; obesity , stress , unhealthy diets ; and smoking[2022 ] have all been demonstrated in young adults . knowledge of the predisposing risk factors is vital in the modification of lifestyle behaviors conducive to optimal cardiovascular health . measuring and appropriately disseminating knowledge of the modifiable risk factors at an early age is an essential preventive educational approach . strategies to achieve even a modest lowering of the levels of blood pressure in the population of children and young adults are therefore important public health goals . an attempt is made in the present study to assess the knowledge of risk factors of hypertension among university students and associate it with the blood pressure , physical activity , family history of cvd , and sociodemographic variables . gulf medical university ajman , united arab emirates ( uae ) has four colleges , namely , college of medicine , college of dentistry , college of allied health sciences and college of pharmacy , with multinational students . for the academic year 2009 2010 it had an annual intake of 160 students ( 60 in mbbs , 40 in dmd , 35 in pharm d , and 25 in bpt ) , in the four programs , out of which 110 students who were present on the day of data collection participated in the study with a response rate of 68.75% . the survey was conducted using a pre - tested , structured , self - administered questionnaire . the questionnaire included sociodemographic variables like age , gender , nationality , ethnicity , and religion , and questions on the knowledge regarding the modifiable and non - modifiable risk factors for hypertension and a 30-day recall of physical activity . the students were categorized as less active if they did not practice any planned regular physical activity for at least 30 minutes / day for five days a week . to assess the knowledge on risk factors of hypertension , eleven modifiable and three non - modifiable risk factors were included . each modifiable risk factor was given a score of one if it was answered correctly and zero for a wrong answer . out of a total score of eleven a score of six and more was considered as good knowledge and a score less than six considered poor knowledge . this was validated by three experts in the area of public health , epidemiology , and internal medicine . the investigators explained to the respective class the purpose of the study and sought verbal consent before distributing the questionnaires . anyone was permitted to opt out of the study , however , all present in the classroom participated . blood pressure was measured by the same team using a mercury sphygmomanometer , twice , with a gap of one minute , and interpreted as per the blood pressure guidelines issued in 2003 , by the national heart , lung , and blood institute as shown in table 1 . blood pressure guidelines , 2003 data was analyzed using pasw-17 ( ibm , chicago , illinois ) . chi square test was done for association between knowledge of risk factors and age , gender , nationality , course , diet , physical activity , smoking , presence of hypertension , and family history of diabetes , hypertension , kidney disease , stroke , and cvd . in addition to the variables that showed a significant p - value ( p < 0.05 ) , gender ( p value 0.09 ) and presence of pre / hypertension ( p value 0.07 ) were included in the binary logistic regression analysis , to identify the crude odds ratio ( or ) . those factors with crude or > 1 were included in the multiple binary logistic regression analysis , to calculate the adjusted odds ratio . the study assessed the knowledge of eleven modifiable and three non - modifiable risk factors of hypertension and its association with study variables in 110 entry level students in one medical university . majority [ 74(69.2% ) ] of the participants was less than 20 years of age , 84 ( 76.4% ) were females , 51 ( 46.4% ) were in the mbbs course , and 44 ( 40% ) were arabs . sociodemographic characteristics of the participants of the 110 participants , stress , high cholesterol , obesity , and smoking were identified as risk factors by 83 ( 75.5% ) , 81 ( 73.6% ) , 81 ( 73.6% ) , and 79 ( 71.8% ) , respectively . seventy - six ( 69.1% ) , considered high salt intake as a risk factor for hypertension , and 69 ( 62.7% ) a high - calorie diet . energy drink and coffee consumption were considered as risk factors by 71 ( 64.5% ) and 51 ( 46.4% ) , respectively . nearly half , 52 ( 47% ) of the respondents were aware of physical inactivity being a risk factor , whereas , only 15 ( 13.6% ) had knowledge of the risk of oral contraceptives in hypertension . the overall knowledge of modifiable risk factors was better than that of non - modifiable risk factors , wherein 97 ( 88% ) did not know the risk related to male gender , 66 ( 60% ) the risk with increasing age , and 56 ( 50% ) the risk of family history of cardiovascular diseases [ table 3 ] . knowledge regarding risk factors of hypertension among the participants table 4 shows the association between the knowledge of risk factors with different demographic and other variables . overall 66 ( 60% ) of the 110 participants had good knowledge of the modifiable risk factors . good knowledge when compared to the arabs ( p < 0.03 ) , students enrolled in the medicine and physical therapy had significantly more than those enrolled in pharmacy and dentistry ( p < 0.02 ) . all eight smokers and 58 ( 56.9% ) of the non - smokers had a good knowledge . the respondents who had pre - hypertension / hypertension showed good knowledge , more than normotensives , although not statistically significant . family history of diabetes mellitus is seen to be significantly associated with good knowledge ( p < 0.04 ) when compared to family history of hypertension , cvd , kidney diseases , and stroke . only the variables that showed significant level p < 0.05 from the chi - square test were included in the binary logistic regression analysis , to identify the crude odds ratio ( or ) and confidence interval as seen in table 5 . however , gender and presence of pre- / hypertension with p - values of 0.09 and 0.07 were included and smoking excluded , because two cells had an expected value of less than five , making the chi square not valid . association between knowledge of risk factors and specific demographic and other variables crude and adjusted odds ratios associating a good level of knowledge of modifiable risk factors with specific demographic and other variables those factors with a crude or > 1 , that is , nationality , course , diagnosis of hypertension , and history of diabetes in the family were included in the multiple binary logistic regression analysis , to calculate the adjusted odds ratio . results in table 5 show that the net effect was not significant and knowledge was not dependent on any of the factors . the present study assessed the knowledge of risk factors of hypertension among entry level students in a medical university and associated with the blood pressure , physical activity , family history of cvd , and sociodemographic variables , so as to identify the areas to be emphasized in the health promotion practice related to hypertension . risk factors of hypertension are not well studied in young adults and public awareness of hypertension in countries undergoing epidemiological transition is dismal . however , the results of the present study indicate that more than 70% of the participants were aware that stress , high cholesterol , and obesity were the risk factors of hypertension . it is interesting to note that all eight smokers had good knowledge of the risk factors . more than 60% were aware of high salt intake and a high - calorie diet being risk factors . however , a gap in knowledge was seen in two modifiable risk factors , namely , physical activity ( 52.7% ) and oral contraceptives ; 86.4 % of the participants were not aware that these were risk factors for hypertension . more than 50% were not aware of the non - modifiable risk factors such as male gender ( 88.2% ) , increasing age ( 60.0% ) , and positive family history of cvd ( 50.9% ) . these findings were similar to that a study done in germany where the overall knowledge of risk factors was good , but less people could tell the association between physical activity ( 58% ) and hereditary factors ( 48% ) with hypertension . in a study done in seychelles , another country in epidemiological transition , it was seen that a high proportion showed good basic knowledge of hypertension , where 96% were aware of the association of hypertension with salt and obesity . the benefit of physical exercise on bp was also well recognized by 79% of the participants , most persons reported that smoking caused high blood pressure . a recent study on knowledge and perceptions about hypertension among neo- and settled - migrants in delhi , india , demonstrated that knowledge about hypertension was only moderate and comprehensive knowledge was lacking . another study in nepal , with regard to the knowledge about heart attack and hypertension , among individuals attending a cardiac camp showed that the respondents were aware of the basic facts regarding myocardial infarction and hypertension . however , lacunae in knowledge were noted , the knowledge scores for hypertension especially were significantly lower among the respondents . an effort to reverse the major risk factors of hypertension is the key aspect of suggested lifestyle changes . primary prevention aims to reduce or modify hypertension risk factors through the implementation of appropriate policies and educative programs , in order to avoid or delay the development of cardiovascular disorders , whereas , primordial prevention focuses on the prevention of the emergence of risk factors , and hence , the importance of the present study . the present study identified gaps in the knowledge regarding both modifiable and non - modifiable risk factors of hypertension among students from one university , which may not be representative of all university students . a larger study in the region is essential to gather such information about hypertension ; as it is crucial to devise sound prevention and control programs , to improve knowledge attitudes and lifestyle practices early in life , to control hypertension .

there are 30 cases reported in the literature between 1982 and 2010 on brain - dead pregnant women whose somatic non - neurological functions were maintained successfully to facilitate fetal maturation in the uterus . however , of the cases reported , 12 viable infants were born and survived the neonatal period . based on previous reports , the gestational age of the fetus was important in deciding to attempt somatic support following brain death . however , this is no longer an important issue especially with the important advances in life - support technology and critical care that enables the maintenance of vital functions . additionally , there are case reports that describe the successful prolongation of pregnancy without regard to gestational age at the time of brain death . spike reported a case in which successful somatic support began at 16 weeks of gestational age . likewise , bernstein et al . reported a similar neonatal outcome when somatic support began at 15 weeks gestation . treatment of maternal physiologic changes and infectious complications due to brain death to maintain somatic survival and close surveillance of the fetus can lead to favorable neonatal outcomes . we report a case of maternal brain death at 16 weeks of gestation with somatic support provided for 110 days and a successful neonatal outcome . our patient was a 35-year - old woman , gravida 2 para 2 , with no previous history of chronic medical illnesses except for gestational diabetes . she presented at 16 weeks of gestation to the emergency department with an acute - onset excruciating headache after waking up in the morning . the patient had tonic - clonic convulsions at home as witnessed by her husband . in the emergency department , the patient had low glasgow coma scores ( gcs ) that required intubation . at the emergency department , a computed tomography ( ct ) scan revealed a left frontal hematoma measuring 5 2.6 3.5 cm with mild edema and mass effect causing a midline shift of 4 mm to the right . there was also bleeding that spread to the left lateral ventricle via the third ventricle into the fourth ventricle . additionally , there was a left subdural hematoma that measured 4 mm in diameter . ct angiography ( cta ) of the brain done on the same day revealed frontal lobe bleeding , carotid bifurcation aneurysm , and a small subarachnoidal bleeding . the patient was then sent to the operating room ( or ) on the same day where a trial of coiling of the aneurysm failed . the patient was then admitted to the intensive care unit ( icu ) for monitoring and supportive measures . she was noted to have 2 mm size midposition pupils with sluggish reaction to light bilaterally . on the second day , a second trial of endovascular coiling failed . on the third day in the icu and upon physical examination , the patient had a sudden dilatation of the left pupil to 3 mm with a sluggish reaction to light , and the right pupil remained 1 to 2 mm in size . additionally , her heart rate dropped to 5559 beats / minute with a blood pressure of 110/60 mmhg that was managed with crystalloid and colloid therapy plus low doses of norepinephrine . urgent ct of the brain was then performed and revealed modest subarachnoidal rebleeding with a spastic reaction of the vessels with extended ischemia in the middle cerebral artery ( mca ) . the third ventricle was well separated which indicated some flow problem at the aqueduct level . on the fourth day , the patient was taken for another trial of coiling but that also was difficult ; there was also an occlusion of the left carotid system by a thrombus . ct of the brain showed deterioration when compared to the previous examination ; the ischemic areas were better demarcated corresponding to the left mca and part from the left anterior cerebral artery with perforating branches to the basal ganglia . the midline shift had increased and there was an increase in the effacement of gyri and sulci . cerebral angiography revealed a thrombosis in the left internal cerebral artery ( ica ) and mca . after the last attempt of failed coiling , the pupils became fixed and dilated at 7 mm bilaterally . therefore , the patient was shifted to the or on the same day for decompressive craniotomy and placement of external ventricular drains ( evd ) for monitoring intracranial pressure ( icp ) . the patient then came from or ventilated and sedated with fixed dilated pupils ( right pupil 4 mm in size , and left was 7 mm in size ) with no cough or gag reflexes and an icp monitor reading of less than 10 mmhg . on the fifth day , medical management for high icp was started and included muscle relaxants , mannitol , hypertonic saline infusions , and thiopental coma without success . as such , icp remained elevated until removal of the monitor on the eighth day of admission . upon physical examination , the patients had a gcs of 3 with no gag or cough reflexes , while the pupils were 5 mm dilated in size and fixed . apnea test was not done due to a viable fetus and the use of vasopressors , whereas eeg was not done because of the local scalp and skull conditions as the brain tissue was visible . upon confirmation of this diagnosis , our patient received full ventilatory and nutritional support , vasoactive drugs , maintenance of normothermia , and other supportive measures required to prolong gestation and improve the survival prognosis of her fetus . the 110-day hospital course of the patient was complicated by medical problems that included severe hypotension managed with vasopressors , and hypertension treated with antihypertensives . intranasal 1-deamino-8-d - arginine vasopressin ( ddavp , rhone - poulenc rore pharmaceutical inc . , collegeville , pa ) and water flushes through the nasogastric tube ( ng ) were initiated for the treatment of diabetes insipidus and hypernatremia . additionally , the patient developed several episodes of sepsis due to pneumonia , urinary tract infection , and line infection that were treated successfully with antibiotics . moreover , the patient developed meningitis that was treated with meropenem 2000 mg intravenously three times daily and vancomycin 1000 mg twice daily with a target trough level of 15 to 20 mg / l . all decisions regarding the treatment of the patient were taken with the consensus of her family . the current status of the patient was discussed with a multidisciplinary approach and involved the adult and infant ethics committees . the decision was to continue somatic support until the patient was 32 weeks gestation and a cesarean section ( cs ) could be done . an ultrasound scan after admission revealed intrauterine growth retardation ( iugr ) with biometry corresponding to 25 weeks gestation , an estimated fetal weight of 650 gm , and oligohydramnios with no other fetal anomalies . subsequently , intrauterine monitoring of the fetus was performed using serial ultrasounds , heart rate monitoring , and amniocentesis . betamethasone therapy was also administered for fetal lung maturity and prophylaxis of fetal respiratory distress syndrome . was delivered with an apgar score of 6/7/9 at 1 , 5 , and 10 min , respectively , and an average weight of 750 gm . application of nasal continuous positive airway pressure ( cpap ) was necessary owing to a mild respiratory distress syndrome . apnea test was done twice on the mother with positive results on both occasions . on the basis of the clinical examinations and these confirmatory technical tests , death was pronounced . brain death of a pregnant woman usually raises a dilemma whether to prolong the maternal homeostasis and ventilatory support to maintain the fetus alive until maturity , or to disconnect life - support measures and terminate life . according to the reported cases in the medical literature , this is the second reported case where prolonged somatic support led to the delivery of a viable child in which the fetus was about 16 weeks of gestation . furthermore , this is the first case in literature with the longest somatic support ( 110 days ) . until this case report , this supports the fact that the gestational age at the time of maternal brain death is not the main consideration in deciding whether to decide somatic support or not . however , gestational age of the fetus can predict the successful delivery of the fetus . showed that the severity of the disability correlates with how extremely children were born as preterm infants . he also showed that at 22 , 23 , 24 , and 25 weeks of gestation , a fetus has about 1% , 11% , 26% , and 44% likelihood of survival with a 0.7% , 5% , 12% , and 23% chances of survival without handicap at 30 months , respectively . parry et al . also showed that the probability of mortality increases in infants born before 32 weeks gestation . however , the improved outcome for preterm infants can be attributed to closer surveillance of the mother and preterm obstetric interventions and hence the decision of prolonged somatic support must be done on an individualized basis . until now , the decision to support brain - dead patients and the duration of support have been controversial ethical issues , becoming more complex with the involvement of pregnancy . moreover , only a few reported cases on managing a brain - dead woman are found in the literature ; so ethical issues in this context remain relatively little publicized . suddaby et al . showed that of 252 brain - dead patients reported , only 5 ( 2.8% ) involved pregnant women . moreover , as very few women are prepared for such a devastating event with advanced directives or prior statements , the decision for prolonging pregnancy should be taken in collaboration with a surrogate decision maker from the family . field et al . concluded that ethical considerations should support the decision of maintaining the brain - dead mother despite the technical difficulties and the economic costs incurred in such a case . additionally , although maternal care during brain death incurs significant costs , increasing the intrauterine time where the brain - dead mother can serve as a natural incubator can positively affect neonatal outcomes and therefore can reduce the duration of stay in the nicu and associated costs . hence , the closer the fetus is to viability , the more justifiable the provision of extended somatic support , especially when considering that the gestational age beyond which therapeutic abortion is permitted has been exceeded . it is noteworthy to say that with extended somatic support , the pregnant mother will serve as an incubator . therefore , this will violate her right to autonomy and bodily integrity , which are mostly cited issues in this context . some professionals suggest that if the mother had a previous inclination to donate her organs , extended somatic support is justifiable to preserve the organs . others argue that such support is still in the experimental stage with suboptimal knowledge about the adverse effects of the medications on the fetus . however , ethical justification for prolonging the vital functions of the mother can be supported better if she is a prospective organ donor as the fetus would be the first to benefit from receiving the organs of the mother . in the systematic review done by esmaeilzadeh et al . , organ donation from the brain - dead mother was carried out in 10 patients with an excellent one - year graft survival . for all of those patients , excellent patient and graft outcomes were reported . of note , maternal somatic support in all the cases ended either after delivery or after organ donation . also , the number of organs harvested was excellent . in the retrospective analysis of organ donors in brain - dead patients done by suddaby et al . , five of seven pregnant women were organ donors for 20 transplant recipients . of 25 donated organs ( 5 hearts , 5 livers , 10 kidneys , 5 pancreases ) in this analysis , consequently , supporting maternal vital functions may be ethically justifiable to support both the birth of a child and possible organ donation . as events of brain death during pregnancy are infrequent , the wishes of the mother are mostly unknown . hence , the family should be engaged in decisions made after extensive counseling regarding life - maintenance strategies , prognosis of the mother , and possible damages they may be caused to the fetus . catlin et al . performed an ethical analysis and suggested discussing issues of brain death during prenatal interviews , and incorporating the fate of the unborn child in the case of such an event in the form of a legal document . from a medical point of view , somatic support should also involve a meticulous multidisciplinary approach from disciplines that can take care of the mother and the fetus until successful delivery as it is associated with many maternal medical complications . feldman et al . summarized complications and management when pregnancies with brain death were prolonged with the goal of achieving delivery of a viable infant . furthermore , hemodynamic changes in the brain - dead mother can induce physiologic stress to the fetus . therefore , ensuring fetal intrauterine growth and maturation should be done through serial ultrasounds , heart rate monitoring , and amniocentesis due to a few reports on iugr . fortunately , despite major complications noted in our case , prolonged somatic support was successful . in the systematic review done by esmaeilzadeh et al . , 12 ( 63% ) of 19 reported cases led to the delivery of a viable child after extended somatic support . of note , all of the 6 remaining cases were less than or equal to 20 weeks gestational age at the time of brain death . however , the true percentage at which the somatic support can lead to the delivery of a viable child can not be successfully determined as not all cases with negative and positive neonatal outcomes are reported . apparently , special medical support and interventions from various clinical disciplines such as intensive care medicine , obstetrics , neonatology , anesthesiology , neurosurgery , and an ethics committee can lead to successful outcomes . adrenal insufficiency can add to hypotension and should be treated with methylprednisolone as it does not readily cross the placenta . however , the initial treatment for hypotension consists of aggressive fluid replacement utilizing crystalloids followed by vasopressors for refractory cases . the endogenously produced vasopressors like norepinephrine and dopamine seem to be safe , whereas vasopressin has the potential to decrease uterine blood flow . powner et al . reviewed case reports of brain - dead pregnant women with successful delivery of their infants after somatic support . from these reports , preservation of uterine / placental blood flow was the most important priority during somatic support due to the absence of autoregulation of the uterine vasculature . this can result in fetal hypoxia and neurological injury to the fetus during any episode of maternal hypotension . maintaining normothermia is an important issue as hypothermia can result in iugr as fetal energy will be directed away from normal growth and maturation . from an infectious point of view , this can lead to septicemia ultimately which represents the greatest risk for maternal somatic functions . such infections should be treated aggressively regardless of safety to the fetus as bacteria can develop resistance to antibiotics over a prolonged period of time in the intensive care unit . until now , due to the low number of cases that reported a successful somatic support in brain - dead pregnant women , there are no definitive guidelines or proven management strategies in this context and all the interventions are considered experimental . mallampalli et al . reviewed the expected physiologic changes and highlighted specific recommendations regarding organ support of brain - dead pregnant women . prolongation of pregnancy until at least 28 weeks of gestation is preferred especially with stable pregnancy . according to current literature , there is no justification for prolonging pregnancy beyond 32 weeks of gestation especially when glucocorticoid - induced fetal lung maturity with cesarean section is the optimum method . maternal neurologic injury has a potential for fetal demise without intensive critical care support to the mother . the outcomes reported in our case are in accordance with previously published case reports on the appropriateness and safety of such a strategy that involved a multidisciplinary approach . prolonging somatic support in a pregnant woman with brain death to allow fetal survival can have favorable outcomes . despite being a tragedy , maternal death can represent an opportunity to save the life of the fetus and organ harvesting if decided by the surrogate decision maker .

a 45 year - old male patient came to the hospital to receive tympanoplasty for chronic otitis media . the patient was 66.6 kg and 164 cm tall , and all physical and general examination results were normal other than the ventricular bradycardia of 58 beats per minute in the ecg . glycopyrrate ( 0.2 mg ) was i m injected as a pre - anesthetic medication , while vital signs before anesthesia were normal other than the ventricular bradycardia of 45 beats per minute . intravenous anesthesia was planned , rather than anesthesia using a volatile agent , in order to perform nerve integrity monitoring during surgery . before inducing anesthesia , the bis sensor was attached while the patient was conscious , and the bis values registered 95 . medication was started with fresofol mct 2% ( fresenius kabi , bad homburg , german ) , with a target site concentration of 6 g / ml and remifentanil target site concentration of 4 ng / ml using orchestra base primea ( fresenius kabi , bad homburg , german ) . after confirming loss of consciousness , rocuronium ( 50 mg ) was iv injected and manual ventilation was maintained with oxygen at 6 l / min . after confirming that there was sufficient muscle relaxation and with bis values decreased to 44 , oral intubation was performed with an endotracheal tube , with a diameter of 7.5 mm ( safety - flex , mallinckrodt ) , using a curved laryngoscope . ventilation was maintained with o2 of 1.5 l / min and air of 2 l / min , and with a target site concentration of 3.5 g / ml and remifentanil target site concentration of 3.0 ng / ml being continuously iv injected , the bis values were maintained between 41 to 60 . subsequently , the nim-2 was attached , and when electricity was applied to measure the resistance in the facial nerve monitoring mode , a severe artifact in the eeg was observed intermittently for 15 seconds on the bis monitor . a message then appeared on the bis monitor indicating that the data unavailable due to poor signal quality , so further bis monitoring was impossible ( fig . when nim-2 monitoring was discontinued , the eeg was monitored normally again and it was observed that the bis monitor was able to read the values . it was impossible to monitor nim-2 and bis simultaneously , but the bis values had been stable during continuous iv injection of the medication before starting nim-2 monitoring , while the surgery itself was a stable surgery , so it was decided not to observe bis values during nim-2 monitoring , and instead , a continuous iv injection speed with target site concentration of fresofol at 3.5 g / ml and remifentanil at 3.0 ng / ml was maintained . the surgery continued without bis monitoring , but when nim-2 monitoring was suspended , before the suturing of the operated area at the completion of tympanoplasty , eeg waves were detected on the bis monitor again . after completing the suture and dressing of the operated area , when spontaneous respiration was observed , following the increase in bis values , pyridostigmine ( 10 mg ) and glycopyrrolate ( 0.4 mg ) were administered in order to reverse muscle relaxation . when spontaneous respiration was sufficiently recovered , bis monitor showed a value of 91 and the patient was able to open his eyes and obey oral commands . at this point , the total surgery time was 3 hours , with the anesthesia time at 3 hours and 45 minutes . the patient was moved to the ward from the recovery room with no special problems , and was discharged after undergoing a recovery period of 5 days in the hospital ward . the bis is a multifactorial parameter obtained from the eeg , which monitors the hynagogue of anesthesia , and is calculated through the general anesthesia experiences of numerous volunteers and patients using several different anesthetic agents . it is expressed as values between 0 - 100 , where patients not under anesthesia show 90 - 100 , while 0 is shown when cortical electrical activity is completely oppressed . values of 40 - 60 imply that the possibility of awakening and cognition during surgery is low . bis monitoring during surgery allows the maintenance of a suitable level of sedation or depth of anesthesia , and can prevent unnecessary deep anesthesia , excessive administration of medications , hypotension , awakening during anesthesia , and memory formation . however , there are research results which report that electrical equipment , the specific clinical state , abnormal eeg patterns , and nmba , as well as a number of other factors , can interfere with bis monitoring . for example , there was a case where the bis increased to 90 when an atrial pacemaker was used during heart surgery , however , this value decreased when the pacemaker was turned off . also , when a forced - airwarming blanket was directly operated on the patient 's face , a false elevation of bis was observed , with bis again returning to normal when the operation of the blanket was stopped . there has also been a report that bis rapidly increased during shoulder arthroscopy when the shaver was started and began generating oscillations . sloane reported a total thyroidectomy case that combined bis and recurrent laryngeal nerve monitoring of the electromyography endotracheal tube . during this surgery , the bis figures rapidly increased , and despite additional administration of midazolam and etomidate , there were no changes in the bis figures . however , when the emg stimulator was turned off , the bis values decreased from 75 to 24 . for this case , sub - dermal electrode 1 was attached to the orbicularis oculi muscle area , and electrode 2 was attached to the orbicularis oris muscle area . when current stimulation was given from the standard monopolar probe , a resulting emg response was detected . when a facial nerve is stimulated , an attached alarm is sounded to notify the operating surgeon . this device , in principle , passes electric currents of 0.8 - 1.0 ma through electrodes in regular intervals to grasp the accurate location of the electrodes . these electric currents seem to flow into the bis electrodes , which receive eegs to cause interference . in effect , as the equipment was activated , an eeg artifact occurred on the bis monitor , which was stably monitoring at the time , while the bis values were substantially interfered and were unable to continue monitoring . not only for this patient , but similar phenomena were observed in other patients using the same monitoring equipment , while receiving the same surgery . in comparison , this phenomenon did not occur in thyroidectomy procedures experienced by the authors using this same monitoring equipment . when performing thyroidectomy , an endotracheal intubation is performed with a nim emg reinforced endotracheal tube ( nim emg endotracheal tube , medtronic xomed , usa ) , electrodes are mounted so that they make contact with the vocal cords , and standard monopolar probes are also used in order to monitor the stimulation of the recurrent laryngeal nerve . in these cases , the eeg waves were continuously monitored without artifacts on the bis monitor , which is in stark contrast to the monitoring of the facial nerve . no large differences were observed , as the same nim-2 equipment was used and the electric current used for stimulation was approximately 1.0 ma , but the different results are considered to be related to the distance of the stimulus electrodes from the bis probe . the electric signal for stimulation enters through each electrode and generally comes out through the grounds attached to the chest , but in facial nerve monitoring , the electric current enters through the electrodes attached next to the eyes , which are close to the front forehead where the bis probes are attached , so it is highly possible that these electric signals can enter into the bis probe . in contrast , when monitoring the recurrent laryngeal nerve , electric currents enter through the vocal cord and the current flows towards the chest where the grounds are located , so there is a low possibility of the electric currents traveling upwards . even when there is an electric current leak , they are greatly decreased when reaching the forehead area , so the effect should be insignificant . therefore , when nim-2 monitoring is used , operating surgeons should be fully aware that the reliability of the bis can decline when monitoring the facial nerves . a method to reduce this unreliability is to accurately install the location of the electrodes so that the least ma of the stimulating current is given to the bis . when the depth of anesthesia is uncertain or in patients who have a high risk of awakening during surgery , methods , such as the stopping of nim-2 monitoring during regular intervals in order to check bis , should be adopted . when monitoring the recurrent laryngeal nerve , there are fewer interruptions from the stimulating electric currents on the bis values so relatively reliable information can be obtained . however , in order to obtain more accurate values , it is necessary to adjust the location of the electrodes so that they have the least possible stimulating electric current . recently , with the increase in interest and information of the general public regarding anesthesia , the use of bis monitoring is vitalized as awakening or cognition during anesthesia emerges as a sensitive issue . bis provides reliable information in objective figures for the depth of anesthesia , but safer anesthesia can be provided to the patient when it is understood that other monitoring devices and surgical equipment used during surgery , such as nim-2 , can have a distinct influence on bis monitoring .

diabetes mellitus is a leading cause of mortality and increased disability - adjusted life years ( dalys ) worldwide . in 2010 , an estimated 1.3 million diabetes - related deaths occurred worldwide , which was twice as many as occurred in 1990 . furthermore , the dalys from diabetes increased by 30% during this same period from 380 per 1,000 persons to 523 per 1,000 persons . although the mortality rate of diabetes in korea decreased from 25.0 per 100,000 persons in 2003 to 21.5 per 100,000 persons in 2013 , diabetes was still ranked as the fifth most common cause of death in 2013 . in addition to its association with mortality , diabetes , and diabetes - related chronic complications such as retinopathy , neuropathy , nephropathy , cardiovascular , and cerebrovascular diseases , peripheral artery occlusive diseases , and amputations are related to significant increases in medical costs . recently , the prevalence of diabetes has increased in conjunction with the incidence of obesity . according the korea national health and nutrition examination survey ( knhanes ) studies in 2001 to 2013 , the age - standardized prevalence of diabetes among adults 30 years of age and older increased from 8.6% to 11.0% and the age - standardized prevalence of adult obesity , defined as a body mass index ( bmi ) of 25 kg / m or higher , increased from 29.2% to 31.8% . during the same period , the rates of diabetes and obesity tended to increase among men , while among women the prevalence of diabetes increased but the prevalence of obesity decreased ( fig . 1 ) . studies have consistently reported an association between obesity and diabetes . in a large representative sample from the united states , the highest prevalence of diabetes was observed in subjects with a bmi 40 kg / m , and menke et al . found that changes in bmi over time are the greatest contributor to the increased prevalence of diabetes in this population . eckel et al . demonstrated that obesity is linked to diabetes through the increased production of adipokines and cytokines , reductions in adiponectin levels , altered ectopic fat deposition , and mitochondrial dysfunction . however , because not all obese individuals develop diabetes , the relationship between obesity and diabetes remains unclear . for example , the influence of abdominal obesity on the risk of diabetes was found to be more important than that of general obesity . given the striking parallel increases in the incidences of these two conditions , obesity is likely an important contributing factor to the manifestation of diabetes and the complications , mortality , and morbidity related to this disease . in recent decades , there has been a dramatic increase in the number of obese people with diabetes in korea . the average bmi of diabetic korean patients increased from 21.9 kg / m in 1989 to 1990 to 24.8 kg / m in 2010 to 2012 . in 2013 , 71.6% of men and 74.2% of women with diabetes were obese or overweight ( fig . national surveys conducted between 2001 and 2013 in korea revealed age - related increases in the prevalence of diabetes in all age groups , except for those between 30 and 39 years of age . because korea is a rapidly aging society , this increase was particularly evident in people aged 70 years and older ; the rate of diabetes in this age group was 27.6% in 2013 , approximately twice as high as in 2001 ( fig . 3 ) . however , the mean age at first diagnosis of diabetes was very similar among men in 2005 ( 49.3 years ) and 2013 ( 50.6 years ) , while the mean age among women decreased slightly from 56.4 to 55.3 years ( fig . , there is roughly a 5-year difference between men and women in terms of age at the first diagnosis of diabetes . according to the centers for disease control and prevention in the united states , the mean and median ages at the first diagnosis of diabetes among american adults aged 18 years or older were 53.7 and 53.8 years in 1997 and 53.8 and 54.2 years in 2011 . the median ages at diagnosis of diabetes were 53.6 years for men and 55.2 years for women in 2011 . in korea , the age at the diagnosis of diabetes is similar to that in the united states , but korean men tend to develop diabetes about 3 years earlier than american men ( unpublished data ) . the knhanes 2013 revealed an inverse linear relationship between bmi and age at diagnosis of diabetes among newly diagnosed subjects ( fig . 5 , unpublished data ) . it has been reported that obesity is independently associated with early onset diabetes and the european prospective investigation into cancer and nutrition - potsdam study found that weight gain in early adulthood ( 25 to 40 years ) is more strongly associated with the risk and earlier onset of diabetes than weight gain after the age of 40 years . a number of studies have reported that the age at onset and duration of diabetes are associated with microvascular events , cardiovascular disease , and all - cause mortality . for example , banerjee et al . suggested that the duration of diabetes is an important determinant of ischemic stroke risk . reported that diabetes duration is independently associated with the risk of macrovascular complications such as cardiovascular death , non - fatal myocardial infarctions or nonfatal strokes , microvascular complications such as nephropathy and retinopathy , and death from any cause . additionally , a longer duration of diabetes is associated with serious diabetes - related long - term complications in midlife . . demonstrated that people who developed diabetes prior to 60 years of age had a risk of coronary heart disease that was approximately twice that of persons who were diagnosed with diabetes after age 60 . because the chronic worsening of hyperglycemia is generally associated with an insulin insufficiency in individuals with a longer duration of diabetes this finding may have important public health and economic implications because individuals with diabetes tend to require sustained health services and often develop early complications during the period of highest productivity . in conclusion , the prevalence of diabetes is increasingly shifting to younger individuals and obese people . as more people develop diabetes earlier and live longer , public efforts should focus on healthy lifestyle changes , primary prevention measures , screening for the early detection of diabetes , and long - term management . following a diagnosis of diabetes , treatment measures should include intensive glycemic control to minimize the risk of microvascular and macrovascular complications . because the treatment of obesity is an important intervention that can reduce the prevalence of diabetes , the management of obesity should be considered for obese people with diabetes .

regional myocutaneous pedicle flaps ( rmf ) are known to be relevant in the reconstruction of major head and neck oncologic defects with pectoralis major myocutaneous pedicle flap ( pmmc ) being the best - known rmf . for over three decades , since first described by ariyan in 1979 , pmmc has continually been used in the reconstruction of head and neck oncologic defects . with time , the free flap came into existence and showed superiority at reconstructing three - dimensional head and neck defects . predictably , it has since grown to become the gold standard for head and neck reconstruction . expectedly , high surgical expertise and expensive equipment are required to achieve this high standard ; conditions not easily met in many - limited opportunity environments . while rmf continues to be relevant in the reconstruction of head and neck defects in many developing countries ; its use is often limited to single - stage salvage reconstructions in more endowed societies . this study institution , despite performing , some free flaps have no defined role for pmmc and other rmfs in head and neck reconstruction . while there are many reports on the indications and outcomes of rmf in the developed environment , the same can not be said for rmf indications in resource - constrained environment . the aim of this study thus , was to review our experiences with rmfs in head and neck reconstructions , with a view to evaluating the indications and outcomes from a resource constrained environment with some free flaps expertise . this was a retrospective case series from records of rmf cases performed for head and neck reconstruction , at the study institution . eligibility for study inclusion comprised cases with advance head and neck diseases requiring ablative surgery and reconstruction ( preferential or salvage ) with pmmc and/or other rmf . these other types of rmf were platysma , trapezius , deltopectoral , and forehead flaps . data collated include ; demographic data , pathology and tnm staging , primary site , indications and types of surgery , types of flap , flap complications , deaths , and follow - ups of survivals . the pmmc was raised using the defensive deltopectoral incision as shown in figure 1 , while other rmfs were raised as simple axial pattern flaps . defensive deltopectoral incision for pectoralis major myocutaneous harvesting with supraclavicular advancement flap indications were classed on the basis of rmf either being the preferential flap of choice ( with the requisite reason stated ) or as a salvage flap after initial flap failure . where rmf was the preferential flap of choice , the decision was made at the institution 's multidisciplinary tumor board meeting . if rmf was used as a salvage flap , the institute 's head and neck cancer unit took the decision . complications of flap loss were classed as total or partial flap loss , flap skin dehiscence , donor site infection and additional co - morbidity . the total flap loss was adjudged as complete necrosis of the skin , subcutaneous tissue and distal muscle paddle while partial flap loss was adjudged as partial necrosis of skin , and subcutaneous tissue . the treating physician , who adjudged patient 's fitness for long reconstruction surgery diagnosed additional co - morbidity due to any underlying medical condition . data were analyzed using ibm statistical package for social science ( spss ) for apple mac os version 20.0 . percentages , mean , and standard deviation were analyzed for age , gender , primary site , histoloathology , surgery type and indications , reconstruction types , complications , deaths , and follow - ups . the impact of the variables age , gender , surgery type and indications , and reconstructions type on postoperative complication were analyzed using univariate and multivariate analyses . the impact variable on 2 years overall survival ( os ) and disease free survival were equally analyzed using kaplan a total of 17 patients were consecutively treated with rmf between 2011 and 2013 with only one case performed in 2011 . all patients were treated for both head and neck malignancies and benign jaw tumors , of which 76.5% ( 13/17 ) were malignancies and 53.8% ( 7/13 ) of the malignancies were squamous cell carcinomas ( scc ) . patient 's demographic data a total of 10 cases had pmmc flaps while 7 had other rmf consisting of : 3 platysma flaps , 2 trapezius flaps , 1 deltopectoral flap ( dpf ) ( combined with a pmmc ) , and 1 forehead flap . the combined dpf and pmmc are shown in figures 2 and 3 . of the two major indications , the preferential indication was 64.7% ( 11/17 ) comprising ; financial constraints 29.4% ( 5/17 ) and medical co - morbidity 23.5% ( 4/17 ) while the salvage indication ( surgery ) following free flap failure constituted 35.3% ( 6/17 ) of the cases . further breakdown of the indications is shown in table 2 . a combined pectoralis major myocutaneous and deltopectoral flap harvesting retromolar trigone ca muco - cutaneous defect reconstruction with pectoralis major myocutaneous and deltopectoral flap primary indications for pmmc the pmmc flaps were used to reconstruct mucosa only in five cases ; mucosa and skin in three cases [ figure 3 ] , and esophageal patch in two cases . for the only pmmc case in 2011 , the the salvage surgeries were mainly for failed radial forearm free flap ( rfff ) reconstructions for buccal mucosa and floor of mouth ( fom ) carcinomas resections and a failed anterior lateral thigh ( alt ) free flap for esophageal patch following total laryngectomy and others as shown in table 3 . in the other flap group , the flaps were used to reconstruct mucosa only defect in four cases , skin only defect in two cases and both skin and mucosa defects for one case . other salvage rmf surgeries were for failed rfff in tongue ca and cancrum nasalis resections and loss of skin paddle in a failed fibula free flap following mandibulectomy . oro - cutaneous fistulation complication that occurred following one of the flap dehiscence was primarily repaired . in the univariate analysis of variables on postoperative complications , only flap indications had a statistical significant impact variable on complication ( p = 0.040 ) . complications following rmf flap variables influencing postoperative complications a total of 53% ( 9/17 ) of the total cases died , and all were adjudged stage iv malignancies . the deaths based on groups comprised ; 80% ( 8/10 ) in the pmmc group and 14% ( 1/7 ) in the other flap group . a total of 8 cases were disease free ( 1 mucoepidermoid ca , 1 mandibular alveolus ca , 1 ca tongue , 1 ca fom , 3 ameloblastomas , 1 cancrum nasalis ) . the cases os ( > 2 years ) for stage iv primary malignancies of the head and neck with curative intent surgery was 23% ( 3/13 ) . the histopathology of these 3-malignant cases with > 2 years os was mucoepidermoid carcinoma and 2 scc of the tongue and fom respectively . of the 77% ( 9/13 ) of the malignancy cases that died , 4 cases failed at the neck , 2 cases had primary site failure and 3 cases died of medical causes . miere 's analysis of variables on os found to have no significant impact factor ( p = 0.673 ) graphically shown in [ figure 4 ] . miere 's analysis of impact of surgery indications on overall survival ( p = preferential indications ; s = salvage indications ) of the 17 cases , 9 cases were scheduled for chemoradiation as part of the initial treatment plan . one of the malignant cases not scheduled for chemoradiation died from other surgical complication while six other cases scheduled for chemoradiation died from their diseases . a total of 17 patients were consecutively treated with rmf between 2011 and 2013 with only one case performed in 2011 . all patients were treated for both head and neck malignancies and benign jaw tumors , of which 76.5% ( 13/17 ) were malignancies and 53.8% ( 7/13 ) of the malignancies were squamous cell carcinomas ( scc ) . a total of 10 cases had pmmc flaps while 7 had other rmf consisting of : 3 platysma flaps , 2 trapezius flaps , 1 deltopectoral flap ( dpf ) ( combined with a pmmc ) , and 1 forehead flap . of the two major indications , the preferential indication was 64.7% ( 11/17 ) comprising ; financial constraints 29.4% ( 5/17 ) and medical co - morbidity 23.5% ( 4/17 ) while the salvage indication ( surgery ) following free flap failure constituted 35.3% ( 6/17 ) of the cases . further breakdown of the indications is shown in table 2 . a combined pectoralis major myocutaneous and deltopectoral flap harvesting retromolar trigone ca muco - cutaneous defect reconstruction with pectoralis major myocutaneous and deltopectoral flap primary indications for pmmc the pmmc flaps were used to reconstruct mucosa only in five cases ; mucosa and skin in three cases [ figure 3 ] , and esophageal patch in two cases . for the only pmmc case in 2011 , the free flap not possible indication was due to lack of free flap expertise . the salvage surgeries were mainly for failed radial forearm free flap ( rfff ) reconstructions for buccal mucosa and floor of mouth ( fom ) carcinomas resections and a failed anterior lateral thigh ( alt ) free flap for esophageal patch following total laryngectomy and others as shown in table 3 . the flaps were used to reconstruct mucosa only defect in four cases , skin only defect in two cases and both skin and mucosa defects for one case . other salvage rmf surgeries were for failed rfff in tongue ca and cancrum nasalis resections and loss of skin paddle in a failed fibula free flap following mandibulectomy . oro - cutaneous fistulation complication that occurred following one of the flap dehiscence was primarily repaired . in the univariate analysis of variables on postoperative complications , only flap indications had a statistical significant impact variable on complication ( p = 0.040 ) . a total of 53% ( 9/17 ) of the total cases died , and all were adjudged stage iv malignancies . the deaths based on groups comprised ; 80% ( 8/10 ) in the pmmc group and 14% ( 1/7 ) in the other flap group . a total of 8 cases were disease free ( 1 mucoepidermoid ca , 1 mandibular alveolus ca , 1 ca tongue , 1 ca fom , 3 ameloblastomas , 1 cancrum nasalis ) . the cases os ( > 2 years ) for stage iv primary malignancies of the head and neck with curative intent surgery was 23% ( 3/13 ) . the histopathology of these 3-malignant cases with > 2 years os was mucoepidermoid carcinoma and 2 scc of the tongue and fom respectively . of the 77% ( 9/13 ) of the malignancy cases that died , 4 cases failed at the neck , 2 cases had primary site failure and 3 cases died of medical causes . miere 's analysis of variables on os found to have no significant impact factor ( p = 0.673 ) graphically shown in [ figure 4 ] . miere 's analysis of impact of surgery indications on overall survival ( p = preferential indications ; s = salvage indications ) of the 17 cases , 9 cases were scheduled for chemoradiation as part of the initial treatment plan . one of the malignant cases not scheduled for chemoradiation died from other surgical complication while six other cases scheduled for chemoradiation died from their diseases . regional myocutaneous pedicle flaps continue to be relevant flaps for head and neck reconstruction because of their ease of accessibility to the surgical field , ease of harvesting , minimal learning curve and reliability . in a limited opportunity environment , the lack of free flap surgery capabilities ( free flap not possible ) would have to be included as an indication . despite the preference for microvascular surgery in developed environment , rmf has continued to remain in use , as cited in reports from centers in europe , asia , and north america . to the best of our knowledge , not much study , in a study center with free flap expertise , has been published in africa ; this being some attempt to undo that . the preferred reconstructive indication in developed environment includes medical co - morbidity , advance neck metastasis , and cases with a high possibility of free flap failure . from this study , the prevalent indications for rmfs were due to failed free flaps ( 35.3% ) , financial constraints ( 29.4% ) and medical co - morbidity ( 23.5% ) . no other study in the literature conducted in both environments reported failed free flaps as the most prevalent indication for pmmc or rmf salvage surgery . the high rate of free flap failures was possibly due to the steep learning curve associated with learning and incorporating microvascular surgery into a practice . a lot of our early cases were undertaken during the start - up of our free flap program . this study environment is a region where head and neck fellowship is undertaken outside the continent of africa , and the availability of free flap expertise is currently low . the report of financial constraints as a second indication in this study was at variance with other studies , in that , it was reported to be the most prevalent indication in a developing environment while studies from developed environment adduced co - morbidities as the major indicator . it can be submitted that in the absence of free flap expertise , financial constraints would be the prevalent indication for rmf , in a developing environment . conversely , while some authors have reported relative comparison between the costs of free and regional flap surgery , the reality in limited opportunity environment is a significantly higher cost for free flaps than regional flap reconstructions prevails . this is due to the low awareness and absence of microvascular surgery expertise and equipment . these implicitly limit the performance of free flap reconstruction in these regions including where this study was conducted . by this implication , free flap surgery would be costly , and rmfs may be the only option left for some of these patients . this was at variance with reports from developed environments , which reported it to be the most prevalent indication . these in clear terms point to the difference in the mitigating factors for indications of rmf in both environments . the overall flap - related complication was been reported to be between 18% and 40% . our flap complications showed a complication incidence of 35% ( 6/17 ) , with pmmc flaps having a higher overall complication incidence of 40% ( 4/10 ) . overall partial flap loss was 17.6% ( 3/17 ) , flap skin dehiscence was 11.8% ( 2/17 ) , and donor site infection was 11.8% ( 2/17 ) . this study did not include postoperative quality of life assessment as an indicator to flap outcome , but the limitations of rmf , especially pmmc are well - documented . they include : excessive tissue bulk , limited pedicle length and arc of rotation , as well as reduced shoulder and neck function . with the superior outcome following the use of rfff and alt for repair of partial or circumferential pharyngeal defect seen in laryngectomy , pmmc use has been criticized for high salivary leakage . in this study , 2 patients underwent pharyngeal defect repair with pmmc following total laryngectomy . one case died at icu 1-week after surgery and could not be assessed for postoperative complications . the salivary leakage recorded following pmmc patch was in line with reports in the literature . bi - paddled pmmc flaps were used in two cases to repair both mucosa and skin for reconstruction of full - thickness cheek defects . free flap failure and financial constraints are the two - main indications for pmmc and other rmf flaps for head and neck reconstruction , in a developing environment with some free flap expertise . in this era of free flap surgery , environments like mine would inevitably imbibe these trends and commence free flap start - up programs . flap failure would be inevitable , and pmmc and other rmf would be a very useful salvage . financial constraints continue to determine treatment choice while co - morbidity is the prevalent indication in our environment . because of the small sample size in this study ,

the abo blood group , which was discovered at the beginning of the 20 century by karl landsteiner remains the most important blood group clinically ( 13 ) . in 1924 , this blood group was classified into four antigens ( a , b , o and ab ) and six genotypes ( aa , ao , bb , bo , oo and ab ) . it is one of the conventional blood group polymorphisms , which are important for genetic markers in linkage analysis , blood transfusion , personal identification and disease detection ( 4,5 ) . a range of techniques have been used for abo genotyping , including polymerase chain reaction ( pcr)-restriction fragment length polymorphism , pcr - single - strand conformation polymorphism , allele - specific - pcr , pcr - amplified product length polymorphism , reverse transcription - quantitative ( rt - q)pcr and dna chip ( 3,68 ) . these methods are , however , time - consuming and disadvantageous due to the requirement for labor - intensive post - amplification procedures , including restriction electrophoresis and enzyme cleavage , or the use of radioactive - labeled dna probes . therefore , a simpler , quicker and more informative method for the genotyping of the abo alleles is required . the present study aimed to improve the abo genotyping method , based on lamp , which directly uses a one - step isothermal reaction to determine the above - mentioned six genotypes . the developed technique was a powerful innovative gene amplification approach as a simple rapid tool for clinical detection and identification . peripheral blood samples were collected from 101 unrelated chinese volunteers into edta - coated tubes at the shaanxi provincial people 's hospital ( xi'an , china ) with informed consent . the study was approved by the ethics committee of the college of life sciences , northwest university ( xi'an , china ) . the abo phenotypes of all blood samples were identified by serological methods , based on the abo blood group antigens present on red blood cells and igm antibodies present in the serum . the genomic dna from the volunteers was isolated from 1 ml blood using a whole blood genomic dna isolation kit ( xi'an goldmag nanobiotech co. , ltd . two single nucleotide polymorphisms ( snps ) at nucleotides 261 and 803 , located on exons 6 and 7 of the abo gene , which cover the most polymorphic sites of the complete abo sequences , as shown in fig . 1 ( 9 ) , were selected . the primer set for lamp amplification includes a set of four primers , comprising two outer and two internal primers , which recognize six distinct regions on the target sequence , designated in fig . all oligonucleotide primers were synthesized by beijing genomics institute ( bgi ; beijing , china ) . each analysis was performed by subjecting samples of prepared blood from each individual to four lamp amplifications . two reactions used the o or b primer set and the other reactions used the non - o or non - b primer set . the amplification was performed in a final volume of 25 l , containing 0.8 m forward inner primer and backward inner primer , 0.2 m f3 and b3 primers , 15 l isothermal master mix ( imm ; optigene , horsham , uk ) and 50 ng genomic dna . the reaction conditions were optimized to ensure that the amplifications were highly specific , including assessment of primer concentration , preparation blood concentration and reaction temperature . the lamp assays were performed in 8-well strips by incubation at 62c for 40 min . simultaneously , the fluorescence intensity of the florescent dye , contained in the imm , was monitored using a genie ii system ( optigene ) . for the samples to be sequenced , a 200 bp fragment for exon 6 and a 740 bp fragment for exon 7 were amplified using the following two primer pairs synthesized by bgi : exon 6 , forward : 5-tccatgtgaccgcacgcctc-3 and reverse : 5-gggtctctaccctcggccacc-3 ; exon 7 , forward : 5-ccgtgtccactactatgtcttcacc-3 and reverse : 5-acaacaggacggacaaaggaaacag-3. the pcr products were sequenced by the bgi . peripheral blood samples were collected from 101 unrelated chinese volunteers into edta - coated tubes at the shaanxi provincial people 's hospital ( xi'an , china ) with informed consent . the study was approved by the ethics committee of the college of life sciences , northwest university ( xi'an , china ) . the abo phenotypes of all blood samples were identified by serological methods , based on the abo blood group antigens present on red blood cells and igm antibodies present in the serum . the genomic dna from the volunteers was isolated from 1 ml blood using a whole blood genomic dna isolation kit ( xi'an goldmag nanobiotech co. , ltd . two single nucleotide polymorphisms ( snps ) at nucleotides 261 and 803 , located on exons 6 and 7 of the abo gene , which cover the most polymorphic sites of the complete abo sequences , as shown in fig . the primer set for lamp amplification includes a set of four primers , comprising two outer and two internal primers , which recognize six distinct regions on the target sequence , designated in fig . 1 . table i lists the sequences of the typing primers used for the lamp reaction . all oligonucleotide primers were synthesized by beijing genomics institute ( bgi ; beijing , china ) . each analysis was performed by subjecting samples of prepared blood from each individual to four lamp amplifications . two reactions used the o or b primer set and the other reactions used the non - o or non - b primer set . the amplification was performed in a final volume of 25 l , containing 0.8 m forward inner primer and backward inner primer , 0.2 m f3 and b3 primers , 15 l isothermal master mix ( imm ; optigene , horsham , uk ) and 50 ng genomic dna . the reaction conditions were optimized to ensure that the amplifications were highly specific , including assessment of primer concentration , preparation blood concentration and reaction temperature . the lamp assays were performed in 8-well strips by incubation at 62c for 40 min . simultaneously , the fluorescence intensity of the florescent dye , contained in the imm , was monitored using a genie ii system ( optigene ) . for the samples to be sequenced , a 200 bp fragment for exon 6 and a 740 bp fragment for exon 7 were amplified using the following two primer pairs synthesized by bgi : exon 6 , forward : 5-tccatgtgaccgcacgcctc-3 and reverse : 5-gggtctctaccctcggccacc-3 ; exon 7 , forward : 5-ccgtgtccactactatgtcttcacc-3 and reverse : 5-acaacaggacggacaaaggaaacag-3. the pcr products were sequenced by the bgi . using this method , base substitutions at two snp sites in the abo gene ( nucleotides 261 and 803 ) were detected simultaneously by four primers , setting the extension reaction to distinguish the six genotypes ( aa , ao , bb , bo , oo and ab ) . in the case of haplotypes , a positive allele - specific reaction excluded the corresponding positive non - allele - specific reaction and vice versa . allele b was distinguished from the non - b primer at nucleotide 803 and allele o was distinguished from the non - o at nucleotide 261 . a summary of all possible patterns are indicated in table ii and representative data are shown in fig 2 . the observed allele frequencies were : 28.71 , 30.20 and 41.09% , for a , b and o , respectively ( calculated from : a : ( aa+ao/2+ab/2)/101 ; b:(bb+bo/2+ab/2)/101 ; o : ( ao/2+bo/2+oo)/101 ) , from which phenotype frequencies were calculated to be 43.56 , 40.59 , 7.92 and 7.92% for a , b , ab and o , respectively ( calculated from : phenotypes/101 ) . the results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing , and no discrepancies were observed . the abo blood group system was the first blood group system to be identified and has been used extensively as a marker in population studies , epidemiology , and forensic investigation ( 10 ) . the abo blood group is fundamental for transfusion medicine , as accurate testing of the blood donor and recipient is essential for the prevention of hemolytic transfusion reactions and hemolytic disease of the newborn ( 11,12 ) . the abo blood group is also critical for assessing the risk of developing certain malignancies ( 13,14 ) and cardiovascular disorders ( 5,15 ) . to date , several methods have been reported for abo genotyping , which rely predominantly on differentiating a , b and o at specific base substitutions . however , technical errors and several clinical conditions or diseases can lead to discrepancies between erythrocytes and sera , resulting in an incorrect genotype being determined ( 3,1618 ) . an emerging novel approach , lamp , is gaining attention as a result of its practicality , speed and usefulness in laboratories and clinical settings ( 1921 ) . lamp is a simple , rapid and accurate gene amplification technique , using a set of four specially designed primers to span six distinct regions on the target gene . the amplification procedure is simple and rapid , wherein the whole procedure can be completed in a single step by incubating all reagents ( samples , primers , dna polymerase with strand displacement activity and substrates ) in a single tube under isothermal conditions , which can be completed in < 1 h , with the dna being amplified 1010 times ( 22 ) . the primary characteristic of lamp is its advantage of reaction simplicity and higher amplification efficiency . in the present study , the dna amplification procedure was < 40 min at a constant temperature of 62c , which allowed no time loss for thermal change . the dna polymerase provides high amplification efficiency as a result of its high specificity and the presence of the target gene sequence is easily detected by judging the presence of amplified products . in addition , no denaturation of double stranded dna into a single stranded dna is required . in addition , the more sensitive signal detector instrument , genie ii , assists in the rapid processing of the data . it takes ~20 min to reach the peak , followed by another 20 min to complete the entire reaction , which is relatively quicker compared with normal pcr - based assays . it has been previously reported that the detection limit of the lamp assay was 10 times lower compared with rt - qpcr and 10 times lower compared with conventional pcr ( 22,23 ) . however , lamp systems with high sensitivity may lead to false positive results . in the present study , the blood samples from 101 chinese individuals were successfully genotyped using peripheral blood dna and no false positive reaction was observed , according to the direct sequencing . with the use of four primers designed to recognize six distinct regions , only the target snp is strictly and specifically amplified , even in coexistence with its homologous gene . these results demonstrated that the four primer sets designed were extremely specific to the two snp sites in the abo gene . also , the application of imm , including florescent dye , reduced the risk of cross contamination due to the reduction of lid opening . in conclusion , the lamp assay developed in the present study has great potential for rapid abo genotyping , which can be applied in laboratories and clinical settings . it is also envisioned that several other known snps may also be detected by this method with corresponding primer sets . considering the advantages of rapid amplification , easy detection and simple operation , lamp may offer more applications for point - of - care testing .

postoperative morbidity and mortality , especially in cancer patients , have been reviewed in a number of studies in the field of gynecology . morbidity after gynaecological surgery ranges from approximately 10% to 20% , whereas mortality is extremely rare . clinical guidelines and recommendations based on the unusual events of patient death are of little importance in medical care of the general population , and therefore information on severe acute morbidity as evidenced by near - miss cases and utilisation of intensive care units ( icus ) may help to audit the quality of care in a more meaningful manner . reports on the utilisation and outcomes of critical care services required in the management of all gynaecological patients are rare . the other side of the coin is that severe morbidity and the need for intensive care poorly reflect the long - term effects leading to mortality after discharge from hospital . as far as we are aware , studies of the need for intensive care in gynaecology have not been recently conducted in the nordic countries , where patient care is organised by public health care units , and thus virtually all women receive modern care . this study was undertaken to evaluate the population - based frequency , causes , costs and outcome of intensive care in gynaecology . we retrospectively reviewed all gynaecological patients treated in the intensive care unit at kuopio university hospital from march 1993 to december 2000 . in this study , by ' gynaecological patients ' we mean patients who were admitted to the hospital primarily on gynaecological indications . our institution is a tertiary care facility dealing with all kinds of gynaecological care from assisted reproduction ( 180 ivf cycles per year ) to outpatient ( 950 operations ) and major surgery ( 1020 patients each year ) , including a gynaecological oncology service , and it is part of a general hospital complex having a surgical / medical intensive care unit . about 10% of the operations are transfers from a provincial referral base of one million inhabitants , mainly because of invasive cancer or pre - existing medical disorder considered to pose a high risk to the woman . our hospital has 24-hour on - site consultants in gynaecology , anaesthesia and intensive care . the intensive care unit at kuopio university hospital is a 22-bed tertiary care and medical / surgical intensive care unit . the anaesthetist or gynaecologist consults the intensive care physician , who makes the decision as to whether or not the patient is to be admitted to the icu . patients undergoing radical cancer surgery are routinely monitored during emergence from anaesthesia in the postanesthesia care unit for the first postoperative night , and , if stable the next morning , are transferred to a ward for further postoperative care . in this study , the time in the postanesthesia care unit was not counted as utilisation of the critical care services , and thus patients were admitted to the icu on specific clinical indication only . subsequent care is primarily in the hands of the icu team in close cooperation with the department of gynaecology . our icu provides all intensive care services for patients aged over 1 year in a primary population of 256,000 inhabitants . all patients in this referral population requiring intensive care are treated in our institution . for the most part , gynaecological patients and women with pregnancy - related complications who were treated in the icu before 16 weeks of pregnancy were enrolled in this study . each case requiring intensive care was documented with respect to the marker of severe acute morbidity , the primary factor ( the initiating event leading to transferral ) and any organ dysfunction or failure ( list of organ systems involved ) . hospital charts were reviewed to determine the length of time in the icu , specific interventions and overall outcome of all patients . for each patient , the acute physiology and chronic health evaluation ( apache ) ii score was calculated at the time of admission . patients ' records for the 6 months following the study were reviewed as regards survival . to conduct an economic analysis , the intensity of treatment during the icu stay was evaluated on a daily basis by using the therapeutic intervention scoring system ( tiss ) for each patient . the costs of intensive care were calculated by dividing the yearly total cost of the department by the total tiss score . thus , the total cost covered all the salaries , materials , full allocation of step - down costs ( e.g. administration , depreciation , rent ) and all secondary expenses such as the use of laboratory and imaging facilities and consultations with personnel of other specialities . overall , the mean cost of one tiss point was us$31 , and based on this figure , the total cost of the intensive care of the 23 gynaecological patients was calculated . there were 23 women who fitted the definition of a critically ill patient and they were admitted to the icu , the frequency of admissions being 2.3 per 1000 women undergoing major surgery over the years studied . for comparison , the equivalent figure for urological patients undergoing pelvic surgery was 5.8 per 1000 during the same period of time . of all gynaecological patients requiring intensive care , two ( 9% ) were received as tertiary referrals from other hospitals . the utilisation rate of the icu varied from two to six admissions per year during the study period . the mean ( sd ) number of days of intensive care required was 4.97 ( 9.28 ) . there was no mortality in the operating theatre or in the icu , but critically ill patients beyond surgical treatment and with progressive terminal disease were not admitted to the icu . intensive care of gynaecological patients accounted for 0.17% of all admissions to the icu and for 0.31% of all care days in the unit , and the equivalent parameter with regard to tiss scores was 0.30% of all care days in the unit during the study period . the clinical characteristics of the women admitted to the icu are presented in table 1 . gynaecological malignancies and cardiovascular diseases leading to respiratory failure were the most prevalent disease categories . in comparison with gynae - cological patients who had benign diseases , the cancer patients were specifically at high risk for icu admission , at an odds ratio of 5.46 ( 95% confidence interval 2.3113.0 ) ( p < 0.01 ) . of the 23 women admitted to intensive care , 20 underwent gynaecological surgery : 3 of the operations were emergencies and 17 were elective . reoperation was carried out in 12 cases , either because of postoperative haemorrhage or procedure - related injuries . women who ended up in the icu for reasons unrelated to surgery were one who was admitted to the hospital because of ovarian hyperstimulation syndrome and two with uterine bleeding problems . clinical characteristics of gynaecological patients ( n = 23 ) transferred to the intensive care unit apache ii , acute physiological and chronic health evaluation ii ; ards , adult respiratory distress syndrome ; bmi , body mass index . the most common reasons were postoperative haemorrhage , disease - related complications and infections , with 43% of the 23 cases in need of intensive care being due to haemorrhage , 39% due to infection , 34% due to respiratory failure or heart failure , 9% due to procedure - related injuries and 8% due to other reasons . causes of haemorrhage included surgical bleeding ( n = 7 ) and use of anticoagulants ( n = 3 ) . infections included six cases of septic infection , two of which were secondary to procedure - related injury ( one to the bowel and one to the ureter ) . anaesthesia was not considered to be the primary cause of or a contributing factor to severe morbidity in any case . reasons for admission of gynaecological patients to the intensive care unit table 3 lists interventions in the icu . we do not have a formal policy concerning the use of pulmonary artery catheters and their use is based on the clinical judgement of the intensivist in charge . however , the clinical practice is to use a pulmonary artery catheter when a patient is haemody - namically unstable or has severe respiratory failure . vasoac - tive drugs such as norepinephrine , epinephrine , dobutamine and dopamine are not usually used without a pulmonary artery catheter . the first patient was a 46-year - old woman who had had insulin - dependent diabetes for 40 years . the other patient was a 70-year - old woman who postoperatively developed sepsis and oliguria . interventions for gynaecological patients in the intensive care unit the overall outcome for women admitted to the icu was favourable among those treated for reasons other than malignancy , but four deaths ( 17% ) occurred in patients with gynae - cological malignancy after they had been transferred to ward care . this in - hospital mortality pattern indicates how extremely ill the group with malignancy was . two of these patients died as a result of unresponsive septic shock in the ward , one died of pulmonary embolism and one of uncontrolled bleeding . in each case , a multidisciplinary team made the decision not to readmit cancer patients having a poor prognosis ( advanced ovarian cancer ) to the icu . the problems that arose were not considered to be a result of poor treatment , but simply beyond the reach of critical care medicine . all but one of the surviving patients returned to their previous social and physical activity . in one case of narcotising fasciitis , clinical improvement followed surgical debridement of superficial fascia and subcutaneous tissue of the abdominal wall . two more cancer patients ( 9% ) died within the 6-month follow - up period . the mean ( sd ) cost of one tiss point was $ 31 ( 2 ) and the total cost of intensive care for the ( 23 surviving ) gynaeco - logical patients was $ 154,969 . this accounted for 0.28% of all costs incurred by the intensive care offered in our hospital . the need for gynaecological critical care was infrequent , and overall the results of this study give a somewhat pessimistic picture of clinical outcome , with 26% mortality within 6 months . most of the women who ended up in intensive care had undergone surgery and were classified as being at high risk , reflecting the fact that the risk of serious morbidity is especially relevant to those judged to be at high risk because of gynaecological malignancy or pre - existing medical disorders . mortality for cancer patients admitted to icus have been reported to range from 40% to 80% as regards solid tumors . a marked association was also observed in our patients between postoperative haemorrhage and need for intensive care . urinary and gastrointestinal tract injuries during gynaecological procedures appeared to be relatively common in cancer surgery and they accounted for almost a tenth of all admissions . the validity of the current results is high , since the study presents a regional outcome , avoiding the selection bias inherent in multicentre studies . another advantage is that all patients in the referral population requiring intensive care were included in the study . the mean length of time of intensive care required was of the order of 5 days and the mean cost of treatment of each critically ill woman in the icu was of the order of $ 7044 , without including ancillary costs incurred by treatment in the hospital ward . in this study , however , in a clinical setting it is difficult to compare frequencies as such , since indications for admission differ considerably according to the availability of facilities offering advanced in - patient gynaecological care . accordingly , whatever the level of care , a severe postoperative haemorrhage may threaten the life of a women , without subsequent need for intensive care . however , we recorded no postoperative mortality with ward care alone , and therefore the level of care appears to have been appropriate . on the other hand , this kind of study can not fully answer the question of what would have happened without the possibility of intensive care . massive postoperative haemorrhage is still a major cause of severe morbidity in gynaecological care , but within the subgroup of critically ill patients , gynaecological malignancy is a major cause of in - hospital death . a delay in the correction of hypovolaemia , in the diagnosis and treatment of defective coagulation or in the surgical control of bleeding are the avoidable factors in most postoperative near - miss cases caused by haemorrhage . a multidisciplinary team involving a gynaecologist , anaesthetist and haematologist early diagnosis and treatment of patients at high risk may also curtail potentially grave consequences of severe infections . primary repair of urinary and gastrointestinal tract injuries is most likely to be successful if done at the time of injury . patients with pre - existing medical conditions , such as cardiovascular and respiratory disorders , might benefit from improved preoperative evaluation of pulmonary function and a pulmonary rehabilitation programme to reduce the risk of respiratory failure . on the other hand , the results of this study indicate that the reasons for and frequency of critical care have not changed greatly over the past few years . on the other hand , we acknowledge that admission and discharge criteria may change over 7 years , even when the same physicians are making decisions . similarly , the decision to not admit a severely ill patient who is beyond surgical treatment or who has progressive terminal disease may change . furthermore , the decision to proceed to surgery in patients with , for example , cardiovascular disease , a decision outside the purview of the intensivists , may well change over time , thereby influencing the frequency of admissions . finally , the current study does not include severely ill patients seen elsewhere who qualified for admission for intensive care at kuopio university hospital but died before they reached the unit . our data show that the need for intensive care in gynaecology often occurs in cancer patients . although the need for intensive care is often unforeseeable and unavoidable , postoperative haemorrhage and pre - existing medical disorders are often related to subsequent severe morbidity . thus , in certain patients presenting with conditions placing them in a high - risk category , clinical suspicion , advanced preparation and timing of operation may decrease the severity of a developing event . when a gynaecological patient needs intensive care , the length of stay is usually short . reported that after discharge from hospital , most general patients who had been admitted to the icu returned to their preadmission physical activity and social status , and this finding also applies to the surviving gynaecological patients in the present study . however , the presence of malignancy may contribute to death in a way not predictable by the apache ii score , but this scoring system has not been validated for gynaecological patients and the accuracy of the score in gynaecological patients needs further clarification . overall , the information obtained in this study may be useful not only for counselling purposes but also when allocating resources . of course , the need for intensive care should not be the only outcome measure considered in the quality of gynaecological care , but it must be one of the most important ones . apache ii = acute physiological and chronic health evaluation ii ; icu = intensive care unit ; ivf = in vitro fertilisation ; tiss = therapeutic intervention scoring system .

stressful situations are not uncommon in everyday life , experienced for example by a doctor in the emergency room , a police officer in action , or a financial trader on a london trading floor . individuals sometimes need to make important decisions when the stakes are high and when not enough information or cognitive resources are available to guarantee a sound choice . however , the high pressure may dramatically change decision making strategies , leading to different choices than would be made without such pressure . for example , individuals may approach situations differently depending on whether decisions are easy to make without far reaching consequences or life - altering and ambiguous . although it is vital to understand decision making under stress , the majority of previous studies on decision making are carried out in non - stressful contexts . only recent years have witnessed a remarkable burgeoning of decision - making research related to stress . but many findings in this field are mixed , leaving the specific effects of stress on judgment and decision making relatively unclear . the purpose of the current review is to summarize evidence from both human and animal studies on decisions under stress and to elucidate the neurobiological mechanisms underlying strategy shifting in the context of stress induced decision making . theories on decision making have proposed that there are two routes to making decisions : a fast route labeled system 1 and a slow route labeled system 2 ( kahneman , 2011 , evans , 2008 , gilovich et al . , 2002 , evans and stanovich , 2013 ) . for example , whenever a snake is detected or believed to be out there , instinctive fear is aroused and avoidance behavior is initiated without much thought . such genetically hard - wired responses can enhance our ability to cope with vital environmental challenges of the type experienced during most of human history . prolonged practice and experience also produce involuntary actions or habits . on the other hand , system 2 runs slowly and in an effortful manner , requiring complex computation . the pros and cons associated with each option are calculated and compared until an optimal choice can be made . comparing both systems , system 2 is thought to be an evolutionarily more recent system and can flexibly check , modify , and override the decisions from system 1 ( tversky & kahneman , 1983 ) . although evidences showing that stress modulates decision making is accumulating ( starcke and brand , 2012 , galvan and rahdar , 2013 , morgado et al . , 2015 ) , there is currently no theoretical framework to explain why stress should influence decisions in certain ways . the evolutionary perspective on stress posits that the stress response has been shaped by natural selection to increase the ability of organisms to cope with situations that require action or defense ( cannon , 1914 ) . when organisms are faced with possible damage or a loss of resources and a fight - or - flight response is required , they can express protective features that allow them to survive adverse conditions and help them mitigate the harmful effects of environmental stresses ( nesse & young , 2000 ) . for example , when under stress , organisms show increases in heart rate and contractility that speed circulation , increases in the rate and depth of breathing that speed gas exchange , increased sweating that cools the body and makes it slippery , increased glucose synthesis associated with spikes in energy , a shunt of blood from gut and skin to muscles , and greater muscle tension that increases strength and endurance ( graham , 1953 ) . based on the evolutionary accounts of stress and the dual - system theories of judgment and decision making , it is reasonable to predict that stress promotes evolutionarily rooted intuitive responses in system 1 . these intuitive responses are fast and require fewer cognitive resources to execute than in system 2 . in normal situations , the intuition system may initiate some default action tendency and the reasoning system checks whether such a tendency is compatible with the current goals and environment . that is , intuition proposes first and reasoning decides whether to approve or to modify it . when under stress , the reasoning system may not check these response tendencies and instead allow individuals to rely on these rigid default actions in response to environmental challenges . grounded on this intuition proposes , reasoning decides type of dual - system , i propose that when under stress , intuitive responses may bypass the examination of reasoning and reach the threshold to become final decisions . thus , stressed individuals may fall back more on intuition and involve less amounts of conscious reasoning . because they are intuitive , automatic processes ought to lead to premature choices , and stress may exacerbate decision biases via shifting decision strategies from deliberation to intuition , labeled as stress induced deliberation - to - intuition intuition is a cognitive process that has multiple layers of meanings and implications . in the sidi model , intuition refers to an automatic , habitual , and evolutionarily based decision making process , corresponding to system 1 . deliberation refers to system 2 , which is a slow , goal - directed , and reasoning based process . this dual system account complements the previous theory that stress promotes a shift from flexible cognitive to rather rigid habit memory systems ( schwabe & wolf , 2013 ) . it is also consistent with the idea that stress impairs prefrontal cortex functions such as working memory and attention regulation , switching from thoughtful top - down control by the pfc to bottom - up control by the amygdala and related subcortical structures ( arnsten , 2009 ) . here , i extend these ideas by explicitly proposing that stress favors intuition versus reasoning in the decision making domain and thus leads to decision biases in some circumstances . the effects of stress on human behaviors are multifaceted ( staal , 2004 ) , including long - term memory ( schwabe and wolf , 2013 , schwabe et al . , 2012 , jols et al . , 2006 ) , working memory ( arnsten , 2009 ) , and learning ( jols et al . , 2006 ) . in this review , i focus only on the effect of stress on reward - based decision making in humans and discuss how the existing empirical findings support the sidi hypothesis . reward - based decision making refers to the process of examining reward magnitudes , probabilities , and risks , comparing options , and choosing a course of action . in the next section , i summarize the findings concerning stress and different decision making components , including the encoding of decision parameters ( e.g. reward / punishment processing and risk analysis ) , executive control , and social decision making ( see table 1 ) . weighing positive and negative aspects of decision options it has been proposed that stress triggers additional reward salience ( stars ) , which may contribute to a stress induced risk preference change ( mather & lighthall , 2012 ) . it is argued that because stress sensitizes dopamine release in reward - processing brain regions ( pruessner et al . , 2004 ) , acute stress may enhance selection of previously rewarding outcomes but impair avoidance of previously negative outcomes . indeed , there is some empirical evidence of reward hypersensitivity under stress . in one study , cortisol responders in the stress group , compared to the no stress group , exhibited differential activity in the ventral striatum to cues predicting high versus low reward ( lewis et al . , 2014 ) . in another , a cold pressor stress enhanced learning about cues that predicted positive outcomes in both younger and older adults ( lighthall et al . , 2013 ) . it has also been found that behavioral preference for sexual rewards increased with long - term cortisol exposure ( 2 months pre - test cortisol derived from a hair sample ) ( chumbley et al . , 2014 ) . however , most studies have shown that acute stress actually reduces reward responsiveness . using the same probabilistic stimulus selection task ( psst ) ( 2013 ) found that stress induced by threat of shock selectively reduced reward sensitivity with no influence on punishment processing in highly stress - reactive individuals ( berghorst et al . , 2013 ) . other research showed that stress induced by threat of shock impaired reward responsiveness , particularly in individuals with anhedonic symptoms ( bogdan & pizzagalli , 2006 ) ; regression analyses indicated that self - report measures of anhedonia predicted stress - induced hedonic deficits even after controlling for anxiety symptoms ( bogdan & pizzagalli , 2006 ) . this is consistent with findings showing that individuals with posttraumatic stress disorder exhibit smaller bilateral striatal activations in response to gains , which are in turn associated with more self - reported motivational and social deficits ( elman et al . , 2009 ) . participants under stress also show decreased differential responses to reward and punishment in the dorsal striatum and ofc ( porcelli et al . , 2012 ) . however , whether such an effect is driven by the reduced sensitivity to reward , enhanced responses to punishment , or both , remains unknown . consistent with these stress studies on reward processing , in a randomized within - subject design , montoya et al . found that administration of a high dose of cortisol ( 40 mg ) down - regulated activity in the brain regions involved in reward - related behavior in male participants ( montoya et al . , 2014 ) . this study provides direct evidence that cortisol acts on brain 's reward circuit , suggesting that stress may modulate reward processing via cortisol . gender differences and individuals ' temperament are also important modulators of stress related reward sensitivity and decision - making . it was found that stress led to greater reward collection and faster decision speed in males but less reward collection and slower decision speed in females ( lighthall et al . , 2012 ) . one study on gender showed that psychological stress , elicited by strongly aversive movie clips , resulted in a significant decrease in reward - related responses in the medial prefrontal cortex ( mpfc ) without affecting ventral striatal responses in women ( ossewaarde et al . individual differences research has shown that higher levels of life stress are associated with lower positive affect for participants with relatively low , but not for those with high , reward - related ventral striatum reactivity ( nikolova et al . , 2012 ) , and high cortisol levels are related to stronger ventral striatum activation ( oei et al . , 2014 ) . taken together , findings regarding whether stress increases or decreases reward sensitivity are still mixed . the sidi account also does not make a simple prediction regarding the direction of reward sensitivity changes under stress . whether stress amplifies or diminishes previous research has shown that individuals tend to strongly prefer avoiding losses to acquiring gains , known as loss aversion ( kahneman & tversky , 1984 ) . loss aversion might be the result of an evolutionary process selecting for the evolutionary most advantageous risk attitude ( mcdermott et al . , according to the sidi model , which emphasizes evolutionary adaptedness of decision - making , stress should potentiate loss aversion . in line with this idea , it has been found that during times of chronic stress , glucocorticoids , acting through the amygdala and hippocampus , promote selective attention to mostly negative precedents and produce a tendency to find threat and risk where none exist ( korte , 2001 , sapolsky , 2000 ) . stress also has been shown to significantly increase ventral striatum aversive ( but not appetitive ) prediction error signals in a learning task ( robinson et al . , 2013 ) . amygdala and insula reactivity to biologically salient stimuli were also exaggerated in a group of combat exposed individuals , suggesting that severe stress exposure sensitizes amygdala and insula reactivity ( van wingen et al . , 2011 ) . under stress as a result of social evaluative threat , low trait - level punishment sensitivity was related to a tendency towards better reward learning and poorer punishment learning ; the opposite pattern was found in highly punishment sensitive individuals ( cavanagh et al . , 2011 ) . stress has also been shown to restore decision - making deficits in heavy drinkers by increasing sensitivity to losses ( relative to gains ) in the iowa gambling task ( igt ) ( gullo & stieger , 2011 ) . only one study found that stress reduces use of negative feedback in reward - based learning tasks ( petzold et al . , 2010 ) . interestingly , one study found that individuals with lower chronic cortisol displayed stronger loss aversion , whereas individuals with higher endogenous cortisol weighted losses and gains more equally ( chumbley et al . , 2014 ) , suggesting that cortisol reduces oversensitivity to potential losses . thus , in the domain of monetary negative feedback processing , the majority of studies consistently demonstrate that stress potentiates punishment detection , supporting the sidi account . learning that certain environment stimuli predict aversive outcomes promotes survival in the face of present and future threats ( maren , 2001 ) . stress may operate as an orchestrated defense that makes innate fight or flight decisions to help animals adapt to threat ( cannon , 1932 , mcnaughton and corr , 2004 ) . consistent with this notion , the sidi model predicts that stress elevates the sensitivity to potential threat in general . it was found that in soldiers , combat stress increased amygdala and insula reactivity to biologically salient stimuli ( i.e. angry and fearful face stimuli ) ( van wingen et al . , such enhanced vigilance to the angry faces was also found in high cortisol responders in a sample of healthy volunteers ( roelofs et al . , 2007 ) . police officers who had larger cortisol increases to the social - stress task subsequently made fewer errors in a threat - related decision making task in which they were deciding whether or not to shoot targets , suggesting that stress may exacerbate vigilance for threat cues ( akinola & mendes , 2012 ) . in additional to the primary physiological fight - or - flight responses to stress for both males and females , it has been proposed that females respond to stress by engaging in nurturing activities designed to benefit both mother and offspring ( the tending pattern ) and by affiliating with social groups to reduce risk ( the befriending pattern ) ( taylor et al . , the sidi model accords with the tend and befriend hypothesis and proposes that stress sharpens threat sensitivity in males in order for the individuals to get ready to engage in fight - or - flight ) and reduces threat sensitivity in females to allow for being tending and befriending . using the fear conditioning paradigm , several studies found gender - dependent effects of stress on fear acquisition and fear extinction . in a typical fear - conditioning procedure , an initially neutral conditioned stimulus ( cs ) acquires emotional properties through predictive pairings with an aversive unconditioned stimulus ( us , e.g. an electric shock ) . during the fear acquisition phase , it was found that stress led to higher conditioned responses in males ( zorawski et al . , 2005 , zorawski et al . , 2006 , jackson et al . , 2006 , merz et al . , 2013a , antov et al . , 2013 ) , but impaired conditioned responses in females ( jackson et al . , 2006 ) . neuroimaging studies using cortisol application , however , revealed reduced conditioned responses in men ( stark et al . , 2006 ) , but enhanced conditioned responses in women in several brain structures ( stark et al . this discrepancy might be due to the fact that exogenous cortisol levels may actually inhibit the stress - induced activation of the hypothalamus when no aversive stimulation was administered , women taking oral contraceptives exhibited higher conditioned responses than men and women in the luteal phase ( merz et al . , 2012 ) , although no effect of cortisol or sex hormones on fear acquisition was found in this study ( merz et al . stress impaired the neuronal correlates of fear learning and expression in men , but facilitated them in oral contraceptives women ( merz et al . , 2013b ) . during the retrieval phase , exposure to stress attenuated fear retrieval in healthy men ( merz et al . , although stressful life events ( sles ) did not have a main effect on fear conditioning , a weak 5-httlpr genotype by sles interaction on fear learning was found ( klucken et al . , 2013 ) . these studies suggest that how stress modulates fear learning depends on sex , current sex hormone availability , and genotypes . taken together , stress seems to boost learning from negative feedback and threat in general , but it also exerts fine - tuned modulation on behaviors depending on the evolutionary demands of male and female homo sapiens . decision makers may fall back on automatized reactions to risk under the influence of disruptive stress . previous research identified the reflection bias , the reversal of risk - attitude when the sign of the outcomes changes ( kahneman & tversky , 1979 ) . this bias emphasizes the inconsistency in risk attitude ( risk aversion in the domain of gains but risk seeking in the domain of losses , the sidi model predicts that stress amplifies this reflection effect since it might be evolutionally adaptive to change risk preference according to domains . consistent with this prediction , using a novel financial decision - making task combined with ice - cold water induced stress , it was found that the reflection effect was significantly increased under stress ( porcelli & delgado , 2009 ) . stress potentially exacerbates behavioral biases in decision - making by inducing more conservative choices for those who are generally risk - averse and more risky choices for those who tend to be risk - seekers . however , using a more complex game of dice task ( gdt ) , it was found that stress reduced the effect of loss framing , such that stressed participants showed less risky behavior compared with non - stressed participants ( pabst et al . , 2013b ) . more studies are needed to further examine how stress modulates domain - specific risk sensitivity . in the domain of mixed gains and losses , previous research on the relationship between stress and risk sensitivity it has been demonstrated that cortisol levels in a group of male traders in london were significantly and positively correlated with financial uncertainty , which was measured by the variance of economic return and the expected variance of the market ( coates & herbert , 2008 ) . thus , financial uncertainty elicits heightened stress , which may in turn shift individuals ' risk preferences . in non - traders , recent research found that individual and aggregate levels of endogenous cortisol predict subsequent risk - taking and administered cortisol shifted investment towards riskier assets ( cueva et al . , 2015 ) . indeed , using a double - blind , placebo - controlled , cross - over protocol , the same research group raised cortisol levels in volunteers over eight days and found that participants became more risk - averse ( kandasamy et al . , 2014 ) . it seems that stress response calibrates risk taking to the circumstances , so that individuals avoid risks in times of prolonged uncertainty , such as a financial crisis . another study found that stress reduced older adults ' but not younger adults ' risk taking in a computer - based driving game ( mather et al . , 2009 ) . thus , stress can lead to risk aversion at least in some circumstances in elderly . on the other hand , it is argued that men should be more likely to take greater risk after stress , analogous to a fight response to stress during competition for territory or other valuable resources , whereas women should be more likely to be conservative after stress , avoiding endangerment of the lives of dependent offspring ( taylor et al . consistent with this view , several studies have found gender differences in response to stress . in one study , acutely stressed ( anticipating a public speech ) men were risk - taking , whereas acutely stressed women were risk - aversive ( preston et al . , 2007 ) . similar gender by stress results were found in research using the balloon analogue risk task in which risk taking is associated with more reward collection ( lighthall et al . , 2012 , exposure to cold pressor stress ( immerse a hand for 1 min into ice water ) also increased neural response to the risky decision task in the dorsal striatum ( putamen ) and anterior insula among men , but decreased the response in these regions among women ( lighthall et al . , 2012 ) . in the igt , the more ( salivary ) cortisol levels were elevated after the tsst the poorer the subsequent performance in the igt in male subjects , whereas in females , slightly elevated levels of cortisol after the tsst improved igt performance and highly elevated levels decreased igt performance ( van den bos et al . , 2009 ) . however , a recent study found only a small but non - significant decrease in performance in the igt task in healthy male subjects ( zhang et al . , 2011 ) . the inconsistent findings regarding the effect of stress on risk taking in males might be due to culture difference ( western vs. eastern ) , which has not been examined yet . the complexity of tasks also plays an important role in modulating the effects of stress on decision making strategies . using the game of dice task , a decision - making task with explicit and stable rules that taps both executive functioning and feedback learning , it was found that stress can lead to disadvantageous decision making ( starcke et al . , 2008 ) . recent studies showed that stressed participants in the single - task group made riskier decisions compared with nonstressed controls , but the effects of stress and a secondary task cancelled each other out ( pabst et al . the authors interpreted these results as showing that stress evokes a shift from serial to parallel processing . thus , task complexity may determine how much executive control is available for making decisions and thus modulate how stress may influence decision - making . these findings , taken together , suggest that stress may amplify risk sensitivity depending on the domain ( win / loss ) , gender , and task complexity . they indicate that the sidi model needs to make more detailed predictions regarding the relationship between stress and risk sensitivity , taking into account contextual situation factors and individual differences . people usually generate judgments and attitudes through automatic processes and then use controlled processes to make necessary adjustments ( kahneman & tversky , 1979 ) . when first being asked is the population of chicago more or less than 200,000 ? , participants automatically anchor their answer on 200,000 when later asked what is chicago 's population ? . if mental resources are diminished , such fine - tuned adjustments may be compromised , according to the sidi model . indeed , a previous study found that under stress , individuals were more likely to make decisions before all available alternatives had been systematically considered , even if no time constraint for the performance of the task was imposed ( keinan , 1987 ) . this study indicates that stress makes decision makers more impulsive and more likely to make unexamined responses . recently , kassam et al . found that challenge stress , which individuals appraise as demanding but manageable , improves adjustment , whereas threat stress , which individuals perceive as a situational demand that outweighs resources , reduces adjustment ( kassam et al . , this study demonstrates that how stress is perceived ( challenge or threat ) dictates how it influences decision making . cognitive control not only inhibits the tendency to make premature responses but also regulates emotional responses to stimuli . a large body of work has shown that responses to emotionally salient stimuli can be flexibly changed and controlled through cognitive regulation ( nolen - hoeksema , 2012 ) . recruiting cognitive strategies to deliberately change the way a stimulus is evaluated , either by reinterpreting ( i.e. , reappraising ) its meaning or focusing on its more positive aspects , has proven effective at reducing the subjective , physiological , and neural components of emotional arousal . it is specifically under stressful conditions that individuals may benefit most from such deliberate forms of emotion regulation . however , the sidi would predict that the efficacy of cognitive regulation attempts after stress exposure would be reduced due to the diminished cognitive control ability under stress . indeed , regulation training has been shown to produce robust fear reduction in non - stressed participants but not stressed participants ( raio et al . , 2013 ) . a recent neuroimaging study also found that acute stress impairs self - control in goal - directed choice by reducing connectivity between ventromedial pfc and dorsolateral pfc regions linked to self - control success ( maier et al . , 2015 ) . these results highlight critical limitations of this technique to control affective responses under stress ; other techniques such as emotion regulation strategies may also have limited power to overcome stress - biased decisions . if stress markedly impairs the cognitive regulation of emotion , it is less likely than individuals can exert cognitive control to overcome stress induced decision biases . another crucial function of cognitive control is to keep individuals on the track of pursuing goals . in the ever - changing environment , individuals need to adjust their behaviors according to the goals they want to accomplish . such goal - directed flexible behaviors demand an effortful control and monitoring of the response . to increase response efficiency , recurring decision processes can be automated to form a rule or a habit . the goal - directed system learns action - outcome associations ( dickinson , 1985 ) , and is believed to be mediated by prefrontal cortex areas and dorsomedial striatum ( balleine & o'doherty , 2010 ) . by contrast , the habitual system learns stimulus response associations regardless of outcomes , and is supported by posterolateral putamen ( balleine and o'doherty , 2010 , yin and knowlton , 2006 ) . a recent study in humans showed that stressed individuals continued to perform the action associated with a particular outcome even after this outcome had been devalued , accompanied by a significant decrease in explicit knowledge of action - outcome contingencies ( schwabe & wolf , 2009 ) . the -adrenoceptor antagonist propranolol , which blocks the action of adrenaline and noradrenline , blocked this stress - induced bias toward habit behavior , suggesting that noradrenergic activation plays a crucial role in such stress elicited decision strategy switching ( schwabe et al . , 2011 ) . importantly , a longitudinal assessment of the stressed individuals showed that both the structural and functional changes triggered by stress are reversible and that decisions become goal - directed again ( soares et al . , 2012 ) . the link between neuroticism and distress has been shown to be strong in individuals with high perseverative response tendency ( i.e. , less switching across consecutive trials ) , suggesting a relationship between distress and response preservation ( robinson et al . , 2006 ) . a recent psychopharmacological study found that cortisol impaired performance in the cognitive reflection test ( crt ) by biasing responses toward intuitive but incorrect answers ( margittai et al . , 2015a ) . the profound influence of stress on individuals ' ability to adjust their behavior to changing circumstances can even be found in infants . recent evidence showed that 15-mo - old infants exposed to stress kept performing a previously effective action , even after the action suddenly became ineffective , suggesting that stressed human infants tend to perform habitual behavior rigidly ( seehagen et al . , 2015 ) . these findings echo the well - established stress - induced switching from goal - directed to habitual control of action in rodents ( see a more comprehensive review in ref . chronic stress caused rats to become insensitive to changes in outcome value ( graham et al . , 2010 ) , accompanied by atrophy of the medial prefrontal cortex and caudate and hypertrophy of the putamen ( dias - ferreira et al . , 2009 ) . stressed rats also relied more on habit memory rather than cognitive memory to guide their actions ( elliott & packard , 2008 ) . taken together , evidence from both human studies and animal research point to the possibility that stress biases decision - making strategies by shifting from goal directed decisions to habitual choices ( schwabe & wolf , 2011 ) . to summarize , previous studies suggest that cognitive control is diminished under stress , leading to premature decision - making . without optimal cognitive control , decision makers are less likely to do fine - tuned adjustments , exhibit weakened cognitive regulation of emotion , and also often fail to choose goal - directed actions . in accord with the sidi model , the deliberation to intuition framework for understanding decision making under stress does not entail the assumption that stress promotes prosocial or antisocial responses . such responses should be adapted in ancestral human environments , thus are ecologically rational ( hammerstein and hagen , 2005 , gigerenzer and selten , 2002 ) . have shown that individuals are more cooperative and altruistic when they have to make choices quickly under time pressure compared with conditions in which they are given enough time to do analytic calculation ( rand et al . , 2012 , rand et al . , 2014 ) . however , whether such a conclusion can be generalized to other social contexts awaits further exploration . if individuals ' innate tendency is to cooperate , the sidi hypothesis would hold that stress promotes cooperation in social situations . the majority of studies on stress and prosociality supports this prediction ( buchanan & preston , 2014 ) , across a range of experimental paradigms . in a study using the dictator game , responders under social evaluation stress allocated significantly more money than controls ( takahashi et al . , 2007 ) . using the trust game , it was found that male participants who experienced acute social stress engaged in substantially more prosocial behavior ( trust , trustworthiness , and sharing ) compared with participants in a control condition ( von dawans et al . , 2012 ) . the authors reasoned that stress triggers social approach behavior , which operates as a potent stress buffering strategy in humans , thereby providing evidence for the tend - and - befriend hypothesis and also extend this model to male individuals . social closeness also modulate prosocial behaviors in males such that stressed males only showed increased generosity towards close but not distant others ( margittai et al . , 2015b ) . survey results have also shown that higher levels of acculturative stress are linked to greater anonymous prosocial tendencies and with fewer costly ( altruistic ) prosocial tendencies ( mcginley et al . , recent studies using complex moral dilemmas have shown that persons under stress show significantly fewer utilitarian responses compared to control subjects ( youssef et al . , 2012 , however , contrary to the above findings that stress promotes prosocial behaviors , another study found that stress decreased men 's tendency to reject unfair offers in the ultimatum game ( ug ) and reduced the amount of money allocated to the other participant in the dictator game ( vinkers et al . , 2013 ) . research using the everyday moral decision - making task ( emdm ) , which seeks to distinguish between altruistic and egotistical behavior , has shown that the cortisol stress response was associated with egoistic decision - making in high - emotional situations ( starcke et al . , 2011 ) . for example , in the ug , rejecting unfair offers is usually interpreted as a costly prosocial behavior since such altruistic punishment costs individuals the potential earning that they could have otherwise earned ( fehr & gachter , 2002 ) . however , they are many social motives may drive such behavior ( pillutla and murnighan , 1996 , sanfey et al . , 2003 ) . recent studies also showed that the tendency to reject unfair offers in the ug is not correlated with individuals ' tendencies to exhibit various prosocial behaviors in other situations ( yamagishi et al . , 2012 ) . also , how individuals believe they will act in hypothetical moral decision situations may differ dramatically from how they actually act in the real world situations ( ajzen et al . another possibility is that the link between stress and prosocial tendency is modulated by social closeness . a recent study found that men tested 20 min after stressor onset indeed showed increased generosity towards close but not distant others compared to non - stressed men or men tested 90 min after stressor onset ( margittai et al . , 2015b ) . thus , the proposal tendency elicited by stress may be limited to close friends or family members but not extended to strangers . nevertheless , majority of these findings do provide support for the notion that stress leads to prosocial behaviors ( buchanan & preston , 2014 ) . stress also influences individuals ' ability to mentalize . in the beauty contest game , which is designed to measure the depth of reasoning , participants under stress chose higher numbers than non - stressed participants , indicating less strategizing ( leder et al . , 2013 ) . in this game , entrants are asked to pick a number between 0 and 100 , with the winner of the contest being the person that is closest to 2/3 the average number picked for all contestants . the lowest , ' level 1 players believe that all other players choose randomly and therefore choose 33 ( 2/3 of 50 ) . similarly , level 2 players would choose 22 ( 2/3 of 33 ) . the smaller the number players choose , the higher level of strategic thinking they are . a recent study found that stress exposure influences individuals ' mind reading abilities , assessed by movie for the assessment of social cognition ( masc ) . high cortisol responses led to elevated masc scores in men but reduced masc scores among women ( smeets et al . , these results partially support the idea of sex differences in biobehavioral stress responses , with men engaging in fight - or - flight responses and women showing tending and befriending behavior ( smeets et al . , 2009 ) . it is possible that in certain situations , the default action for men to respond to threat is to fight rather than flee . overall , there are only few studies examining the effects of stress on social decisions , although social decisions are the most important choices we make in our daily life . the lack of neuroimaging data in this field also hinders our understanding of the neural mechanisms mediating stress and social decision making . how human brains respond to stress is a topic that has been investigated extensively in both animal studies and human research , see reviews in refs . ( schwabe and wolf , 2013 , arnsten , 2009 , roozendaal et al . the stressful event leads to activation of two biological systems : the autonomic nervous system ( ans ) , involving release of ( nor)adrenaline , and the hypothalamo - pituitary - adreanal ( hpa ) axis ( arnsten , 2009 ) . immediate fast responses are mostly mediated by the sympathetic nervous system ( sns ) and the associated release of epinephrine from the adrenal medulla ( joels & baram , 2009 ) . a more delayed response - release of cortisol emanates from the adrenal cortex ( de kloet et al . , 2005 , droste et al . , 2008 ) this is initiated by neural signals to the hypothalamus , which releases corticotropin - releasing hormone ( crh ) , resulting in secretion of adrenocorticotropic hormone ( acth ) from the anterior pituitary gland on the bottom of the brain . stress evokes high concentrations of catecholamines such as noradrenalin and dopamine , and an increased concentration of the glucocorticoid cortisol . the changes of these hormones may alter functioning of neural correlates of decision making , such as the dorsal pfc , ventromedial pfc / anterior cingulate cortex , striatum , hippocampus , and amygdala . the sidi model predicts the existence of two neural circuits that support system 1 and system 2 respectively . stress should enhance the system 1 intuition related neural activity ( e.g. , in subcortical regions ) and decrease system 2 reasoning associated brain activity ( e.g. , in prefrontal cortex ) . here , i selectively review neuroimaging studies that examine both the prefrontal control system and subcortical emotional response system in relation to stress . previous neuroimaging research has demonstrated a shift in activity from the newly developed prefrontal cortex to phylogenetically older midbrain regions when threat stress draws closer . using a pac - man like computer game in which volunteers were pursued by a virtual predator , mobbs et al . found that as the virtual predator grew closer , brain activity shifted from the ventromedial prefrontal cortex to the periaqueductal gray ( mobbs et al . , 2007 ) . in another study , phylogenetic threat was introduced by making participants believe that a tarantula was placed close to their foot , and the experience of fear coincided with augmented activity in a cascade of fear - related brain networks including the periaqueductal gray , amygdala , and bed nucleus , as well as diminished activity in the orbitofrontal cortex and posterior cingulate cortex ( mobbs et al . , 2010 ) . in a series of studies , social evaluative threat was also shown to cause activity increases in a more dorsal pregenual cingulate region , whose activity was coupled with heart rate increases ; conversely , social evaluative threat caused activity decreases in a right ventromedial / medial orbital region , which were coupled with heart rate increases ( wager et al . numerous studies have identified structural and functional connectivity between prefrontal cortex and amygdala , a key region in emotion processing ( kim et al . , 2011 , kober et al . , 2008 ) . functional coupling between the prefrontal cortex and the key reward region striatum is also well established ( van den bos et al . , 2012 , haber and knutson , 2010 ) . if stress interferes with cognitive systems , it is plausible that reduced cognitive control ability would lead to an exaggerated reliance on lower - level automatized systems ( masicampo & baumeister , 2008 ) . a recent study , using both pet and fmri , demonstrated that in the montreal imaging stress task ( mist ) , stress induced significant deactivation of the limbic system including hippocampus , hypothalamus , medio - orbitofrontal cortex and acc in subjects who reacted to the stressor with increased cortisol ( pruessner et al . , 2008 ) . large - scale network analysis has provided similar results . in a recent study , responsiveness and interconnectivity within a network including cortical ( frontoinsular , dorsal acc , inferotemporal , and temporoparietal ) and subcortical ( amygdala , thalamus , hypothalamus , and midbrain ) regions increased during exposure to a fear - related acute stressor ( hermans et al . , 2011 ) . importantly , b - adrenergic receptor blockade , but not cortisol synthesis inhibition , diminished the increase , suggesting that the neuromodulator noradrenaline drives this network reorganization . this study not only identified two key networks involved in stress but also highlighted the importance of the balance between the two networks . recently , these researchers proposed that there are two brain networks , salience ( e.g. emotional reactivity ) vs. executive control ( e.g. working memory ) , in governing stress ( hermans et al . , 2014 ) . the two stress - related networks play differential roles in the neurobiology of decision making under stress and may be analogous to the system 1 and system 2 framework in the sidi model . taken together , these neuroimaging studies show that stress diminishes activity in prefrontal cortex and augments activity in subcortical regions including amygdala , hippocampus , and midbrain . this activity profile supports the notion that stress evokes a switch from deliberation that is supported mainly by prefrontal cortex to intuition that involves phylogenetically older brain regions such as subcortical areas ( see fig . this perspective , referred to as the stress induced deliberation - to - intuition model , posits that decision making under stress is influenced by a combination of immature cognitive control and heightened intuitive response tendency , which are tied to the prefrontal reasoning system and the subcortical intuition related regions . it is acknowledged that not all evidence mentioned above fits the simple dichotomy the sidi model assumes . for instance , studies often yield mixed results regarding whether stress increases or decreases sensitivity to reward . there are several methodological caveats that may account for such a discrepancy , such as the time between stress induction and task performance ( schwabe & wolf , 2014 ) , the number of subjects , and the power of the stress induction task used ( allen et al . , 2014 ) . moreover , the stress effects are modulated by age ( galvan & rahdar , 2013 ) , gender , personalities ( lempert et al . , 2012 , richards et al . , 2014 ) , the nature of the experimental tasks , appraisal of threat ( van wingen et al . , 2011 ) , individuals ' basal cortical level , and cortisol responses during stress ( roelofs et al . , 2005 ) . the effects of stress on decision - making are also multidimensional , ranging from reward / punishment sensitivity and risk tendency to adjustment and emotion regulation . although previous studies tried to isolate these dimensions , it is worth noting that stress may influence some or all of these decision - making stages . however , despite all the factors that complicate the relationship between stress and decision making , it is also worth noting that the majority of studies do provide convergent evidence that stressed individuals are more sensitive to threat / punishment , less likely to exert cognitive control to examine their responses , and more prosocial in social contexts . at the neural level , accumulating evidence suggests that stress diminishes activity in the evolutionally new prefrontal cortex and exaggerates evolution rooted subcortical regions . thus , both behavioral and neuroimaging studies so far support the sidi model , although further investigation of stress and decision making is urgently needed . the sidi can serve as a simple but useful tool to guide further research on stress and decision - making . first , a large number of decision biases are believed to be a result of the imbalance between system 1 and system 2 . in addition to the several biases mentioned before ( e.g. , the framing effect and reflection effect ) , there are many other biases that should be examined to further test the sidi model , such as social conformity ( huang et al . , 2014 , asch , 1955 ) , the decoy bias ( hu and yu , 2014 , huber et al . , 1982 ) , the default bias ( yu et al . , 2010 ) , delay discounting ( yu , 2012 , mischel et al . , 1989 , haushofer et al . , 2013 , haushofer and fehr , 2014 ) , and availability ( tversky & kahneman , 1974 ) . for example , it is reasonable to predict that individuals would be more vulnerable to social influence if they rely more on system 1 to make decisions , as the sidi model posits . moreover , the sidi may also predict how stress interacts with psychiatric disorders such as addiction , depression , and bipolar mood disorder ( zhang et al . for example , stress may amplify the decision - making deficits in addictive patients ( zhang et al . , 2011 ) . stress may also interact with aging to determine decision making in the elderly , whose prefrontal cortex based cognitive control ability is already compromised ( peavy et al . , 2009 ) . the sidi model would also predict that the effect of stress on decisions should mimic effects found in patients with prefrontal lesions or individuals after brain stimulation , which diminishes activity in cognitive control related brain regions . the distinction between deliberative and intuitive systems has enjoyed considerable popularity in social cognition and decision making domains ( kahneman , 2011 , evans , 2008 , gilovich et al . , 2002 , evans and stanovich , 2013 , evans , 2003 ) , although it has also been challenged ( kruglanski & gigerenzer , 2011 ) . across different sub - fields of psychology , a diverse set of dual - process models have been proposed , including analytic vs. heuristic ( kahneman , 2011 , evans , 2008 , evans and stanovich , 2013 , evans , 2003 ) , conscious vs. unconscious ( dijksterhuis et al . , 2006 , lassiter et al . , 2009 ) , associative versus rule based ( ashby & o'brien , 2005 ) , goal - directed vs. habitual ( dickinson , 1985 ) , cognitive vs. affective ( bechara et al . , 1997 ) , and reflective ( c - system ) vs. reflexive ( x - system ) models ( lieberman , 2007 ) . recently , there have been discussions about how stress modulates leaning and decision making through the lens of duality models . based on research with non - human animals , arnsten posited that stress impairs pfc function and strengthens functions mediated by amygdala and other subcortical regions , thus creating a vicious cycle ( arnsten , 2009 ) . in response to stress , the amygdala activates stress pathways in the hypothalamus and brainstem , which produces high levels of catecholamine release ( e.g. noradrenaline and dopamine ) . on the one hand , this disrupts the top - down control by the pfc and allows the bottom - up control by sensory cortices to be dominating . on the other hand , high levels of catecholamine strengthen amygdala functioning which biases individuals towards reflexive and habitual responding ( arnsten , 2009 ) . for example , whether stress always exerts opposite influence on pfc and amygdala system or stress only alters one system and leaves the other intact remains to be examined . schwabe and wolf proposed that stress influences instrumental behavior by favors habitual over goal - directed memory systems ( schwabe and wolf , 2013 , schwabe and wolf , 2011 ) . extending previous these accounts , the current sidi model is not limited to specific forms of decision making ( e.g. spatial navigation and instrumental learning ) and attempts to integrate decision parameters encoding , risk evaluation , cognitive control , and social emotions . the sidi model extends these proposals by integrating a range of decision - making components that allow more precise predictions of behavior . importantly , the proposed model describes how the two systems interact at various stages of processing in a synergistic or antagonistic fashion . the sidi model differs from previous accounts on stress and decision - making on how the two systems interact . previous accounts seem to take the parallel - competitive forms of dual - process theory , based on the notion that two systems work in parallel and produce two forms of decisions that may lead to competing attempts to control the behaviors . the sidi model corresponds more closely to the default - interventionist assumption that intuition system supplies rapid default responses ( intuition proposes ) and deliberation system may approve or intervene upon ( deliberation decides ) ( evans , 2008 ) . the key differences between the two approaches relate to the order and dominance of different cognitive processes . whether the two systems operate in parallel or the intuition system is fast and the deliberation is slow to decide can be tested using high temporal resolution neuroimaging methods combined with computational models such as granger causality analysis ( goebel et al . , 2003 ) . how the pfc - mediated deliberation system exerts control over the subcortical - based intuition system can also be delineated using functional connectivity analysis methods such dynamic causal modeling ( friston et al . , 2003 ) . the challenge for researchers in this field is to delineate the exact neuropsychological processes of the switching from deliberation to intuition system and tease apart factors that modulate such switching . neuroimaging methods can be applied to elucidate the biopsychological processes of decision making under stress . the arbitrary between intuitive and deliberative responses has not been directly investigated in the context of stress . one possibility is that there is an arbitrator that keeps track of the degree of reliability of the two systems and uses this information in order to proportionately allocate behavioral control . another possibility is that the arbitrator deems that the intuition system should be relied on , it allows all decisions bypass the deliberation processes . in this case , the arbitrator modulates only one of the two systems due to reasons of computational efficiency . a recent computational neuroimaging study provides evidence for the existence in the human brain of an arbitrator mechanism that determines the extent to which model - based ( goal - directed ) and model - free ( habitual ) learning systems control behavior ( lee et al . , 2014 ) . , found that inferior lateral prefrontal and frontopolar cortex encode both reliability signals of both systems and the output of a comparison between those signals , implicating these regions in the arbitration process ( lee et al . , 2014 ) . importantly , instead of modulating either model - based or model - free systems depending on which one has the most reliable estimate , the controller appears to work by selectively gating the model - free habitual system . whether stress works in a similar way in determining which system should control behavior remain to be examined using computational modelling and neuroimaging methods . although some believe that system 2 is superior to system 1 and always leads to rational decisions , this is not always the case , especially for experts . for example , the recognition - primed decision model proposes that people use their experience in the form of patterns ( klein , 1999 ) . these patterns help the decision makers to recognize the relevant cues , identify plausible goals , and remember typical types of reactions in certain type of situation . when people need to make a decision , they can quickly match the situation to the patterns they have learned and experienced in the past . thus , if the first workable option turns out to be satisfactory and deliberative analysis does not produce a significantly better one , system 1 can produce even better and more effective outcomes than system 2 ( dijksterhuis et al . , 2006 , reyna , 2004 , johnson and raab , 2003 ) . for example , chess experts rapidly retrieve a schema that usually provides a solution to the current problem rather than exhaustively comparing all options ( chase & simon , 1973 ) . intuitive responses are not necessarily associated with disadvantageous outcomes and more cognitive control is not always more adaptive . whether a response or tendency is advantageous or not is context dependent . risk seeking may be advantageous if the task is structured to favour risk - seeking strategies . thus , stress does not necessarily degrade decision quality , and it can improve decisions in some circumstances . the sidi model is agnostic with respect to whether the stress induced changes in decision making strategies produce desirable or undesirable consequences . it is worth noting that the sidi model does not imply that stress is always detrimental for decision - making . in certain circumstances , moreover , it might not be obvious that which responses are our rapid intuitive responses and which are not , especially in social contexts . thus , without a clear definition of what an innate response is , the argument that stress promotes intuitive processes is a circular one . humans may have default tendency to be risk taking in certain situations and be risk aversion in other situations ( e.g. the reflection bias ) . it is arbitrary to simply assign a general tendency ( e.g. risking taking , loss aversion , and prosocial ) as the default or an innate response . research on stress should be integrated with studies in which time constraints , physical fatigue , hunger , and cognitive loads are manipulated in order to better define what intuitive responses are . otherwise , the hypotheses of the sidi are derived post - hoc and therefore represent a common factor to many stress phenomena . first , stress level is a matter of degree . intuitively , too much as well as too little stress is often detrimental for cognitive performance . in most studies mentioned above , researchers assume that the elicited stress levels are well above the optimal level . if individuals reach the ultimatum level of stress that they can not handle , they may crash and give up . lack of stress , such as boredom and apathy , may also hinder individuals ' performance due to lack of motivation . thus , the relationship between he stress level and performance might be inverted u - shape ( yu , 2015 ) . second , the time dependency of cortisol effects might also modulate when and how stress shapes behaviors . previous studies have revealed time - dependent effects on working memory processing , emotional memory , and brain function in general ( henckens et al . the rapid , non - genomic effects of cortisol in combination with noradrenaline increase subcortical and decrease prefrontal functioning , whereas the aftermath of stress is associated with upregulated prefrontal functioning ( hermans et al . the sidi model applies to the immediate rapid effect of stress and may make opposite predictions for the late or recovery stage of stress . the sidi model would predict that when under time pressure , the greater the psychological stress , the greater the tendency to make a premature choice ( hence the shorter rt ) or seek premature closure . fourth , different ways in which stress is induced in the laboratory ( using social evaluation , cold - pressor tasks , pharmacological methods , and stressful life events ) may also impact the stress effect . for example , cold - presssor tasks may produce physical challenges whereas social evaluation tasks may tap more into psychological and social stress , although they all elevate cortical levels . individuals may develop various compensatory strategies and coping responses to deal with stress over a long time period . environmental factors such as social support and societal interventions may also play a role in determining the effects of chronic stress on behaviors . finally , individuals ' characteristics such as age ( galvan & rahdar , 2013 ) , gender , and personalities ( lempert et al . , 2012 , richards et al . , 2014 ) may also interact with stress and determine to a large degree how stress modulates decision - making . facing similar stressful events , some may cope well but others may develop psychological disorders such as anxiety or even posttraumatic stress disorder ( ptsd ) , depending on individuals ' life experience and personal traits . at this stage , it is hard to develop a model that can explain all existing findings in the literature given the complexity of this issue . despite that the sidi model may have oversimplified the evidence or overlooked inconsistencies in the literature , the framework offers a useful model for understanding how stress modulates decision making in humans . specially , in the absence of an alternative theoretical account , the sidi model provides a useful guideline for the formulation of specific and testable hypotheses and an integrated account for the observed effects of stress on behavioral outcomes and the structural and functional neural changes in the human brain . there are findings that run counter to predictions derived from the sidi model , suggesting that more nuanced analyses and theory modification are needed . further specification of the model will become possible as the field develops new advanced research methods and refined ways to integrate behavioral and neuroscientific sources of evidence . the current review provides an overview of the recent research on how stress shapes decision - making . stress may interfere in this competition between emotion and cognitive functioning and thereby impair decision - making . the framework presented in this review serves as a new way to revive the discussion on how stress influences decisions and may encourage researchers to adopt a more theory - based and hypothesis - driven approach to their investigations . observing some behavior effects of stress in isolation is not sufficient to allow us to make conclusions about how stress perpetrates decision - making processes in general . the current review provides a relatively comprehensive summary of stress and economic decision - making and proposes that stress may potentiate decision biases . the stress induced deliberation - to - intuition model provides testable hypotheses and can guide future research in this direction . although what constitutes intuitive decisions is debatable , integrating findings from studies that manipulate reasoning abilities may inform research on stress and decision - making . this field is still in its infancy and requires more research to fully understand the phenomenology of decision - making under stress . understanding how stress biases decisions would be highly useful to those people working or living under extreme stress , such as military soldiers in the war zone , plane pilots , and emergency responders . the authors declared that they had no conflicts of interest with respect to their authorship or the publication of this article .

note : in order to minimize pcr carryover contamination , it is important to keep several important key steps in the pcr physically - separate from one another : 1 ) template ( dna ) preparation , 2 ) pcr setup , and 3 ) gel electrophoresis . it is also recommended to use barrier tips in order to prevent culture or reagent contamination of pipetters . inoculate 1 ml of luria bertani broth or other complex medium ( brain heart infusion , superbroth , etc . ) with salmonella isolate from a single isolated colony . transfer 1 ml to a sterile , 1.5 ml capacity microfuge tube , centrifuge the bacterial cell suspension at 4,500 xg for 2 min . to pellet cells . decant supernatant , resuspend the cell pellet in 1 ml of 100% ethanol and set at room temperature for 10 min . pellet cells as before ( 4,500 xg , 2 min . ) , remove the supernatant and resuspend cells in 1 ml pbs . centrifuge cells ( 4,500 xg , 2 min . ) , discard supernatant and resuspend cells in 1 ml of sterile dh2o . dilute the final cell suspension 1/20 in dh2o ( 5 l/95 l dh2o ) and store at -20c . the shelf - life of the whole cells as pcr template at -20c is about one month . note : preparation and dispensing of the pcr reaction mix ( template , buffers , oligonucletoideprimers , nucleotides and taq dna polymerase ) needs to be performed in a physically separate room dedicated and preferably done in a pcr / uv workstation . note : the pcr set up room must also be self - contained with a designated -20c freezer for storage of pcr reagents ( buffers , oligonucleotide primers , and nucleotides ) , enzymes and template , dedicated pipetter set for pcr setup , disposable latex gloves , laboratory coat , barrier tips , microtiter plates , glass capillaries , microfuge tubes , and 10% bleach for decontaminating surfaces . use a dedicated pipetter set ( p10 , p100 , and p1000 ) to dispense pcr reagents . this pipette set must never leave the set - up workstation . note : obtain molecular biology or pcr grade dh2o and aliquot 1 ml into 1.5 ml capacity centrifuge tubes . note : decontaminate the work area before use with uv illumination and/or wiping down surfaces with 10% bleach . minimize back and forth traffic from pcr set - up to areas where the pcr reactions are incubated in the thermocylcer and pcr products ( amplicons ) are separated on agarose gels . if you go back into the pcr set - up area , dispose of the pair of latex gloves you are currently wearing and put on a new pair of gloves . make a master primer stock in dh2o to a concentration of 5x10 m. dilute the primers 1/20 in dh2o ( 5 l/95 l dh2o ; 25 m ) . retrieve pcr reagents and template from -20c freezer , and allow reagents and samples to thaw out on ice . dispense 9l of the pcr reaction mix into each of 10 round bottom wells in a sterile , 96-well , polystyrene microtiter plate , starting at the top of the column ( e.g. a1 ) and moving down the column to the top of the next . add 1 l of dh2o to the 9 l of pcr mix ( no dna control ) to the first well ( a1),0.5l of the positive control templates ( i / g , m typing primers- typhimurium and enteritidis ; r / z10 typing primers- heidelberg and hadar ; and 1,2/e , n , x typing primers- typhimurium and hadar ) to 9 l of the pcr mix in the 2 well ( b1 ) , and 1 l of escherichia coli k12 dh5 to the 9 ml of pcr mix in the third well ( c1 ) . for each additional well ( d1-h1 ; a2 , b2 ) , add 1 l of the thawed sample template to the 9 l of the pcr reaction mix . for the i / g , m allelotyping pcr , s. enterica serovar typhimurium ( i ) and enteritidis ( g , m ) serve as positive controls , and salmonella serovars heidelberg ( r ) and hadar ( z10 ) are the positive controls for the r / z10 allelotyping multiplex pcr . place 10 l capacity glass capillaries into designated holders for the idaho technology rapidcycler ( 8) . once secured in the holder , load each glass capillary with the pcr reaction mix by touching the capillary to the bottom wells of the microtiter plate . tilt the holder to allow the liquid to move down the tube and provide space between the ends and the pcr mix before heat sealing the glass tubes with a butane torch to seal both ends of the capillaries . place the capillary tubes and holder into the idaho technology rapidcycler and use thermocycler programs described in table 1 for the different allelotyping primers to run the pcr . note : wear a different lab coat designated for use in electrophoresis lab and a new pair of disposable latex gloves . note : wear uv protective eye goggles or face shield and always were gloves when handling ethidium bromide , especially agarose gels . prepare 100 ml molten 1.5% ( w / v ) molecular biology grade agarose in 1xtae ( or 1x tbe ) electrophoresis buffer ( 7 ) by repeatedly heating the agarose suspension in a microwave set at 20% power for 2 - 5 min intervals with periodic visual inspection . set the molten agarose in 60c water bath until it equilibrates to the temperature of the water bath . choose a gel comb with sufficient teeth to produce enough wells for controls , samples , and at least 3 molecular weight standards for their placements at the ends and the middle of the agarose gel . add 2 l of ethidium bromide ( 10 mg / ml ) to 100 ml of molten 1.5 % ( w / v ) agarose in 1xtae ( or tbe ) , gently swirl bottle to mix , avoid producing bubbles , and gently pour the contents of the bottle into the middle of the gel mold for 15 by 10 cm agarose gel . use the edge of tissue paper or paper towel to remove any bubbles in the agarose gel . let the gel mold set at room temperature until agarose solidifies ( 30 - 45 min ) . transfer the gel tray with gel & comb to submersible , horizontal electrophoresis chamber containing 1x tae ( or 1x tbe ) electrophoresis buffer . gently remove the comb from the agarose gel . check placement of the gel tray relative to the cathode or positive pole of the electrophoresis chamber to be sure this pole is at the bottom of the gel . note : remember the negatively charged dna migrates towards the cathode ( i.e. , " run to red " ) . premix 200 l of dna molecular weight markers ( 0.25 g/l ) with 40 l of 6x dna loading dye . load 4.8 l of the premixed dna molecular weight marker xiv to the first , middle and last wells . load each of the pcr samples starting with well 2 and advancing to each subsequent well , moving from left to right . avoid loading the sample in any of the wells containing the molecular weight standard . to load samples contained in each glass capillary : remove each capillary from its holder and etch both ends of the capillary with a glass cutter.place thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary.place the capillary dispenser on the end opposite of the pcr reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube.place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the ficoll - weighted sample into the agarose well . be careful not to puncture the well with the capillary or introduce an air bubble into the well by turning too much past when the last of the liquid leaves the capillary . remove each capillary from its holder and etch both ends of the capillary with a glass cutter . place thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary . place the capillary dispenser on the end opposite of the pcr reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube . place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the ficoll - weighted sample into the agarose well . be careful not to puncture the well with the capillary or introduce an air bubble into the well by turning too much past when the last of the liquid leaves the capillary . once all the samples and standards are loaded , set the constant voltage of the power supply to 90 v ( 9 volts / cm agarose gel ) . discontinue electrophoresis once the yellow dye ( taurazine ) front is 1 cm from the bottom of the gel . once electrophoresis is completed , transfer the gel to a uv transilluminator to visualize dna fragments and molecular weight standard . capture image on polaroid film using a polaroid camera with orange glass filter ( settings : 2 x , and 4.5 y ) or as a digital image using a ccd camera and print image with a thermal printer . rinse 3 times with dh2o and place back in pcr setup room to air dry . results for allelotyping pcr of salmonella isolates 1 - 10 are shown in figure 1 ( lanes 3 - 12 ) and interpreted as follows . an o allele designation is given to salmonella isolate based on amplicon ( pcr product ) matching in size to the pcr control and as predicted for the o allele primer sets used ( 3 ) : b- 560bp , c1- 340bp , c2- 400 bp , d1- 620 bp , and e1- 280 bp . for isolates tested with the o allelotyping pcr ( fig . 1a ) , we assigned o alleles b ( lanes 10 - 13 ) , c1 ( lanes 7 - 9 ) , c2 ( lanes 4 , 5 ) , d1 ( lane 3 ) , and e1 ( lane 6 ) to the ten salmonella isolates tested in lanes 3 - 12 . 1a , for h1 alleles i ; g , m ; r ; and z10 ( fig . again , a h1 allele is assigned to each isolate dependent on the presence of an amplicon matching in size to the pcr control and as predicted for the 4 h1 allele primer sets used ( 3 ) : i- 510 bp ( fig . 1b , lane 2 ) ; g , m- 310 bp ( fig . 1b , lane 2 ) ; r- 170 bp ( fig . 1c , lane 2 ) ; and z10- 360 bp ( fig . 1c , lane 2 ) . alleles were assigned to one of the ten salmonella isolates : i - isolates 3 and 8 ( fig . 1b , lanes 5 & 10 ) ; g , m- isolates 1 and 5 ( fig . 1b , lanes 3 & 7 ) , r to isolates 6 and 9 ( fig . 1c , lanes 8 & 11 ) ; and z10 to isolates 2 , 7 , and 10 ( fig . finally , salmonella isolates were tested by pcr for h2 alleles associated with the 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex and e , n , x ; e , n , z15 antigen complex . the primer sets for the h2 1,2 ; 1,5 ; 1,6 ; 1,7 complex and h2 e , n , x ; e , n , z15 complex produce 290 bp and 150 bp amplicons , respectively ( 3 ) . the primer sets can not distinguish specific h2 alleles within either h2 antigen complex to assign a definitive allele type , ex . salmonella isolates 4 , 6 , 8 - 10 were positive for h2 alleles associated with h2 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex , while isolates 2 and 7 were positive for h2 alleles associated with e , n , x ; e , n , z15 antigen complex ( data not shown ) . while salmonella isolates 1 , 3 , and 5 were negative for the two h2 antigen complexes , only isolates 1 and 5 were negative for h2 flagellin , as determined using a generic h2 flagellin pcr ( 1 ) ( data not show ) . these results are summarized in table 2 along with the presumptive salmonella serovar assigned to each isolate . multiplex pcr for identifying specific o and h alleles associated with s. enterica serovars enteritidis , hadar , heidelberg , and typhimurium . ( b , c ) h1 allele multiplex pcr for identifying flagellin alleles : i / g , m ( b ) and r / z10 ( c ) . lanes 1and 13 : 100 bp ladder ( promega ; madison , wi ) ; lane 2 : multiplex pcr controls for o alleles b , c1 , c2 , d1 , and e ( a ) , h1 alleles i / g , m ( b ) , and h1 alleles r / z10 ( c ) ; and lanes 3 - 12 : salmonella isolates 1 - 10 ( table 2 ) . protocol for salmonella flagellinallelotyping pcr : composition , conditions , and expected results . * allelotyping pcr results ( fig . 1 ) and salmonella serovar designation based on identification of o , h1 , and h2 alleles > h2 pcr produces a 1.5 kb amplicon for all salmonella possessing h2 flagellin , regardless of h2 allele ( 1 ) . h2 multiplex pcr can not distinguish among the different alleles for the h2 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex ( 3 ) . phage conversion of s. enterica serovar hadar alters lps o antigen to produce serovar istanbul . table 3.salmonella enterica serovars identified by allelotyping pcr the serovars highlighted in bold are the ones more commonly encountered by diagnostic or food microbiology laboratory . h2 pcr produces a 1.5 kb amplicon for all salmonella possessing h2 flagellin , regardless of h2 allele ( 1 ) . phage conversion of s. enterica serovar hadar alters lps o antigen to produce serovar istanbul . note : in order to minimize pcr carryover contamination , it is important to keep several important key steps in the pcr physically - separate from one another : 1 ) template ( dna ) preparation , 2 ) pcr setup , and 3 ) gel electrophoresis . it is also recommended to use barrier tips in order to prevent culture or reagent contamination of pipetters . inoculate 1 ml of luria bertani broth or other complex medium ( brain heart infusion , superbroth , etc . ) with salmonella isolate from a single isolated colony . transfer 1 ml to a sterile , 1.5 ml capacity microfuge tube , centrifuge the bacterial cell suspension at 4,500 xg for 2 min . to pellet cells . decant supernatant , resuspend the cell pellet in 1 ml of 100% ethanol and set at room temperature for 10 min . pellet cells as before ( 4,500 xg , 2 min . ) , remove the supernatant and resuspend cells in 1 ml pbs . centrifuge cells ( 4,500 xg , 2 min . ) , discard supernatant and resuspend cells in 1 ml of sterile dh2o . dilute the final cell suspension 1/20 in dh2o ( 5 l/95 l dh2o ) and store at -20c . the shelf - life of the whole cells as pcr template at -20c is about one month . note : preparation and dispensing of the pcr reaction mix ( template , buffers , oligonucletoideprimers , nucleotides and taq dna polymerase ) needs to be performed in a physically separate room dedicated and preferably done in a pcr / uv workstation . note : the pcr set up room must also be self - contained with a designated -20c freezer for storage of pcr reagents ( buffers , oligonucleotide primers , and nucleotides ) , enzymes and template , dedicated pipetter set for pcr setup , disposable latex gloves , laboratory coat , barrier tips , microtiter plates , glass capillaries , microfuge tubes , and 10% bleach for decontaminating surfaces . use a dedicated pipetter set ( p10 , p100 , and p1000 ) to dispense pcr reagents . note : obtain molecular biology or pcr grade dh2o and aliquot 1 ml into 1.5 ml capacity centrifuge tubes . note : decontaminate the work area before use with uv illumination and/or wiping down surfaces with 10% bleach . minimize back and forth traffic from pcr set - up to areas where the pcr reactions are incubated in the thermocylcer and pcr products ( amplicons ) are separated on agarose gels . if you go back into the pcr set - up area , dispose of the pair of latex gloves you are currently wearing and put on a new pair of gloves . make a master primer stock in dh2o to a concentration of 5x10 m. dilute the primers 1/20 in dh2o ( 5 l/95 l dh2o ; 25 m ) . retrieve pcr reagents and template from -20c freezer , and allow reagents and samples to thaw out on ice . dispense 9l of the pcr reaction mix into each of 10 round bottom wells in a sterile , 96-well , polystyrene microtiter plate , starting at the top of the column ( e.g. a1 ) and moving down the column to the top of the next . add 1 l of dh2o to the 9 l of pcr mix ( no dna control ) to the first well ( a1),0.5l of the positive control templates ( i / g , m typing primers- typhimurium and enteritidis ; r / z10 typing primers- heidelberg and hadar ; and 1,2/e , n , x typing primers- typhimurium and hadar ) to 9 l of the pcr mix in the 2 well ( b1 ) , and 1 l of escherichia coli k12 dh5 to the 9 ml of pcr mix in the third well ( c1 ) . for each additional well ( d1-h1 ; a2 , b2 ) , add 1 l of the thawed sample template to the 9 l of the pcr reaction mix . for the i / g , m allelotyping pcr , s. enterica serovar typhimurium ( i ) and enteritidis ( g , m ) serve as positive controls , and salmonella serovars heidelberg ( r ) and hadar ( z10 ) are the positive controls for the r / z10 allelotyping multiplex pcr . place 10 l capacity glass capillaries into designated holders for the idaho technology rapidcycler ( 8) . once secured in the holder , load each glass capillary with the pcr reaction mix by touching the capillary to the bottom wells of the microtiter plate . tilt the holder to allow the liquid to move down the tube and provide space between the ends and the pcr mix before heat sealing the glass tubes with a butane torch to seal both ends of the capillaries . place the capillary tubes and holder into the idaho technology rapidcycler and use thermocycler programs described in table 1 for the different allelotyping primers to run the pcr . note : wear a different lab coat designated for use in electrophoresis lab and a new pair of disposable latex gloves . note : wear uv protective eye goggles or face shield and always were gloves when handling ethidium bromide , especially agarose gels . prepare 100 ml molten 1.5% ( w / v ) molecular biology grade agarose in 1xtae ( or 1x tbe ) electrophoresis buffer ( 7 ) by repeatedly heating the agarose suspension in a microwave set at 20% power for 2 - 5 min intervals with periodic visual inspection . set the molten agarose in 60c water bath until it equilibrates to the temperature of the water bath . choose a gel comb with sufficient teeth to produce enough wells for controls , samples , and at least 3 molecular weight standards for their placements at the ends and the middle of the agarose gel . add 2 l of ethidium bromide ( 10 mg / ml ) to 100 ml of molten 1.5 % ( w / v ) agarose in 1xtae ( or tbe ) , gently swirl bottle to mix , avoid producing bubbles , and gently pour the contents of the bottle into the middle of the gel mold for 15 by 10 cm agarose gel . use the edge of tissue paper or paper towel to remove any bubbles in the agarose gel . let the gel mold set at room temperature until agarose solidifies ( 30 - 45 min ) . transfer the gel tray with gel & comb to submersible , horizontal electrophoresis chamber containing 1x tae ( or 1x tbe ) electrophoresis buffer . gently remove the comb from the agarose gel . check placement of the gel tray relative to the cathode or positive pole of the electrophoresis chamber to be sure this pole is at the bottom of the gel . note : remember the negatively charged dna migrates towards the cathode ( i.e. , " run to red " ) . premix 200 l of dna molecular weight markers ( 0.25 g/l ) with 40 l of 6x dna loading dye . load 4.8 l of the premixed dna molecular weight marker xiv to the first , middle and last wells . load each of the pcr samples starting with well 2 and advancing to each subsequent well , moving from left to right . avoid loading the sample in any of the wells containing the molecular weight standard . to load samples contained in each glass capillary : remove each capillary from its holder and etch both ends of the capillary with a glass cutter.place thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary.place the capillary dispenser on the end opposite of the pcr reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube.place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the ficoll - weighted sample into the agarose well . be careful not to puncture the well with the capillary or introduce an air bubble into the well by turning too much past when the last of the liquid leaves the capillary . remove each capillary from its holder and etch both ends of the capillary with a glass cutter . place thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary . place the capillary dispenser on the end opposite of the pcr reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube . place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the ficoll - weighted sample into the agarose well . be careful not to puncture the well with the capillary or introduce an air bubble into the well by turning too much past when the last of the liquid leaves the capillary . once all the samples and standards are loaded , set the constant voltage of the power supply to 90 v ( 9 volts / cm agarose gel ) . discontinue electrophoresis once the yellow dye ( taurazine ) front is 1 cm from the bottom of the gel . once electrophoresis is completed , transfer the gel to a uv transilluminator to visualize dna fragments and molecular weight standard . capture image on polaroid film using a polaroid camera with orange glass filter ( settings : 2 x , and 4.5 y ) or as a digital image using a ccd camera and print image with a thermal printer . rinse 3 times with dh2o and place back in pcr setup room to air dry . results for allelotyping pcr of salmonella isolates 1 - 10 are shown in figure 1 ( lanes 3 - 12 ) and interpreted as follows . an o allele designation is given to salmonella isolate based on amplicon ( pcr product ) matching in size to the pcr control and as predicted for the o allele primer sets used ( 3 ) : b- 560bp , c1- 340bp , c2- 400 bp , d1- 620 bp , and e1- 280 bp . for isolates tested with the o allelotyping pcr ( fig . 1a ) , we assigned o alleles b ( lanes 10 - 13 ) , c1 ( lanes 7 - 9 ) , c2 ( lanes 4 , 5 ) , d1 ( lane 3 ) , and e1 ( lane 6 ) to the ten salmonella isolates tested in lanes 3 - 12 . 1a , for h1 alleles i ; g , m ; r ; and z10 ( fig . again , a h1 allele is assigned to each isolate dependent on the presence of an amplicon matching in size to the pcr control and as predicted for the 4 h1 allele primer sets used ( 3 ) : i- 510 bp ( fig . 1b , lane 2 ) ; r- 170 bp ( fig . 1c , lane 2 ) ; and z10- 360 bp ( fig . alleles were assigned to one of the ten salmonella isolates : i - isolates 3 and 8 ( fig . 1b , lanes 5 & 10 ) ; g , m- isolates 1 and 5 ( fig . 1b , lanes 3 & 7 ) , r to isolates 6 and 9 ( fig . 1c , lanes 8 & 11 ) ; and z10 to isolates 2 , 7 , and 10 ( fig . finally , salmonella isolates were tested by pcr for h2 alleles associated with the 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex and e , n , x ; e , n , z15 antigen complex . the primer sets for the h2 1,2 ; 1,5 ; 1,6 ; 1,7 complex and h2 e , n , x ; e , n , z15 complex produce 290 bp and 150 bp amplicons , respectively ( 3 ) . the primer sets can not distinguish specific h2 alleles within either h2 antigen complex to assign a definitive allele type , ex . salmonella isolates 4 , 6 , 8 - 10 were positive for h2 alleles associated with h2 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex , while isolates 2 and 7 were positive for h2 alleles associated with e , n , x ; e , n , z15 antigen complex ( data not shown ) . while salmonella isolates 1 , 3 , and 5 were negative for the two h2 antigen complexes , only isolates 1 and 5 were negative for h2 flagellin , as determined using a generic h2 flagellin pcr ( 1 ) ( data not show ) . these results are summarized in table 2 along with the presumptive salmonella serovar assigned to each isolate . multiplex pcr for identifying specific o and h alleles associated with s. enterica serovars enteritidis , hadar , heidelberg , and typhimurium . ( b , c ) h1 allele multiplex pcr for identifying flagellin alleles : i / g , m ( b ) and r / z10 ( c ) . lanes 1and 13 : 100 bp ladder ( promega ; madison , wi ) ; lane 2 : multiplex pcr controls for o alleles b , c1 , c2 , d1 , and e ( a ) , h1 alleles i / g , m ( b ) , and h1 alleles r / z10 ( c ) ; and lanes 3 - 12 : salmonella isolates 1 - 10 ( table 2 ) . protocol for salmonella flagellinallelotyping pcr : composition , conditions , and expected results . * allelotyping pcr results ( fig . 1 ) and salmonella serovar designation based on identification of o , h1 , and h2 alleles > h2 pcr produces a 1.5 kb amplicon for all salmonella possessing h2 flagellin , regardless of h2 allele ( 1 ) . h2 multiplex pcr can not distinguish among the different alleles for the h2 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex ( 3 ) . phage conversion of s. enterica serovar hadar alters lps o antigen to produce serovar istanbul . table 3.salmonella enterica serovars identified by allelotyping pcr the serovars highlighted in bold are the ones more commonly encountered by diagnostic or food microbiology laboratory . h2 pcr produces a 1.5 kb amplicon for all salmonella possessing h2 flagellin , regardless of h2 allele ( 1 ) . phage conversion of s. enterica serovar hadar alters lps o antigen to produce serovar istanbul . most commercially available pcr tests for the detection of salmonella targets genes unique to the genus ( ex . . however , not all salmonella are equal in their ability to cause disease in humans or prevalence in animal populations . early recognition of antigenic differences in salmonella lps o - antigen and flagellinh1 and h2 antigens has lead to the differentiation of salmonella into over 2,500 serovars based on these antigenic differences . there are 46 distinct o antigens and 86 distinct flagellin ( h ) antigens identified in the genus salmonella . the different combination of o , h1 , and h2 antigens account for the 2,500 salmonella serovars recognized today . a serovar is assigned based on the antigenic formula derived from the identification of the individual o and h antigens . the antigenic formula is written as o : h1 : h2 ; and where " - " , refers to the absence of h1 or h2 flagellin and " nt " ( non - typeable ) for salmonella negative for the o - antigen . for example , the serovar typhimurium is assigned to a s. enterica isolate with the antigenic formula b : i : 1,2 while enteritidis is given to an isolate with the formula d1 : g , m : - . this antigenic variation in the salmonella population is the outward manifestation of differences at the nucleotide level that can therefore be easily exploited by pcr . we have identified the genetic differences associated with specific o and h alleles to develop an allelotyping pcr for identifying s. enterica serovars : enteritidis , hadar , heidelberg , and typhimurium ( 3 ) . the correct assignment and reporting of salmonella serovar requires testing for all alleles associated with o : h1 : h2 antigenic formulas for enteritidis ( d1 : g , m : - ) , hadar ( c2 : z10 : e , n , x ) , heidelberg ( b : r : 1,2 ) , and typhimurium ( b : i : 1,2 ) . without knowledge of o allele or antigen , the finding of the h1 g , m allele alone does not necessarily identify the salmonella isolate as enteritidis as other salmonella serovars : agona , california , and montevideo , also possess this same allele . while the allelotyping pcr was originally designed to detect the fore mentioned salmonella serovars , this test can identify an additional 19 serovars ( table 3 ) . , it has become more practical and cost effective for us to identify the o - antigen by slide agglutination test , using o - specific antisera , rather than performing the o allelotyping pcr when working with isolated salmonella cultures . however , we do recommend using the o allelotyping pcr if your lab uses this pcr as a screen ( 2 ) or for typing salmonella isolates submitted on fta cards ( 6 ) , and include a salmonella - specific pcr with the first screen of samples ( 4 ) . limit the allelotyping pcrs to only those samples that are identified as salmonella by pcr or biochemical / antigenic tests . the protocol described in this article has been optimized for use with the idaho technology rapidcycler , a hot - air thermocycler that allows one to scale down reaction volumes and runs reaction conditions in less time than many heating block thermocyclers . to adapt this protocol for use with a heating block thermocycler may require optimizing reaction conditions empirically by lowering / raising annealing temperatures ; adjusting primer concentrations ; or adjusting mgcl2 concentrations . for example , we have had to adapt the protocol for the mj research ptc-200 thermocycler as follows . working with the same reaction volumes described in table 1 , the working stock concentrationof the i primer set was diluted to 2.5 m , the annealing temperature was lowered from 55c to 45c , and incubation times at the denaturation , annealing and extension steps was lengthened to 20 s for the i / g , m allelotyping pcr . having the appropriate positive and negative controls are essential in the initial start up and optimization for any pcr , including published protocols . we recommend acquiring known salmonella serovars , specifically anatum ( e1 : e , h : 1,6 ) , enteritidis ( d1 : g , m : - ) , hadar ( c2 : z10 : e , n , x ) , heidelberg ( b : r : 1,2 ) , montevideo ( c1 : g , m , s : 1,2,7 ) and typhimurium ( b : i : 1,2 ) ; and a laboratory escherichia coli k12 strain ( dh5 , le393 , jm109 , etc . ) as positive and negative controls respectively for the allelotyping pcr tests described in this article . several of these salmonella serovars will serve as either positive or negative controls , depending on which allele is tested . certain precautions and guidelines need to be followed while performing this and any other diagnostic pcr . first , the physical separation of template preparation , pcr set - up , and gel electrophoresis is critical in preventing possible pcr carry - over contamination and erroneous reporting of false - positives . in addition , the inclusion of appropriate controls is necessary in any routine pcr as these controls identify problems as they arise and assist in the interpretation of results ( 5 ) . most importantly , consistency is essential , especially with regards to the electrophoretic conditions for amplicon ( pcr product ) detection andestimation of amplicon size . to illustrate this point , we purposely suspended electrophoresis earlier than intended in figure 1a . the molecular weight standards ( lanes 1 and 13 ) and positive control ( lane 2 ) are not sufficiently separated to accurately estimate sizes or observe separation of dna fragments where there are small differences ( 50 bp ) in sizes . adequate separation of dna fragments , especially the molecular weight standards , is essential as reporting positives is dependent on the accurate estimation of amplicon size and distinguishing " true positive " from non - specific amplicons that are sometimes observed in the daily running of any pcr ( 5 ) . for example , there is a non - specific amplicon in figure 1b , lane 7 ( 700 bp in size ) . had electrophoresis been discontinued too early , when the dye front was only at the half way point in the agarose gel , there would be little differentiation between 500 bp and the 700 bp dna fragments . therefore , sample in lane 7 would have been erroneously reported as positive for both i and g , m alleles . several of the h1/h2 allelotyping pcr do not have the discriminatory power to discern subtle genetic differences in alleles belonging to the h2 1,2 ; 1,5 ; 1,6 ; 1,7 antigen complex ; e , n , x / e , n , z15 antigen complex and the h1 g antigen complex , which includes the g , m allele . one or two amino acid changes account for the different epitopes present in these flagellar antigens . it was therefore difficult for us to design primers that could exploit these sometimes single base pair differences in the flagellin gene sequence ( 3 ) . for example , salmonella serovars dublin ( d1 : g , p : - ) and berta ( d1 : f , g , t : - ) are also pcr positive with our g , mallelotyping primers . the h2 allelotyping primers can not distinguish typhimurium ( b : i : 1,2 ) from lagos ( b : i : 1,5 ) , heidelberg ( b : r : 1,2 ) from bradford ( b : r : 1,5 ) , winneba ( b : r : 1,6 ) , or remo ( b : r : 1,7 ) , or hadar ( c2 : z10 : e , n , x ) from glostrup ( c2 : z10 : e , n , z15 ) . while the likelihood of encountering some of these serovars : lagos , bradford , winneba , remo or glustrup is remote , it is a possibility and requires either providing disclaimer on the tests ' limitation or another confirmatory test . in conclusion , this pcr - based serotyping rapidly identifies s. enterica serovars enteritidis , hadar , heidelberg and typhimurium , and o , h1 , and h2 alleles associated with 19 additional serovars . this assay is a rapid , cost - effective alternative to traditional microbiological approach to serotyping salmonella .

a systematic literature search was conducted in september 2013 . to find all articles describing experimental huts , the following databases were searched : pubmed ( national center for biotechnology information , national library of medicine , national institutes of health ) , the armed forces pest management board literature retrieval system , ibecs , lilacs , horizon pleins texts ( database of the institut de recherche pour le dveloppement ) , the web of science , the institutional repository for information sharing of the world health organization ( who ) and the cochrane library . in addition , reference lists in the selected papers were screened to find additional articles . the following search terms were used : experimental hut , experimental huts , case pige , cases piges , veranda hut , verandah huts , veranda hut , verandah huts , window trap hut , window trap huts , case exprimentale , cases exprimentales , veranda trap hut , veranda trap huts , verandah trap hut and verandah trap huts . studies were selected if they met one of the following criteria : ( i ) an experimental hut study in which nets with different sizes or numbers of holes were used and ( ii ) an experimental hut study in which holed and unholed nets were used . the search identified 1,051 articles of which 13 discussed experimental hut trials using holed bednets and met the criteria listed above ( experiments described in report and article form or reported in two different languages were treated as one report ) . studies with different size holes - very few studies used nets with different sizes of holes . table presents details of the experimental hut studies evaluating different numbers of holes and displays blood feeding , mortality and exophily rates . port and boreham ( 1982 ) evaluated the protection afforded by untreated nets owned by the men who were employed to sleep in the experimental huts in the gambia . they measured the physical integrity of nets by calculating the sum of the circumference of all holes and the lengths of any splits , which ranged from 0 - 195 cm . interestingly , the proportion of ano- pheles gambiae s.s . feeding on volunteers using nets with few holes was not very different than those where unholed nets were used ; indeed , the lowest proportion of feeding occurred on a volunteer under a net with nets with a hole measure of 16 cm , though the relative attractiveness of the sleepers may have been important . similarly , blood feeding on volunteers using nets with high hole measures was similar to blood feeding on volunteers not using nets . tablestudies conducted in experimental huts evaluating mosquito nets with different sizes and numbers of holesreferencecountryspeciesresistance status of wild mosquitoestype of hutnet typeinsecticidedose ( mg / m)washesnumber of holessize of holes24 h mortality ( % ) blood feeding ( % ) exophily ( % ) port and boreham ( 1982)jiboroh , the gambia anopheles gambiae s.s . unksimilar to east africanused netnonen / aunk -- n / a24n / aused netnonen / aunk1unreportedn / a17n / aused netnonen / aunkunk7 + tearn / a15n / aused netnonen / aunkunk16 n / a0n / aused netnonen / aunkunk54 + tearn / a38n / aused netnonen / aunkunk57 n / a40n / aused netnonen / aunkunk121 + tearn / a85n / aused netnonen / aunkunk133 + tearn / a44n / aused netnonen / aunkunk195 n / a71n / airish et al . ( 2008)ladji , benin culex quinquefasciatus pyrethroid resistantwest africancontrolnonen / a00 - 143645controlnonen / a064 x 4 cm136029controlnonen / a0802 x 2 cm136927itn dippedalphacypermethrin4000 - 222947itn dippedalphacypermethrin40064 x 4 cm234434itn dippedalphacypermethrin400802 x 2 cm195725ngufor et al . m form)91% frequency of kdrwest africancontrolnonen / a064 x 4 cm55647controlnonen / a0802 x 2 cm35440permanet 2.0deltamethrin55064 x 4 cm501270permanet 2.0deltamethrin550802 x 2 cm373268ngufor et al . quinque- fasciatus pyrethroid resistantwest africancontrolnonen / a064 x 4 cm23442controlnonen / a0802 x 2 cm24928permanet 2.0deltamethrin55064 x 4 cm15567permanet 2.0deltamethrin550802 x 2 cm131259malima et al . ( 2008)zenet , tanzania anopheles arabiensis fully susceptible to permethrin 0.75% and deltamethrin 0.05%east africancontrolnon / a064 x 4 cm42791itn dippedalphacypermethrin20064 x 4 cm729951.5 year old itn dippedalphacypermethrin20 ( original dose)unk64 x 4 cm701997olysetpermethrin1,000064 x 4 cm631698seven year old olysetpermethrinunkunknatural > 4 x 4 cm405097malima et al . ( 2008)zenet , tanzania anopheles funestus fully susceptible to permethrin 0.75% and deltamethrin 0.05%east africancontrolnon / a064 x 4 cm7.93291itn dippedalphacypermethrin20064 x 4 cm71101001.5 year old itn dippedalphacypermethrin20 ( original dose)unk64 x 4 cm582996olysetpermethrin1,000064 x 4 cm741693seven year old olysetpermethrinunkunknatural > 4 x 4 cm893695a : total circumference of all holes ; itn : insecticide - treated nets ; n / a : not applicable / available ; unk : unknown . : total circumference of all holes ; itn : insecticide - treated nets ; n / a : not applicable / available ; unk : unknown . 2011 ) both evaluated mosquito nets with different numbers and sizes of holes in southern benin . in both studies , nets with six holes of 4 x 4 cm [ now a standard for who pesticide evaluation scheme ( whopes ) ( who 2013 ) ] were compared with nets with 80 holes of 2 x 2 cm which had been used in previous studies ( asidi et al . 2004 , nguessan et al . 2007 ) . six holes of 4 x 4 cm is equivalent to 96 cm of holes [ 96 cm of the port and boreham ( 1982 ) measure ] , while eighty 2 x 2 cm is equivalent to 320 cm ( 640 cm of the hole measure ) . 2008 ) , working in ladji , near cotonou , used alphacypermethrin - treated nets ( fendona 6 sc ) , conventionally treated at a dose of 40 mg / m , while ngufor et al . ( 2011 ) , working in akron , near porto - novo , used a long - lasting insecticidal net with a target dose of 55 mg / m of deltamethrin . both studies observed significantly higher rates of culex quinquefasciatus blood feeding with 80 holes compared with six holes , for both treated and untreated nets ( in ladji , the difference was not significant for untreated nets ) . quinquefasciatus was not significantly different between nets with different numbers of holes and was significantly higher with treated nets . in both studies , mortality was low , due to pyrethroid resistance in cx . gambiae ( increased blood feeding with more holes , no difference in mortality ) , however , mortality of the pyrethroid - resistant an . gambiae ( yadouleton et al . 2010 ) was 37 - 50% , slightly higher than with cx . ( 2008 ) reported greater blood feeding and less mortality of pyrethroid - susceptible an . gambiae and funestus under a seven - year old olyset net ( originally 2% permethrin w / w ) , which had developed holes ( the size and number of holes is not reported ) , than under an olyset which had six 4 x 4 cm holes cut into it . the seven - year old olyset still had some killing effect , as demonstrated in bioassays , but as the insecticidal effects were significantly less than the new olyset , it is difficult to separate the effects due to reduced insecticide and those due to the holes in the net . studies with holed and intact nets - in experimental hut studies comparing results from holed and unholed nets , a general pattern emerged with increased blood feeding in huts with holed nets , as might be expected . as shown in fig . 1 , the majority of trials collected more blood - fed mosquitoes from huts where holed mosquito nets were used . however , the difference in blood feeding was coupled with very small or no difference in rates of mortality of mosquitoes exposed to treated nets with or without holes ( lines et al . the minimal effect of holes in nets on the mortality of mosquitoes in experimental huts can be seen in fig . note that certain references found by the literature search are not included in figs 1 , 2 , due to data presented in other publications , insufficient information , or holed and unholed nets being tested in separate locations or at different times ( port & boreham 1982 , pan et al . 1995 , curtis et al . fig . 1 : differences in blood feeding between mosquitoes collected in experimental huts where holed and unholed nets were compared ( percent blood - fed with intact nets - percent blood - fed with holed nets ) . fig . 2 : differences in mortality between mosquitoes collected in experimental huts where holed and unholed nets were compared ( percent dead with intact nets - percent dead with holed nets ) . however , there were several exceptions to this pattern , which occurred when intact nets were ripped during a study , when nets were untreated , or when nets were treated with a non - pyrethroid insecticide . 1984 ) , in one of the first experimental hut study on pyrethroid - impregnated bednets , compared bednets treated with permethrin at a concentration of 80 mg / m with untreated nets , with both types of nets holed for approximately 5% of the total area of the net ( 0.5 m for one - person nets and 0.675 m for two - person nets ) . for both treated and untreated nets , the blood feeding rates of an . funestus were quite high ( 75 - 97% ) and slightly higher in holed nets than unholed nets ( never more than 6% higher ) , with the exception of an . funestus where the blood feeding rate was 85% under holed nets and 91% under intact nets . the authors explained these small differences by noting that the nets that began the trial as intact nets were quickly torn by the users over the course of the trial . it should also be noted that the dose of permethrin on the nets ( 80 mg / m ) was lower than currently recommended levels of 200 - 500 mg / m ( whopes 2007 ) . there was almost no mortality of an . gambiae in the huts with untreated nets , while the mortality between huts with holed and unholed nets was nearly the same for one - person nets ( 18% under intact nets , 22% under holed nets ) . ( 1996 ) present a large amount of experimental hut data on mosquito nets with different insecticides , fabrics , months of prior use and number of washes . they found fairly low blood feeding rates by anopheles mosquitoes on sleepers under nets treated with permethrin ( 200 - 500 mg / m ) , deltamethrin ( 3 - 25 mg / m ) and lambdacyhalothrin ( 3 - 15 mg / m ) , with geometric means of less than 45% for all three treatments , holed or unholed . when comparing new nets with the same insecticide dose and fabric , nets with six holes ( 4 x 4 cm ) had between three and nine times more blood feeding than nets without holes . mortality was higher in nets with holes , except for permethrin ( 500 mg / m ) , which had higher mortality under intact nets ( 38% ) compared with holed nets ( 29% ) . it is difficult to compare these results as it seems they were not conducted at the same time ( hut work was done over 4 years ) . ( 2005 ) , working in cote divoire , found only small differences in blood feeding or mortality when holes ( 80 holes of 2 x 2 cm ) were added to nets treated with chlorpyrifos methyl ( 100 mg / m ) . however , they found increases in blood feeding when holes were added to untreated nets . as shown in studies with different sizes or numbers of holes , or in studies with and without holes , the general and unsurprising , observation is that with increased holes there is increased blood feeding by mosquitoes in experimental huts . before continuing , it should be noted that the presence of blood - fed mosquitoes in an experimental hut do not mean that the mosquitoes have fed on the person inside the hut ( kitau et al . 2014).there are several factors that influence a mosquito s ability to feed on a human protected by a net , including the presence of holes in the net , the resistance of the mosquito to the insecticide on the net and the proximity of the human to the net ( determined by the size of the net , the number of people inside the net , the sleeping location of the human , etc . ) , which could allow feeding through the net . no record of the roofs of nets being holed was noted , but in recent behavioural studies , it was found that much of the activity of an . gambiae occurs at the roof ( lynd & mccall 2013 , sutcliffe & yin 2014 ) . it may be that holes in the roof are more important than holes on the sides of a net , which would have obvious importance for net manufacturers . despite the trend for increased feeding on people under nets when holes were present , a small number of holes did not render a net completely ineffective . indeed , in phase ii tests of llins , nets are holed ( ie . , 6 holes of 4 x 4 cm ) to replicate the conditions of used nets . all currently approved llins are able to provide over 70% blood feeding inhibition even with holes in the net , sometimes maintaining this effect even after 20 washes ( whopes 2004 , 2008 , 2009a , b , 2011 , 2012 , pennetier et al . the second characteristic that was evaluated in this review was the mortality of mosquitoes in relation to holes in nets . it was found that often the killing effect of a net was not reduced by perforation of the net . indeed , with the exception of one result in an untreated net ( lines et al . 1987 ) , the mortality of an . gambiae s.l . in huts with holed nets was never more than 6% different from that of huts with intact nets ( fig . some mosquitoes do not leave the bednet and remain inside the bednet until they are collected or die . ( 1985 ) reported mortality and blood feeding results for mosquitoes collected under treated and untreated nets as well as the results for mosquitoes collected in other parts of the hut . they found that for the untreated nets , mosquitoes collected under the net had higher blood feeding than mosquitoes collected elsewhere , both when nets were holed and unholed . the mortality of mosquitoes collected inside the untreated nets was less than mosquitoes collected elsewhere , both for holed nets and unholed nets . however , although the addition of 200 mg / m of permethrin did not change the finding of a higher proportion of blood - fed mosquitoes under the net , the mortality of mosquitoes under nets was greater than that of those outside the net . blood- fed mosquitoes may have different levels of insecticide susceptibility and this effect is not fully understood , as certain factors such as the age of the mosquito or the time after blood feeding can affect susceptibility ( davidson 1958 , rajatileka et al . it should be noted that defensive movements of humans sleeping under a net may also result in some increased mortality . therefore , a holed net might not provide the same personal protection as an intact net , but may continue to kill mosquitoes . killing mosquitoes , even in the absence of personal protection , should be considered when making decisions about vector control ( killeen et al . an important effect of holes which can not be measured in experimental huts is the impact that holes have on the use of the net . in some situations , nets become so torn or holed that homeowners no longer consider them useful ( maxwell et al . net use has also been strongly correlated with net condition ( kilian et al . 2011 ) . the importance of holes in nets in experimental huts is of obvious importance for entomologists , but the larger question is what effect do holes have on the biting and malaria transmission in the field . ( 2011 ) found reduced odds of malaria when nets had no or few holes . however , as shown by successful whopes trials , the effectiveness of a net is not determined uniquely by its physical integrity . a net s success is determined by three factors : the insecticidal activity on mosquito vectors , the physical separation a net provides between mosquito vectors and the human and the way in which the net is used ( time of use , frequency of use , factors such as tucking in the net , etc . ) . would be one that no longer provides any protection to the sleeper or any insecticidal effect which might provide protection to someone else . of major concern to national malaria control programs is the timing of net distribution campaigns . it is clear that nets should be distributed and sensitisation activities undertaken before all 100% nets fail , but as shown by malima et al . it seems likely that homeowners would discard a net before the insecticide on the net is reduced to a level where it has no effect on mosquitoes and the physical structure of a net provides no protection . the distribution of mosquito nets and accompanying sensitisation activities should occur at a rate to reduce transmission of malaria . it seems reasonable to create a matrix of factors which influence the success of nets to determine when these activities take place and it is very likely that these activities will have to be conducted at the national scale , if not at a more detailed level , to ensure success of malaria control programs based on distribution of llins .

we have previously found that multistep renal carcinogenesis occurs in eker rats8 and reported that erc , a rat renal carcinoma gene , is highly expressed in renal cancer.9 rat erc is a homolog of the human mesothelin gene10 , 11 that is localized to chromosome 16p13.3.12 mesothelin is expressed in normal mesothelial cells , mesotheliomas , nonmucinous ovarian carcinomas , and pancreatic ductal adenocarcinomas . although the human mesothelin gene encodes several proteins , its primary product is a 71kda precursor protein that is cleaved physiologically by a furinlike protease into a 40kda cterminal fragment that remains membrane bound and a 31kda nterminal fragment that is secreted into the blood.13 the resultant cterminal 40kda fragment is classified as a mesothelin , and its presence in the serum has been reported to be a useful tumor marker in patients with mesothelioma . we have since developed 22a31 , a cerc / mesothelinspecific mouse mab directed against tumors derived from the human mesothelioma cell line accmeso4 . to characterize this antibody , we carried out both in vitro and in vivo cell proliferation assays . interestingly , the antibody did not inhibit the proliferation of the accmeso4 cell line in vitro , but it dramatically inhibited cell linederived tumor growth in vivo.14 thus , the cerc / mesothelinspecific mouse mab 22a31 may be a potentially useful new tool for treating cerc / mesothelinexpressing cancers , including mesothelioma . in contrast , the secreted nterminal 31kda fragment , which has been cloned as a megakaryocytepotentiating factor , has not been reported to be a useful serum marker for mesothelioma . therefore , we developed an original sandwich elisa system using specific antibodies raised against the 31kda nterminal fragment of erc , nerc / mesothelin . to test this elisa system , we planned a 5year , largescale research screening program in collaboration with the tokyo doken national health insurance association ( tokyo , japan ) , which had worked in japan from 2007 to 2012 at construction sites with a risk of asbestos exposure . as of 2007 , approximately 220 000 blood samples were collected , and nerc / mesothelin levels were determined using the elisa kit . together with the participant 's medical history and related data , elisa analysis identified approximately 60 highrisk individuals from among 30 000 participants . based on the screening , we identified three participants with mesothelioma and 60 participants who were at high risk but had yet to develop mesothelioma . from these results , we predicted that nerc / mesothelin is a blood tumor marker that may be useful for largescale population screening and the early diagnosis of mesothelioma following mass examination.15 moreover , to more precisely measure nerc / mesothelin , we have developed a new 720 elisa system . this system is clinically useful for the precise diagnosis of pleural mesothelioma.16 over the next 4 years , we plan to carry out annual followup on all the participants in the highrisk population and to prospectively assess how many participants ultimately develop mesothelioma , while continuing to screen for any early signs of mesothelioma.17 the results from another study showed that members of a highrisk population who failed to undergo annual nerc / mesothelin screening were diagnosed with mesothelioma in the following year . therefore , we strongly recommended that participants who are identified as being at high risk for developing mesothelioma receive an additional health checkup and nerc / mesothelin test at least annually , even if they have no clinical symptoms . in addition , a new potential marker for mesothelioma was recently reported . in that study , a rat model of peritoneal mesothelioma transcriptome analysis of the rats with epithelioid and sarcomatoid types of mesothelioma defined connective tissue growth factor as a marker for malignant mesothelioma.18 in parallel , our research group has continued to seek a specific clinical marker for sarcomatoid , a rare form of mesothelioma with a particularly poor prognosis , and has already identified several specific markers for this tumor type . although additional research is needed , the results of our recent study and other published reports indicate the future direction for establishing new markers for the diagnosis and treatment of mesothelioma . asian countries , such as thailand , have experienced successful economic development in recent decades . with these advances , large populations have gradually begun to face many of the difficulties that large economies around the world have also faced in the past , such as air pollution and environmental issues . we now have to address these difficulties to protect our people 's lives and living environments . , the proportion of all illnesses represented by cancer has been increasing recently . a significant portion of this increase is assumed to be the result of environmental changes , such as urbanization and air pollution . it is an aggressiveand and a malignant tumor that is chiefly attributed to asbestos exposure . in several asian countries , including japan , rapid economic growth has brought many changes , but asbestos is still widely used . we previously reported that nerc / mesothelin might be a useful blood tumor marker for mesothelioma.6 , 19 , 20 furthermore , we developed and tested a unique diagnosis system in the largescale screening of construction workers in japan . we have also predicted that the incidence of mesothelioma will continue to increase in asia in the near future . we intend to share this diagnosis system and all knowledge gained pertaining to mesothelioma and to continue to establish early diagnosis and treatment systems . finally , our group will work to organize international seminars on environmental carcinogenesis once a year and an education program on the skills required for mesothelioma diagnosis . environmental carcinogenesis causes not only an asbestosrelated mesothelioma but also lung cancer resulting from air pollutants . throughout life , we are exposed to many environmental pollutants , and the environmental conditions of our surroundings vary between individuals . thus , epidemiology research is also needed to help solve the issue of environmental carcinogenesis in asia . through asian epidemiology research and advanced baseline studies in japan in 1775 , the british surgeon percival pott reported that the incidence of scrotal cancer was increasing among chimney sweeps and predicted that the cancer was caused by chimney soot . the industrialization that followed the industrial revolution in the latter half of the 18th century marked the largescale introduction of environmental pollutants . regarding asbestos , our preventative measures and practical responses are decades behind , despite the fact that there has been ample pathological and epidemiological evidence establishing the risks with exposure . future carcinogen research must include risk evaluation along with risk management and establish a good communication system for the people who have a relationship with the patient . in reality , almost everyone who has lived in an industrialized area has asbestos fibers in their lungs , and many can recall a specific incidence of incidental exposure ( e.g. , school teachers and students who handled asbestos samples , mats , or blankets and people in many other life situations ) . asbestos fibers are long and thin and can penetrate the lung , repeatedly scratching the mesothelium surface and causing prolonged cycles of damage , repair , and local inflammation . the length of exposure and the amount of asbestos are positively associated with the risk of mesothelioma . our recent largescale research study that screened for mesothelioma in japan was effective for early diagnosis . to confirm this screening method , we have begun an international largescale research screening program in japan and other asian countries . since we established the international environmental carcinogenesis research center last year at juntendo university ( tokyo , japan ) , japanese researchers have provided technical guidance on the use of this screening method as well as education on mesothelioma to asian doctors and researchers . in the near future , we will confirm that this method is effective in another country and gather reliable evidence for blood diagnosis of mesothelioma . from this center , we have established early diagnosis and treatment systems not only for mesothelioma but also for other diseases resulting from environmental carcinogenesis . moreover , the people living in asian countries , including japan , will understand and care about the risks of environmental carcinogenesis . dr . hino opened the asbestos mesothelioma clinic in 2005 at juntendo university and since then has continuously taken care of patients with mesothelioma and people who fear asbestos exposure . thus , we should establish holistic methods of caring for mesothelioma , including diagnosis , treatment , and endoflife counseling .

gattii species complex , which is composed of two separate species , c. neoformans and c. deneoformans , and five species within c. gattii . the most clinically relevant complex species were recently revised based on phenotypic and genotypic diversity , supported by the presence of distinct and consistent lines , and it proposes to recognize c. neoformans var . grubii ( represented by genotypes vni and vnii ) and c. neoformans ( vniii and vniv ) as separate species , as well as five species of c. gattii ( represented by genotypes vgi , vgii , vgiii , and vgiv ) [ 1 , 2 ] . c. neoformans has a worldwide distribution and has been recovered from pigeon droppings ( columba livia ) , urban environments , and soil . many reports have shown the presence of c. neoformans in the hollows of different tree species , proposing that trees play a major role in cryptococcus infection [ 3 , 4 ] . c. gattii has been proposed to have a specific ecological association with a number of eucalyptus species . e. camaldulensis is a well - known tree in egypt since it was imported by mohamed ali , the governor of egypt ( 18051848 ) , for fixing the river nile banks in the 19th century . it is one of the most widely distributed trees in most of the arid and semiarid areas . this kind of tree exists on almost every roadside in egypt , but there are no data about its role as potential carrier of fungal elements . in egypt , the incidence of cryptococcus neoformans from eucalyptus trees and pigeon droppings has been reported . in this report , the author depended on the conventional differentiation methods to determine c. neoformans varieties . there is no recent noticeable information about the eucalyptus tree role in the ecology of c. neoformans in egyptian environment . therefore , the present study was aimed at determining the possible role of this tree as a potential dispersing source of c. neoformans in delta region 's environment . the study area in delta region covered 240 kilometers ( 150 mi ) of mediterranean coastline of egypt . a total 311 samples out of 200 eucalyptus trees , including leaves , flowers , and woody trunks , were collected from four different governorates in the delta region ( cairo , giza , elmenofia , al - sharqia ) by the rate of fifty samples for each region ( table 1 ) . the samples were stored on ice in clean , sterile plastic bags and transferred to the microbiology department laboratory , faculty of veterinary medicine . the samples were rinsed in sterile distilled water , then immersed in sterile saline solution supplemented with chloramphenicol ( 10.0 mg / ml ) , and homogenized with ultrahomogenization for 4 min . the bottle was left for 30 min at room temperature to settle the sediment . from the supernatant fluid of each homogenized sample , a loopful was streaked onto plates of sabouraud dextrose agar with chloramphenicol and incubated at 30c for 48 hours . the collected pellets were mixed into a microtube with 500 l tes ( 100 mm tris , ph 8.0 , 10 mm edta , and 2% sds ) ; 50100 g proteinase k from an appropriate stock solution was added and then incubated for 30 min ( minimum ) up to 1 h at 5560c with occasional gentle mixing . the lysate mix salt concentration was adjusted to 1.4 m with 5 m nacl ( = 140 l ) ; 1/10 vol ( = 65 l ) of 10% ctab was added and incubated for 30 min at 65c . the lysate was mixed gently and then incubated for 30 min at 0c ; finally , the mix was centrifuged for 10 min at 4c , at 15000 rpm . the supernatant was transferred to a 1.5 ml tube followed by the addition of 0.55 vol isopropanol ( = 510 l ) to precipitate dna followed by immediate centrifugation for 5 min , at 15000 rpm . the primers for cap64 were designed on the basis of dna sequences ( table 2 ) . this was applied by subjecting genomic dna of identified strains by cap64 gene to multiplex pcr amplification using a set of four primers of the laccase gene ( lac1 ) ( table 2 ) which were used for differentiating four major serotypes , a , d , b , and c , of c. neoformans . in this study , e. camaldulensis trees acted as a potential refuge for c. neoformans . a total of 13 ( 4.2% ) c. neoformans isolates out of 311 examined samples in delta region were recovered during the flowering season of eucalyptus tree . all the recovered cryptococcus isolates were identified as c. neoformans strains based on all conventional and physiological characters of c. neoformans ( figure 1 ) . among these , 7 isolates ( 7.9% ) were recovered from 88 eucalyptus flowers , 5 isolates ( 3.6% ) were recovered from 138 eucalyptus tree leaves , and 1 isolate ( 1.1 ) was recovered from 85 woody trunks ( table 3 ) . all tested isolates and reference strain were produced ( 400 bp ) by cap64 specific capsular gene primer ( figure 2 ) . molecular typing of c. neoformans isolates was done by four primers for lac1 gene ( table 2 ) which were used for amplification ; serotype a strains produced three dna fragments with sizes of 0.88 , 0.76 , and 0.25 kb ( figure 3 ) . in egypt , eucalyptus trees are in abundance mainly as windbreaks and for afforestation of the drains and canals or other watercourses , plus the highways and roads in rural or urban areas . ( home country of e. camaldulensis ) , ellis and pfeiffer , 1990 reported the first environmental isolation of c. gattii from wood , bark , leaves , and plant debris of eucalyptus trees . although eucalyptus is present in many of the areas known to have c. gattii cryptococcosis , the actual isolation of c. gattii from eucalyptus trees is rare outside australia . moreover , imported eucalyptus has not been associated with the environmental presence of c. gattii in spain , central africa , canada , papua new guinea , egypt , and italy . on the african level , the isolation of c. gattii from the environment is somewhat limited in comparison to the isolation of c. neoformans . only two cases were recorded in isolation of c. gattii from e. camaldulensis in african countries , egypt and tunisia . in egypt , earlier report of mahmoud ( 1999 ) depended on canavanine - glycine - bromothymol blue ( cgb ) agar to determine c. neoformans variety , which evoked a high need to investigate the environmental ecology of this fungus , depending on molecular techniques to determine the actual variety of c. neoformans in relation to e. camaldulensis in order to establish a real surveillance program and applying the preventive measures for this pathogen infection . this study was applied on eucalyptus trees during the flowering season , as most c. neoformans and c. gattii reported cases were associated with eucalyptus showing strong seasonality in its occurrence , which coincides with the periods of flowering . the results show that the isolation of c. neoformans from eucalyptus flowers is more frequent than from leaves and woody trunk ( table 3 ) . grubii as the global distributed isolate responsible for cryptococcal infection [ 1 , 57 ] . also , it is commonly the recovering of c. neoformans from pigeon droppings , soil , and decaying wood in hollow trees . most of the previous reports stated that c. grubii association with eucalyptus trees or other types of trees is interpreted in one sentence : c. neoformans presence might represent fecal contamination by birds inhabiting these trees [ 9 , 14 , 16 ] . globally , many reports are recorded for isolation of c. grubii from eucalyptus tree parts or other types of trees . in india , more than one report states that c. grubii tree association and its distribution differ from each part of tree or season or time of the study . the prevalence of c. grubii ( 5.56% ) and c. gattii ( 9.26% ) from decayed wood inside trunk hollows of diverse tree species was reported . recently , c. grubii was isolated from the bark of eucalyptus trees followed by flower , bud , fruit , and detritus . the prevalence of c. grubii in this study ( 4.2% ) is somewhat near to the rate of nawange et al . 's ( 2006 ) study ( 5.5% ) , while the recovery rate of c. grubii is the highest from flowers ( 7.9% ) , then leaves ( 3.6% ) , and finally woody trunks ( 1.1% ) ( table 3 ) . in sunny countries , c. neoformans can escape from lethal effects of sunlight and drying by taking trees as a good natural habitat in the environment because these pathogens can live inside woody debris as well as trunk hollows.the result of the present study highlighted the potential role of tree parts of e. camaldulensis in environmental ecology of c. grubii . eucalyptus flowers were the best natural habitat and a suitable transporting means for these pathogen infectious propagules in the surrounding environment . flowering season of eucalyptus tree is mainly in winter and spring from november to february . at this time of year in egypt , the temperature is slightly low to temperate which gives a potential chance for isolation of this pathogen . the association between c. grubii and tree is controlled by many environmental factors including humidity , temperature , and solar radiation [ 13 , 17 ] . in egypt , the eucalyptus tree exists almost along every roadside , especially in the delta region around river nile and its tributaries . these study results confirm the potential role of eucalyptus trees as a major source for c. grubii in egyptian environment which act as a high risk for immunocompromised patients . cryptococcal meningitis in egypt is rarely diagnosed , but this may be due to inadequate investigation rather than absence of definite epidemiological data about the organism in egypt . more attention should be considered for human cases of unexplained chronic meningitis that is not responding to conventional therapy as c. neoformans could be the main cause of such fetal meningitis [ 2023 ] . the only survey for fungal meningitis was done in egypt at namru-3 during the period of 1998 to 2001 of 1000 csf samples , where 10 c. neoformans were recovered at a rate of 0.01% . c. neoformans serotype a is the most common variety in association of pet birds droppings in the egyptian environment . this study 's findings come in the same direction with the previous surveillance of the main causes of cryptococcal infection in egypt and it confirmed that c. neoformans var . grubii is the main etiological agent of cryptococcal infection in egypt . conclusively , this is the first record describing isolation of c. neoformans var . the results highlighted the potential role and risk of eucalyptus tree as a carrier reservoir of one of the high pathogenic fungal elements in egypt .

gastrointestinal ( gi ) bleeding is a common emergency and bleeding from the upper gi tract and small intestine account for 75% and 210% of all gi bleeds , respectively . we describe a 29-year - old man who presented with obscure anemia due to small intestine bleeding . vascular abnormalities account for 7080% , followed by small intestinal bleeds that account for 510% , respectively . adenocarcinoma is the most common primary malignant tumor of the small bowel ( pmtsb ) [ 4 , 5 ] . however , our patient had a gastrointestinal stromal tumor ( gist ) , which accounts for < 1% of all gi neoplasms . the peak age for developing gist is 5060 years [ 6 , 7 ] , and thus gist is rarely the source of small intestinal bleeds . this report describes obscure gi bleeding due to gist of the jejunum of a young adult . a 29-year - old man presented to our hospital with a 2-week history of gross hematochezia and anemia . laboratory findings were as follows ( values are shown with range in parentheses ) : aspartate aminotransferase , 11 ( normal , 535 ) u / l ; alanine aminotransferase , 11 ( 530 ) u / l ; alkaline phosphatase , 185 ( 115359 ) u / l ; -glutamyl transferase , 49 ( 050 ) u / ml ; lactate dehydrogenase , 96 ( 106211 ) u / l ; albumin , 3.0 ( 3.75.5 ) g / dl ; total bilirubin , 0.26 ( 0.21.0 ) g / dl ; and c - reactive protein , 1.2 ( 00.3 ) mg / dl . other than c - reactive protein , almost all blood biochemistry findings were essentially within normal ranges . hematological findings were as follows ( values are shown with range in parentheses ) : white blood cells , 11,600 ( 5,0008,000)/l ; red blood cells , 165 ( 400530 ) 10/l ; and hemoglobin , 4.5 ( 1418 ) g / dl . tumor markers were as follows ( values are shown with range in parentheses ) : cea < 0 ( 05 ) ng / ml ; ca19 - 9 , 3 ( 037 ) u / ml ) ; and il-2 , 280 ( 145519 ) u / ml . all tumor markers were within normal ranges . abdominal computed tomography revealed a well - delineated 45 40-mm mass in the jejunum ( fig 1 ) . the lesion in the jejunum was resected and removal of a possible tumor was assured by lymphadenectomy ( fig 2 ) . the resected specimen initially appeared similar to diverticulosis with abscess , so we diagnosed infected diverticulosis ( fig 3 ) . histopathological assessment revealed a submucosal tumor comprising proliferative atypical spindle or epithelioid cells arranged in fascicle whorls with a dense acute inflammatory infiltrate , abscess and necrosis . immunohistochemical findings showed that the atypical cells were positive for c - kit , and some were faintly positive for cd34 and s-100 ( fig 4 ) . these features were compatible with those of low - risk gist [ 8 , 9 , 10 ] . accurate , prompt diagnosis of a bleeding source is crucial because such bleeding can result in death . the source of 35% of gi bleeds can not be identified by esophagogastroduodenoscopy and/or colonoscopy , but bleeding from the upper and lower gi tracts accounts for 75 and 15% , respectively , of all gi bleeds [ 1 , 7 ] . vascular abnormalities and small intestinal tumors account for 7080% and 510% , respectively , of small intestinal bleeds and pmtsb accounts for only 12% of all primary gi tumors [ 11 , 12 ] . the most frequent symptoms are abdominal pain ( 67.4% ) , abdominal mass ( 31.2% ) and bowel obstruction ( 24.1% ) , followed by hematochezia ( 21.3% ) , jaundice ( 16.3% ) , and fever ( 14.2% ) . leiomyoma and leiomyosarcoma in the small intestine are associated with massive bleeding , whereas lymphoma , gist and carcinoids are associated with relatively slow bleeding [ 4 , 7 ] . our patient had a 2-week history of gross hematochezia and his condition roughly corresponded to gist . mazur et al . originally described gist in 1983 . less than 1% of all gi neoplasms are gist , and they are defined as specific , generally kit ( cd117)-positive and/or platelet - derived growth factor receptor- mutation - driven tumors [ 9 , 10 , 13 ] . among mesenchymal tumors of the gi tract , gist are the most common , and primary gist can occur anywhere along the gi tract between the esophagus and the anus . between 50 and 70% of clinically manifested tumors arise in the stomach , 2030% are found in the small bowel , 515% are located in the large bowel , and < 5% are located in the esophagus and elsewhere . therefore , to initially consider gist in a differential diagnosis of gi bleeding in a younger patient is unlikely . meckel 's diverticulum and crohn 's disease are common when considering only gi bleeds in younger patients . meckel 's diverticulum is the most common congenital anomaly of the gi tract , with an incidence of 24% in the general population . meckel 's diverticulum occurs twice as often in men than in women , and acute diverticulitis can occur at any age , but the incidence peaks in children [ 15 , 16 ] . meckel 's diverticulum is always located within 1 m of the oral side of the ileocecal valve and straying ectopic stomach tissue is the key pathological feature in 27% of patients with this disease . the specimen resected from our patient macroscopically resembled diverticulitis , but it was not obtained from a location within 1 m from the oral side of the ileocecal valve . small intestinal diverticula other than the meckel type can be differentially diagnosed , but the incidence ranges from only 0.06 to 1.3% . finally , microscopic assessment ruled out these types of diverticulosis . first described this condition in 1932 as a chronic inflammatory disease of unknown origin that can occur not only in the terminal ileum but anywhere in the gi tract . it commonly affects men and women aged in the early 20s and late teens , respectively [ 18 , 19 , 20 ] . preoperative upper and lower gi endoscopy and pathological findings excluded crohn 's disease as the bleeding source in our patient . we described hemorrhage due to gist of the small intestine in a young adult male . the possibility of neoplastic disease must be considered even among young adults who present with gi bleeding . written informed consent was obtained from the patient for publication of this paper and any accompanying images .

various methods , such as allografting [ 1 , 2 ] , pasteurized autografting , and bone elongation , have recently been employed for the reconstruction of limbs affected by malignant bone tumors . tumor prosthesis use is advantageous in that it facilitates stable function of the affected limb and an early return to social activities . the survival rate of patients treated with tumor prosthesis was satisfactory in a recent report . however , there are potentially serious complications , including infection , breakage , and loosening . we recently experienced a case in which tumor prosthesis of the knee joint ( howmedica modular reconstruction system ( hrms ) broke 13 years after surgery . reportedly , tumor prostheses breakage is caused by increased patient activity and loosening of the stem . when wide resection is performed for a malignant bone tumor around the knee joint , the surrounding soft tissue is also resected . thus , a hinge - type prosthesis is inevitably needed to stabilize the knee joint . the causes of breakage are thought to include the stem diameter , the length of resected bone , and prosthesis design . forty patients with primary malignant tumors around the knee joint underwent limb salvage by reconstruction with prosthesis use . to investigate the causes underlying breakage of prostheses , we divided these patients into those with ( 7 ) and without ( 33 ) revision of a broken or loose stem . the prosthesis model , stem diameter , length of resected bone , and international symposium on limb salvage ( isols ) x - ray evaluation were determined in each case . in the 7 revision cases , elements assumed to be causative were analyzed in detail to identify problems and possible countermeasures . the 40 subjects had primary malignant tumors of the knee joint and underwent limb salvage by reconstruction with a prosthesis in our department between 1979 and 2008 . the subjects comprised 20 women and 20 men , ranging in age from 7 to 82 years ( mean age , 27.5 years ) . the pathological diagnosis of the primary lesion was osteosarcoma in 28 , chondrosarcoma in 5 , bone malignant fibrous histiocytoma ( mfh ) in 3 , and a giant cell tumor gct ( grade 3 ) , synovial sarcoma , ewing s sarcoma , and a primitive neuroectodermal tumor ( pnet)in one each . the duration of follow - up ranged from 1 year and 5 months to 19 years ( mean : 11 years and 2 months ) . the 7 patients who underwent replacement were 6 women and 1 man , ranging in age from 7 to 44 years ( mean age , 26.5 years).the primary lesion was osteosarcoma in 4 , and pnet , gct ( grade 3 ) , and synovial sarcoma in one each . the duration of follow - up after the initial examination ranged from 12 to 19 years ( mean : 16 years ) . the prosthesis model , replacement site , stem diameter , length of resected bone , resection rate , and isols x - ray evaluation were investigated in all 40 cases . in the 7 cases undergoing revision , conditions and background factors at the time of breakage , breakage site , time of revision , models of previous and new prostheses , stem diameters before and after revision , details of the revision ( blood loss , operative time , surgical procedure ) , and the presence or absence of adjuvant therapy were also investigated . in addition , isols x - ray and functional evaluations were performed before revision and at the final follow - up . the prosthesis replacement site was the distal femur in 28 and the proximal tibia in 12 cases . among the 7 cases requiring revision due to stem breakage or loosening , the replacement site was the distal femur in 5 and the proximal tibia in 2 . the shortest and longest times until revision for breakage and loosening , after the initial wide resection followed by reconstruction of the tumor affected limb or an elongation - type prosthesis placement , were 10 months and 11 years , respectively , with a mean of 6 years and 2 months . at the initial replacement in our 40 cases , the howmedica modular reconstruction system ( hmrs ) was used in 22 , and the rotating hinge type in two . the kotz modular femur and tibia reconstruction system ( kmftr ) was used in 6 , the growing kotz in 10 , the kyocera custom - made prosthesis in one , and the kyocera ph1 ( physio - hinge type 1 ) in one . the broken models in the 7 revision cases were the kmftr in 4 , and hmrs and physio - hinge type i in one each . revision for loosening of the stem was performed in a 7-year - old female with pnet who had undergone reconstruction of the distal femur with a growing kotz ( case 2 ) . the model used for revision was the hmrs in 3 cases reconstructed with a kmftr at the initial replacement excluding a proximal tibial case ( case 2 ) . in case 38 , a ph type 1 with an 11-mm stem diameter was changed to the slightly thicker ph type 2 with a 12-mm stem diameter . in case 39 , the stem diameter ( 12 mm ) of the new prosthesis was the same as that before breakage . however , the new stem , at 15 cm , was longer . in case 2 , a new component was prepared , considering that the tibial component of the hmrs employed for adults is too large . the new tibial component was designed with proximal and distal diameters of 15 and 10 mm , respectively , and a stem length of 14 cm , and the stem was entirely covered with a porous coating ( tables 1 , 2).table 1list of cases with reconstruction of regions around the knee joint using prosthesescaseagegenderpathological diagnosismodelreplacement siteresected length ( cm)stem diameter ( mm)resection rat ( % ) 17mosgrowing kotzdf14836.8027fpnetgrowing kotzdf21953311mosgrowing kotzdf131133416fosgrowing kotzdf131031510fosgrowing kotzdf15945612mosgrowing kotzdf17104478mosgrowing kotzpt121050812mewing sarcomagrowing kotzpt151035916fosgrowing kotzpt1010331012fosgrowing kotzpt1610321134moshmrspt1612471260foshmrsdf1614381357fchondrosarcomahmrsdf1211351418foshmrspt1212341557fchondrosarcomahmrs ( rotating)df1212261627foshmrsdf1813391725moshmrsdf1713341824moshmrspt1311371912foshmrsdf18.512422020mmfh of bonehmrspt14.511472125moshmrs ( rotating)df1212272227moshmrsdf1612332356fchondrosarcomahmrspt1812472416moshmrsdf1210302513moshmrsdf1612272618moshmrsdf1412432720moshmrsdf1213292866fmfh of bonekmftrdf1211272982mchondrosarcomakmftrdf1612343027moshmrspt1711453152foskyocera ( cement)df1611353250mchondrosarcomahmrsdf1811483341mgcthmrspt1312403444foskmftrdf1210233531foskmftrpt1210343626foskmftrdf2310543740fsynovial sarcomakmftrdf1410483815mosph type1 ( cement)df1811553928foshmrsdf2212484066fmfh of bonehmrsdf181238df distal femur , pt proximal femur , hmrs howmedica modular resection system , kmftr kotz modular femoral and tibia replacement , ph type 1 physio - hinge type 1 , ph type 2 physio - hinge type 2table 2revision cases managed by our departmentcaseagesexlocationtime of revision ( mon ) ( m)typediameter ( mm)screw breakagetype of new prosthesisdiametercase 3444fdistal femur48kmftr10hmrs12 mmcase 3531fproximal tibia84kmftr10kmftr10 mmcase 3626fdistal femur10kmftr10hmrs12 mmcase 3740fdistal femur28kmftr10+hmrs12 mmcase 3815mdistal femur108ph type111ph type 212 mmcase 3928fdistal femur132hmrs12+hmrs12 mmcase 27fproximal tibia113growing kotzproximal : 12distal : 9growing kotzproximal : 15 mdistal : 10 mm list of cases with reconstruction of regions around the knee joint using prostheses df distal femur , pt proximal femur , hmrs howmedica modular resection system , kmftr kotz modular femoral and tibia replacement , ph type 1 physio - hinge type 1 , ph type 2 physio - hinge type 2 revision cases managed by our department the stem diameters ranged from 8 to 14 mm ( mean : 11.1 mm ) in our 40 cases . in the non - revision cases , they ranged from 8 to 14 mm ( mean : 11.2 mm ) . in the revision cases , the diameters ranged from 9 to 12 mm ( mean : 10.2 mm ) , the diameter of the broken kmftr was 10 mm in all 4 cases . the prosthesis was changed to one with a stem diameter of 12 mm in 3 of these 4 cases . in case 39 , the stem diameter ( 12 mm ) of the new prosthesis was the same as that of the broken one , but the new stem was longer ( 15 cm ) . in case 2 , the stem region of the tibial component of the growing kotz employed in the initial replacement had proximal and distal stem diameters of 12 and 9 mm , respectively , and a length of 10 cm . on revision , it was replaced with a new porous - coated tibial component with proximal and distal diameters of 15 and 10 mm , respectively , and a length of 14 cm . the respective maximum and minimum lengths of resected bone including the tumor region were 12 and 23 cm , with a mean of 15.4 cm . those in the 33 non - revision cases were 10 and 18.5 cm , respectively , with a mean of 14.6 cm . in the 7 revision cases , these lengths were 12 and 22 cm , respectively , with a mean of 17.7 cm ( table 1 ) . the resected region accounted for 2750% ( mean : 36 ) in the 33 non - revision cases , and 2353% ( mean : 45% ) in the 7 revision cases . thus , the ratio of the resected region was greater , comprising nearly half of the affected bone , in cases undergoing revision for breakage or loosening ( table 1 ) . five had experienced sudden pain in the femoral or knee joint regions , while walking . they visited our hospital , and breakage was identified on x - ray examination . in the other patient ( case 39 ) , dull pain appeared in the proximal femoral region and had become severe about 4 months later . only this patient was actively engaged in activities such as dancing and mountain climbing , while the other 6 were not especially involved in athletic activities . regarding the social backgrounds of these 7 patients , cases 3437 were housewives , case 38 was a clerical employee , mainly working at a desk , case 39 was a speech therapist , and case 2 was a student . x - ray evaluation was performed at the final follow - up in all 40 cases , and all items pertaining to bone remodeling , interface , and anchorage were graded as excellent in about 70% of these patients . however , in the revision group , the interface before revision was graded as poor and fair in cases 2 and 39 , respectively , and anchorage was graded as fair in all cases . after revision , bone remodeling was graded as poor only in case 38 , in whom the bone cortex around the stem was thinned by more than 1/3 . a radiolucent line was also noted in the interface , resulting in a grading of fair . the evaluation was 5380% ( mean : 70.1% ) before and 6386% ( mean : 76.2% ) after revision ( table 3).table 3functional and radiological assessments and the presence / absence of adjuvant therapy before and after replacementfunctional assessmentradiographical assessmentcasebefore replacement ( % ) after replacement ( % ) before replacementafter replacementbone remodeling / interface / anchoragebone remodeling / interface / anchoragecase 347880e / g / fg / g / ecase 357578e / g / fg / g / ecase 367673e / e / ff / e / ecase 377374g / g / fg / g / ecase 388063g / g / fp / f / gcase 396080g / f / fg / g / ecase 25386g / p / fe / e / ee excellent , g good , f failure , p poor functional and radiological assessments and the presence / absence of adjuvant therapy before and after replacement e excellent , g good , f failure , p poor the shortest and longest operative times in the 7 cases were 3 h and 20 min and 6 h and 11 min , respectively , with a mean of 4 h and 7 min . the minimum and maximum blood losses were 155 and 600 g ( mean : 369 g ) , respectively ( table 4).table 4adjuvant therapy and details of revision surgerycaseadjuvant therapyblood loss ( g)operation time ( min)case 34 + 430200case 35370260case 36 + 282212case 37 + 330240case 38 + 155210case 39 + 600220case 2 + 420371 adjuvant therapy and details of revision surgery case 33 in the revision group was diagnosed with a grade 3 gct of the bone , and underwent surgery alone . pre- and postoperative chemotherapies were administered in the other 6 cases . in case 2 , radiotherapy ( 50 gy ) was additionally performed for local control , after the completion of preoperative chemotherapy ( table 4 ) . the prosthesis replacement site was the distal femur in 28 and the proximal tibia in 12 cases . among the 7 cases requiring revision due to stem breakage or loosening , the replacement site was the distal femur in 5 and the proximal tibia in 2 . the shortest and longest times until revision for breakage and loosening , after the initial wide resection followed by reconstruction of the tumor affected limb or an elongation - type prosthesis placement , were 10 months and 11 years , respectively , with a mean of 6 years and 2 months . at the initial replacement in our 40 cases , the howmedica modular reconstruction system ( hmrs ) was used in 22 , and the rotating hinge type in two . the kotz modular femur and tibia reconstruction system ( kmftr ) was used in 6 , the growing kotz in 10 , the kyocera custom - made prosthesis in one , and the kyocera ph1 ( physio - hinge type 1 ) in one . the broken models in the 7 revision cases were the kmftr in 4 , and hmrs and physio - hinge type i in one each . revision for loosening of the stem was performed in a 7-year - old female with pnet who had undergone reconstruction of the distal femur with a growing kotz ( case 2 ) . the model used for revision was the hmrs in 3 cases reconstructed with a kmftr at the initial replacement excluding a proximal tibial case ( case 2 ) . in case 38 , a ph type 1 with an 11-mm stem diameter was changed to the slightly thicker ph type 2 with a 12-mm stem diameter . in case 39 , the stem diameter ( 12 mm ) of the new prosthesis was the same as that before breakage . however , the new stem , at 15 cm , was longer . in case 2 , a new component was prepared , considering that the tibial component of the hmrs employed for adults is too large . the new tibial component was designed with proximal and distal diameters of 15 and 10 mm , respectively , and a stem length of 14 cm , and the stem was entirely covered with a porous coating ( tables 1 , 2).table 1list of cases with reconstruction of regions around the knee joint using prosthesescaseagegenderpathological diagnosismodelreplacement siteresected length ( cm)stem diameter ( mm)resection rat ( % ) 17mosgrowing kotzdf14836.8027fpnetgrowing kotzdf21953311mosgrowing kotzdf131133416fosgrowing kotzdf131031510fosgrowing kotzdf15945612mosgrowing kotzdf17104478mosgrowing kotzpt121050812mewing sarcomagrowing kotzpt151035916fosgrowing kotzpt1010331012fosgrowing kotzpt1610321134moshmrspt1612471260foshmrsdf1614381357fchondrosarcomahmrsdf1211351418foshmrspt1212341557fchondrosarcomahmrs ( rotating)df1212261627foshmrsdf1813391725moshmrsdf1713341824moshmrspt1311371912foshmrsdf18.512422020mmfh of bonehmrspt14.511472125moshmrs ( rotating)df1212272227moshmrsdf1612332356fchondrosarcomahmrspt1812472416moshmrsdf1210302513moshmrsdf1612272618moshmrsdf1412432720moshmrsdf1213292866fmfh of bonekmftrdf1211272982mchondrosarcomakmftrdf1612343027moshmrspt1711453152foskyocera ( cement)df1611353250mchondrosarcomahmrsdf1811483341mgcthmrspt1312403444foskmftrdf1210233531foskmftrpt1210343626foskmftrdf2310543740fsynovial sarcomakmftrdf1410483815mosph type1 ( cement)df1811553928foshmrsdf2212484066fmfh of bonehmrsdf181238df distal femur , pt proximal femur , hmrs howmedica modular resection system , kmftr kotz modular femoral and tibia replacement , ph type 1 physio - hinge type 1 , ph type 2 physio - hinge type 2table 2revision cases managed by our departmentcaseagesexlocationtime of revision ( mon ) ( m)typediameter ( mm)screw breakagetype of new prosthesisdiametercase 3444fdistal femur48kmftr10hmrs12 mmcase 3531fproximal tibia84kmftr10kmftr10 mmcase 3626fdistal femur10kmftr10hmrs12 mmcase 3740fdistal femur28kmftr10+hmrs12 mmcase 3815mdistal femur108ph type111ph type 212 mmcase 3928fdistal femur132hmrs12+hmrs12 mmcase 27fproximal tibia113growing kotzproximal : 12distal : 9growing kotzproximal : 15 mdistal : 10 mm list of cases with reconstruction of regions around the knee joint using prostheses df distal femur , pt proximal femur , hmrs howmedica modular resection system , kmftr kotz modular femoral and tibia replacement , ph type 1 physio - hinge type 1 , ph type 2 physio - hinge type 2 revision cases managed by our department the stem diameters ranged from 8 to 14 mm ( mean : 11.1 mm ) in our 40 cases . in the non - revision cases , they ranged from 8 to 14 mm ( mean : 11.2 mm ) . in the revision cases , the diameters ranged from 9 to 12 mm ( mean : 10.2 mm ) , the diameter of the broken kmftr was 10 mm in all 4 cases . the prosthesis was changed to one with a stem diameter of 12 mm in 3 of these 4 cases . in case 39 , the stem diameter ( 12 mm ) of the new prosthesis was the same as that of the broken one , but the new stem was longer ( 15 cm ) . in case 2 , the stem region of the tibial component of the growing kotz employed in the initial replacement had proximal and distal stem diameters of 12 and 9 mm , respectively , and a length of 10 cm . on revision , it was replaced with a new porous - coated tibial component with proximal and distal diameters of 15 and 10 mm , respectively , and a length of 14 cm . the respective maximum and minimum lengths of resected bone including the tumor region were 12 and 23 cm , with a mean of 15.4 cm . those in the 33 non - revision cases were 10 and 18.5 cm , respectively , with a mean of 14.6 cm . in the 7 revision cases , these lengths were 12 and 22 cm , respectively , with a mean of 17.7 cm ( table 1 ) . the resected region accounted for 2750% ( mean : 36 ) in the 33 non - revision cases , and 2353% ( mean : 45% ) in the 7 revision cases . thus , the ratio of the resected region was greater , comprising nearly half of the affected bone , in cases undergoing revision for breakage or loosening ( table 1 ) . five had experienced sudden pain in the femoral or knee joint regions , while walking . they visited our hospital , and breakage was identified on x - ray examination . in the other patient ( case 39 ) , dull pain appeared in the proximal femoral region and had become severe about 4 months later . at this time , breakage was identified . only this patient was actively engaged in activities such as dancing and mountain climbing , while the other 6 were not especially involved in athletic activities . regarding the social backgrounds of these 7 patients , cases 3437 were housewives , case 38 was a clerical employee , mainly working at a desk , case 39 was a speech therapist , and case 2 was a student . x - ray evaluation was performed at the final follow - up in all 40 cases , and all items pertaining to bone remodeling , interface , and anchorage were graded as excellent in about 70% of these patients . however , in the revision group , the interface before revision was graded as poor and fair in cases 2 and 39 , respectively , and anchorage was graded as fair in all cases . after revision , bone remodeling was graded as poor only in case 38 , in whom the bone cortex around the stem was thinned by more than 1/3 . a radiolucent line was also noted in the interface , resulting in a grading of fair . the evaluation was 5380% ( mean : 70.1% ) before and 6386% ( mean : 76.2% ) after revision ( table 3).table 3functional and radiological assessments and the presence / absence of adjuvant therapy before and after replacementfunctional assessmentradiographical assessmentcasebefore replacement ( % ) after replacement ( % ) before replacementafter replacementbone remodeling / interface / anchoragebone remodeling / interface / anchoragecase 347880e / g / fg / g / ecase 357578e / g / fg / g / ecase 367673e / e / ff / e / ecase 377374g / g / fg / g / ecase 388063g / g / fp / f / gcase 396080g / f / fg / g / ecase 25386g / p / fe / e / ee excellent , g good , f failure , p poor functional and radiological assessments and the presence / absence of adjuvant therapy before and after replacement e excellent , g good , f failure , p poor the shortest and longest operative times in the 7 cases were 3 h and 20 min and 6 h and 11 min , respectively , with a mean of 4 h and 7 min . the minimum and maximum blood losses were 155 and 600 g ( mean : 369 g ) , respectively ( table 4).table 4adjuvant therapy and details of revision surgerycaseadjuvant therapyblood loss ( g)operation time ( min)case 34 + 430200case 35370260case 36 + 282212case 37 + 330240case 38 + 155210case 39 + 600220case 2 + 420371 adjuvant therapy and details of revision surgery case 33 in the revision group was diagnosed with a grade 3 gct of the bone , and underwent surgery alone . pre- and postoperative chemotherapies were administered in the other 6 cases . in case 2 , radiotherapy ( 50 gy ) was additionally performed for local control , after the completion of preoperative chemotherapy ( table 4 ) . recent advancements in surgical approaches and chemotherapy for primary malignant bone tumors have increased survival rates . the usefulness of reconstruction methods for affected limbs , including prostheses , has also been confirmed . however , complications associated with prostheses , such as infection , loosening , and breakage , remain problematic . regarding stem breakage , in 1994 , capanna et al . reported that stem breakage occurred in 6 ( 6.3% ) of 95 cases treated with modular uncemented tumor prostheses . in 2001 , mittenmayer et al . reported that major complications occurred in 19 of 100 cases with uncemented tumor prostheses , 11 of these involved aseptic loosening , and septic loosening and implant fracture occurred in 4 each in 2006 , gosheger reported that stem breakage occurred in 4 ( 1.6% ) of 250 cases with uncemented tumor prostheses . in our department , breakage occurred after 5 years and 6 months on average , with the earliest being 10 months and latest 11 years . the models used were the kmftr in 4 and the hmrs and ph type 1 in one case each . the stem diameters of 10 mm in 4 and 11 mm in one case were relatively thin for distal femoral stems . after revision , the stem diameter was 10 mm in only one , being thicker in all other cases . although increased activity of patients and stem loosening were considered to be the causes of stem breakage , the design of the prostheses may have contributed to breakage , because the prostheses generally had a hinge - type structure . in 2005 , griffin et al . reported that the incidence of kmftr stem breakage involving the proximal tibia rose when the stem diameter was small , and the length of resected bone increased . they also described cases of distal femoral replacement : the 5-year survival rates were 35 , 85 , and 71.2% in patients in whom the distal femoral stem sizes were 1012 , 13 , and 1416 mm , respectively . these observations showed that the stem diameter , rather than the resection length , was related to breakage in cases undergoing distal femoral replacement . the mean stem size of 10.2 mm in the revision cases and 11.2 mm in the non - revision cases confirmed that a thin stem was used in the revision cases . currently , 1115-mm straight types and 1215-mm curved types of diaphysis - fixing pieces are available for hmrs . all stems with a 10-mm diameter are of the growing kotz and kmftr types . in cases reconstructed with the hmrs , stems with a relatively small diameter , 11 or 12 mm , were used in 80% . the mean resection rate was 34.7% in the non - revision and 45% in the revision cases . the length of resected bone was thus greater in the revision than in the non - revision cases . when the resection rate is almost 45% in the clinical setting , possible reconstruction methods other than the use of a tumor prosthesis include total femoral replacement and biological reconstruction [ 3 , 9 , 10 ] when a tumor prosthesis is used , attention should be paid to the bone resection rate . comparison by region , such as the femoral and proximal tibial regions , was not possible because of the small number of cases . however , we would not expect more stress to be loaded on a thin stem used for a region from which a large amount of bone was excised . griffin et al . also reported that stem breakage occurred in 6 ( 6.1% ) of 99 cases reconstructed with the kmftr . they stated that the ideal design of a prosthesis may be a strong and thick stem without holes to stop lateral movement , which facilitates bone ingrowth comparable or superior to that around the kmftr stem . aseptic loosening of the stem may be another cause of stem breakage . in our 40 patients , loosening was apparently present at the interface , being graded as poor in cases 2 and 8 with a growing kotz . it was also graded as poor in case 15 , a 57-year - old female with distal femoral chondrosarcoma in whom a rotating hinge - type hmrs was applied . the incidence of aseptic loosening varies among reports , ranging from as low as 1 and 5% up to 26 and 29% [ 1118 ] . in 2001 , mittermayer et al . reported that aseptic loosening occurred in 27% of cases with complications involving stems . in 1990 , they developed an anatomically curved stem , which fit in the femoral bone marrow cavity to avoid stress shielding generated by firm fixation around the stem the incidence of aseptic loosening in cases receiving this type of prosthesis was approximately 10% during a 42134-month follow - up period . however , only in our case 39 ( 28-year - old female ) in the revision group had a high activity level , i.e. , such a tendency was not apparent in our patients . functional and x - ray evaluations following revision were favorable over the short , medium , and long term in various reports [ 6 , 11 , 14 , 17 ] . a similar tendency was noted in our patients . however , a 40-mm leg length discrepancy remained after surgery in our growing kotz - revised cases , resulting in a functionally unsatisfactory outcome . the shortest and longest operative times in the 7 revision cases were 3 h and 20 min and 6 h and 11 min , respectively , with a mean of 4 h and 7 min , and the minimum , maximum , and mean blood losses were 155 , 600 , and 369 g , respectively . the levels of surgical stress may have been similar to that in the first wide resection with regard to the operative time and blood loss . tang and sim reported the revision procedures for stem breakage [ 19 , 20 ] . the goals of distal femoral revision are to cut - off the femoral bone cortex using a surge airtome or drill following the shape of the stem . this requires great care to avoid breakage of the cut - out bone cortex upon removal of the broken stem . a new stem must also be inserted , followed by returning the cut - off bone cortex block to its original position . concerning reaming , we ream the femoral medullary cavity to a diameter 1 mm larger than that determined by preoperative measurement in principle . however , when the medullary cavity is narrow , reaming is performed to the stem diameter selected based on preoperative measurement . when a trial stem can be inserted , the real stem is inserted . when a trial stem can not be inserted , over - reaming by 1 mm is performed . in revision surgery , since a thicker stem is inserted , over - reaming by 1 mm is always performed . firm fixation is then with a cable . at this point , it is also necessary to add autologous or artificial bone grafting to assure sufficient future strength ( figs . 1 , 2).fig . 1the residual stem in the femur was carefully cut off using a surge airtome or chisel . attention should also be paid to avoiding breakage of the fenestrated bone fragment and to return it to the original position after placement of the new stemfig . 2after placement of the new stem , bone grafting is performed around the stem as shown . the use of a cable should also be considered for achieving stronger fixation the residual stem in the femur was carefully cut off using a surge airtome or chisel . attention should also be paid to avoiding breakage of the fenestrated bone fragment and to return it to the original position after placement of the new stem after placement of the new stem , bone grafting is performed around the stem as shown . the use of a cable should also be considered for achieving stronger fixation based on the above observations , stem size , shape , and porous coating serve as countermeasures against stem breakage , as does bone grafting to the bone stump and preservation of the periosteum [ 5 , 11 ] . however , no ideal prosthesis has as yet been established , though many researchers have investigated and developed various promising models . based on this study , we consider the following points to be important for avoiding prosthetic stem breakage : ( 1 ) minimizing the length of bone resected , i.e. , it is desirable not to exceed one third of the affected bone by employing a limited operation , and ( 2 ) selection of a stem diameter of at least 12 mm . for the femur , the use of a curved stem should be investigated in consideration of the anatomical shape of this bone . regarding limb salvage for malignant bone tumors in children , an elongation - type prosthesis can be lengthened to correspond to the predicted leg length discrepancy , when employed for wide resection of a periarticular tumor including the joint [ 2230 ] . a characteristic of the elongation - type growing kotz is the porous coating on the diaphysis - fixing piece of the elongation region . in contrast , non - porous processing is added to the non - elongation region . however when a pediatric patient grows more than expected , particularly , in the transverse axis of the proximal tibia , loosening and burying of the stem start and slowly progress . although the growing kotz can be elongated with growth , the prosthetic design , particularly the width of the tibial component , should be sufficiently investigated in consideration of the child s development . prosthesis use facilitates the early acquisition of stable functioning of the affected limb , but several complications have yet to be overcome . methods considered to reduce the risk of prosthesis breakage , include limiting resection of the affected bone to no more than 30% and adoption of as thick a stem as possible , i.e. , with a diameter of at least 12 mm , fitting the anatomical shape of the femur . the unchanged function of the affected limb after revision and instructing of patients to avoid excessive exercise in daily activities are also important for maintenance of prostheses . although only the elongation - type of growing kotz is covered by the national health insurance system in japan , this prosthesis should also be selected with care , taking the child s future development into consideration . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

secondary organic aerosol ( soa ) , formed in situ from the conversion of gas - phase volatile organic compounds ( vocs ) to oxidized products that partition preferentially to the particle phase , is an important source of aerosol mass both regionally and globally . it is estimated that approximately 70% of total global organic aerosol production is secondary ( 150 tgc / yr ) , but formation mechanisms remain uncertain . a large fraction of total organic aerosol is thought to originate from the oxidation products of biogenic voc emissions . these naturally emitted compounds constitute the majority of voc emissions to the atmosphere globally , with a diverse and regionally varied mixture of isoprenoid chemical structures , depending on regional plant species distributions . the gas - phase oxidation of vocs to condensable products is initiated by reaction with o3 or the oh or no3 radicals . the relative importance of each competing oxidant depends on the relevant rate constants ( which depends on the voc molecular structure ) and ambient concentrations . for example , no3-initiated reactions with biogenic alkenes have large rate constants , and will be most important at night ( when photolysis and no reaction sinks for no3 radical are minimized ) , and in regions that contain both high biogenic emissions and elevated nox such as downwind of urban areas or industrial point sources . soa yields from oh and o3 oxidation of bvoc have been studied extensively , but less is known about reactions involving no3-induced oxidation . because partitioning to the aerosol phase depends on molecular weight and polarity , the precursor voc generally needs to contain a minimum number of carbon atoms ( or undergo condensed phase oligomerization reactions ) to produce condensable oxidation products and thus form soa . isoprene ( c5h8 ) is the globally dominant bvoc , comprising 50% of total global nonmethane voc emissions by mass with total emissions estimated at approximately 500 tg yr . however , isoprene and its no3-induced oxidation products are quite volatile , and thus have relatively low no3 soa yields of 424% . the larger monoterpenes ( c10h16 ) and sesquiterpenes ( c15h24 ) , which are much more prevalent in coniferous forests , are more efficient soa precursors via their production of extremely low - volatility organic compounds . the no3 oxidation of some terpenes has been studied in laboratory experiments , summarized in table 1 . wngberg et al . , hallquist et al . , and spittler et al . all observed substantially higher ketone yields and lower organonitrate yields for -pinene than for the other terpenes ; where soa yield measurements are available they roughly correlate with organonitrate yields , showing lower yield for -pinene . we note that these chamber experiments may preclude further oxidation of initial products , which could produce additional soa in the real atmosphere ( e.g. , photo - oxidation of pinonaldehyde ) . in hallquist et al . , these ketone yields refer to the dominant single ketone product , e.g. in the case of -pinene , pinonaldehyde . a systematic controlled chamber study of soa production from six mono- and sesquiterpenes is the focus of the work presented here . we measured the soa yield from no3 reacting with the individual monoterpenes -pinene , -pinene , -3-carene , limonene , sabinene , and with the sesquiterpene -caryophyllene ( structures shown in supporting information ( si ) figure s6 and in the abstract ) . since the nitrato - peroxy radicals produced from these terpenes are all tertiary , we expect no ho2 production in the chamber . hence , these experiments explore ro2ro2 and ro2no3 chemistry only , omitting ro2ho2 chemistry that would contribute in the real nighttime atmosphere . these experiments were performed in continuous - flow mode in a dark chamber at the atmospheric chemistry division of the national center for atmospheric research ( ncar ) in fall 2011 . we monitored particle size distributions and total alkyl nitrate concentrations to enable determination of soa mass yields and alkyl nitrate yields . a detailed description of the ncar chamber facility instrumentation and cleaning and operating procedures for the experiments described herein are provided in the si . figure si shows the chamber diagram , and table si the instrumentation employed in these experiments . briefly , the 10 m chamber was run with all reagents input continuously under dark conditions , with an average chamber residence time of 4 h. the no3 and n2o5 concentrations were alternately measured either before entering the chamber , or from the chamber itself . flows were adjusted to give initial [ n2o5 + no3 ] and [ bvoc ] of 10100 ppb in dry zero air . soa experiments were initiated by injecting bvoc ( 50 ppm in n2 ) into the n2o5 + no3 filled chamber , delivered from the standard cylinders using flow restrictors and added to the zero air flow to obtain the final desired mixing ratios ( table 2 ) . a series of 11 experiments were conducted with the six bvoc , in most cases with two levels of oxidant and bvoc concentrations : a nominally high ( 50 ppb ) and a low ( 10 ppb ) concentration of each ( see table 2 ) . for the high concentration experiments only ( expts . 1 , 2 , 4 , 6 , and 9 ) , organic nitrate measurements are available . in these experiments , total alkyl + peroxy nitrate mixing ratios ( ans + pns ) , subsequently referred to collectively as organonitrates , are determined in the gas and aerosol phases by sampling directly or through a charcoal denuder . except -pinene experiments , the ratio of n2o5 to bvoc in the inlet line was approximately 1:1 ; for the high experiments , the oxidant was generally in slight excess ( with the exception of expt # 9 ) . the highest purity sabinene source available was 80% , and contained 20% -pinene . the following measurements were made during each experiment : ( 1 ) no3 and n2o5 concentrations on both inlet and outlet flows by cavity ringdown spectroscopy ( crds , wagner et al . ; dube et al . ) , and on the outlet side only : ( 2 ) no and no2 by chemiluminescence , ( 3 ) o3 by absorption spectroscopy , ( 4 ) particle number and size distribution by a scanning mobility particle sizer ( smps ) , and ( 5 ) organonitrates by thermal dissociation laser - induced fluorescence ( td - lif , rollins et al . ) . the specifications of each instrument used in these experiments are shown in table s1 in the si . in order to interpret observed aerosol formation , it is essential to understand the effect of chamber wall losses on measured aerosol size distributions . following the methods of van reken et al . , we measured the size - dependent wall loss of ammonium sulfate aerosol at steady - state flow . results and additional analysis details are shown in the si ( figure s2 ) . average input and output size distributions were analyzed to derive the first - order size - dependent wall loss coefficient , (dp ) , with the chamber considered to behave as a continuously stirred tank reactor:1 the losses are applied cumulatively , so that corrected aerosol mass timeseries include all particles lost since the start of the experiment , following the method of mcmurry and grosjean , and we find values consistent with previous chamber studies . recent work has shown that vapor - phase wall losses may also substantially contribute to underestimation of chamber soa yields , suggesting that the yields reported here may be lower limits . the implications of this unconstrained process for observed relative organonitrate yields is discussed in below . particle formation and growth was observed for all bvocs except -pinene , although with widely varying yields . the observed time evolution of the particle size distribution function of a representative experiment ( experiment 3 , -pinene at low concentration ) is shown in figure 1 , illustrating the new particle formation and growth that occurred in each experiment ( except -pinene ) . wall loss corrected soa growth curve for experiment 3 ( -pinene low concentration ) . figure 2 shows the time evolution of aerosol number ( ntot , figure 2a ) and volume concentration ( vtot , figure 2b , c ) . these plots show the comparison of soa growth from different bvoc precursors , including both concentration conditions for each precursor ( nominally 50 and 10 ppb for both no3 and bvoc ) . generally , we observe the largest peaks in ntot from the oxidation of the bvocs with two double bonds , -caryophyllene and limonene , regardless of low or high concentration . hence , for example , the peak ntot from limonene at 10 ppb initial concentration is larger than the peak ntot for all the monoterpenes with a single double bond , even at the higher concentration initial conditions . in contrast , the vtot curves grow fastest for the higher concentration experiments of all terpene precursors . in the case of -caryophyllene , the faster volume growth at low loading may be due to differing chemical regime ; higher no3/bvoc ratio could lead to more second - generation products . time series of total wall - loss corrected aerosol ( a ) particle number concentration , and ( b ) volume and mass concentration , from all experiments producing soa . panel ( c ) repeats ( b ) , zoomed in on the lowest 30 g m of the y axis to show that the same pattern in volume growth is observed for the low - concentration series as for the high . low and high indicate the nominal concentrations of bvoc and oxidant in each experiment ; bpin denotes -pinene ; dcar , -3-carene , limo , limonene ; bcary , -caryophyllene ; and sabi , sabinene . we determined the unitless time - dependent soa mass yields over the course of the experiments , defined as y = m/voc , based on wall - loss corrected change in aerosol mass m ( g m ) divided by amount of voc reacted voc ( g m ) , referenced to the beginning of the experiment when both aerosol mass ( m ) and voc were zero . since each experiment was initiated with voc injection into a chamber already filled with oxidant , the voc began to react immediately , preventing measurement of its concentration during initial reaction . since there are uncertainties in both the reaction kinetics and chamber mixing , we employ two separate strategies to bracket the possible range of voc : ( 1 ) a simple observationally constrained method that more accurately captures mixing dynamics due to the high precision and time resolution measurement of [ no3 ] , and ( 2 ) a free - running kinetics box model assuming a well - mixed chamber , that may more accurately capture subsequent ro2 chemistry , but is subject to large uncertainties in ro2 rate constants for these systems and can not capture mixing . for the first method , we use the known amount of voc injected and the observed [ no3 ] in each time step ( t ) to determine the amount of voc consumed via eq 2:2where rate constants k are taken from calvert et al . ( 2000 ) , [ no3 ] is the instantaneous observed concentration ( and hence includes no3 produced from n2o5 added at each time step ) , and [ voc ] is the sum of concentration added to the chamber and the unreacted concentration in the chamber from the previous time step . if this calculation of voc results in a reacted amount less than the voc injected during that time step , then the residual voc builds up in the chamber and contributes to the instantaneous rate calculation for the next timsestep . in this way , a voc timeseries is derived that depends only on observed no3 consumption , assuming a one - to - one no3 to voc stoichiometry . this method ignores any reactions of no3 with first - generation oxidation products ( no3 + ro2 reactions ) , thus rendering it an upper limit on the amount of voc reacted , which leads to lower limit soa yields . however , it has the advantage of allowing our most precise , highest time - resolution measurement to directly incorporate the time scale of chamber mixing into the calculation of voc . thus , for each experiment we determine a complete voc time series that is constrained until no3 concentration decreases to zero , as shown in the si ( figure s3 ) . often , the usable smps data is truncated before this occurs , because the aerosol size distribution grows to diameters beyond the measurement range ( 350 nm ) . in the second method of voc calculation , we employ a free - running kinetics box model constrained only by the known addition rates of n2o5 and voc . the cumulative voc reacted over time is determined , by iterative solution of coupled kinetic equations for all x molecules:3where [ x]in is the concentration of x in the inlet flow ( only n2o5 and voc are added , at known concentrations shown in table 2 ) , [ x]ch is the concentration in the chamber , q is the flow rate through the chamber ( 40 lpm ) , and v the chamber volume ( 10 000 l ) , y are all chemical losses ( for no3 , reactions with voc , ro2 , and no2 ; for voc , only reaction with no3 ) , z are all chemical sources ( n2o5 decomposition for no3 ; none for voc ) , and kwall , x is the rate of loss of x to chamber walls . the complete set of reactions and rate constants used are described in the si figure s4 and table s2 . this method is superior in its chemical complexity , explicitly including ro2 + no3 , ro2 + ro2 , and ro2 + no2 reactions after the initial no3 + voc ro2 . however , because these reaction rates are poorly known for monoterpene nitrato - ro2 , this introduces some uncertainty . further , since we are no longer constrained by no3 data , we must invoke wall losses for no3 and n2o5 . in order to run this model consistently across experiments and with the minimum number of arbitrarily tunable variables , we spun up the model with continuous n2o5 addition to the initial conditions of each chamber experiment , determining no3 and n2o5 wall loss rate constants that best fit all available data . each experiment then proceeds by continuous addition of voc and n2o5 at the known injection rates , which we model free - running , with no tuning to match observed no3 decay . this model assumes a well - mixed chamber at all times , so we interpret discrepancies with observed no3 decay to be due to mixing inhomogeneity or uncertainties in ro2 rate constants ( see si figure s5 for an exemplary model fit of no3 decay ) . because this method incorporates additional losses of no3 , resulting voc consumption is slower , leading to higher apparent soa yields . both methods of voc determination ignore reaction with the second double bonds in limonene or -caryophyllene , which would deplete no3 further and enhance soa yields . the rate of no3 reaction at the second , exocyclic double bond is predicted to be 30-fold slower in the case of limonene , so this is not expected to be a large effect , although it may be more competitive in the low -caryophyllene experiment , where n2o5 concentration greatly exceeded bvoc . each wall - loss corrected smps size distribution in the time series was converted to m as follows . first , we iteratively applied the size - dependent wall loss corrections to the full smps time series to derive a complete time series of the size distributions that would have been observed had no particles been lost the walls . then , number concentration data from each size bin were converted to soa mass concentration in g m using the mean bin diameter and assumed organic aerosol density of 1.4 g cm . since no seed particles were used , all observed aerosol is organic , and the wall - loss corrected aerosol mass gives cumulative m . the ratio of this m to cumulative voc consumed is the unitless mass yield , ( y = m/voc ) . we observe differences between high and low concentration mass yield curves ( figure 3 ) that vary with bvoc , suggesting that the experiments differ in more than simply the total aerosol mass loading . we note from the kinetics modeling that in all experiments , the ratio of ro2no3 to ro2ro2 rates is highest initially and decreases , in the case of the low concentration experiments crossing over so that after several hours of reaction , ro2ro2 reactions are predicted to be dominant , while in the high concentration experiments , ro2no3 reactions dominate throughout . because these rate constants are quite uncertain and not known for specific bvoc precursors , we simply conclude that some of the explanation of the higher yields for the high concentration experiments may lie in the differences in ro2 radical fate . we note here again an important difference between these experiments and what would be observed in the atmosphere : ho2 radicals were nonexistent in these chamber experiments , but are expected to be a significant reaction partner for ro2 in the real atmosphere . yield curves for high and low no3 + bvoc soa experiments , using ( a ) observed no3 decay , or ( b ) complete kinetics model to determine reacted bvoc . bvoc labeling is as in figure 2 . in all low concentration experiments , due to the slower reaction , the voc is better constrained for longer periods , and the mixing time scale is faster relative to reactions , resulting in yield curves with less uncertainty but with the same inter - bvoc trend observed at both concentration conditions . due to these differences , subsequent discussion will focus only on these low concentration experiments . as is apparent in figure 3 , the high concentration experiments ( and early stages of some low concentration experiments ) produce unrealistically high yields , especially in the full kinetics model , suggesting that in these cases , reactions were faster than chamber mixing and that this box model may be inaccurate . according to absorptive partitioning theory , yields should increase with aerosol mass loading , assuming a constant product distribution over time . if , however , the product distribution is changing over time as ro2 reactive fate changes , then mass yields could appear to decrease as an artifact of a shift from early high molecular weight products to later lower molecular weight products . as a result of these uncertainties at early times , we do not fit the mass - dependent yield for these experiments , but rather report yields at 10 g m for each low concentration experiment ( table 3 ) . we choose 10 g m because it is sufficiently late in the low concentration experiments ( generally 1.5 h ) that irregularities in the yield curve have subsided . method 1 represents a lower limit yield , since no other ro2 reactions are included , while method 2 s uncertainty lies in the uncertainty of rate constants ( see si discussion around figures s3s6 ) . for -caryophyllene , extremely rapid modeled ro2 + no3 reactions result in predicted very slow bvoc consumption and thus unreasonably high yields . this may be indicative of the rate constants being different for sequiterpene ro2 . because the predicted relative excess of ro2 + no3 reactions is greatest for -caryophyllene , it is likely that the reported yield from the first method ( which neglects these pathways entirely ) is an underestimate . although we observe different yields for the high and low concentration experiments for each bvoc ( figure 3 ) , the inter - bvoc trend of volume growth observed at the higher oxidant + bvoc concentrations follows a similar pattern seen in the lower concentration experiments ( figure 2 ) . this suggests that the mechanism responsible for this behavior corresponds to the chemical nature of each individual bvoc . among the monoterpenes with one double bond , -3-carene s initial volume growth ( figure 2b ) exceeds both -pinene and sabinene for both high and low concentration experiments , not matching the trend in reaction rate constants ( sabinene s rate of reaction with no3 is faster than -3-carene s ) . sabinene was included in these experiments to assess whether the instability of the three - membered ring was responsible for -3-carene s high soa yield ; sabinene s relatively low a possible mechanism for the observed soa formation trends emerges when examining the structural differences of these monoterpenes . the no3 radical preferentially adds to the double bond in such a way as to make the more substituted alkyl radical , which will quickly react with o2 to become an alkyl peroxy radical , and then upon reaction with another no3 or ro2 , will produce the alkoxy radical ( mechanism shown the si figure s6 ) . in the subsequent reaction forming a ketone , the bond that is broken can be predicted via structure activity relationships ( e.g. , table 3 in vereecken and peeters , shown in black in si figure s6 ) . because a -alkyl substitution destabilizes the adjacent bond more than a -nitrate , the ketones predicted to be formed from breaking the weakest c c bond in -pinene and -3-carene are structurally very different . in -pinene , the predicted product has the radical at the opposite end of the molecule from a terminal nitrate . in -3-carene , limonene , and -caryophyllene , the radical ends up adjacent to the nitrate group , a structure which will stabilize the radical , possibly making it more likely to undergo subsequent radical - initiated oligomer formation rather than decomposition , and explaining high organonitrate and aerosol yields . however , we note that the predicted pathway of -pinene bond scission is inconsistent with the observed dominant product channel of pinonaldehyde ( table 1 ) , which is consistent with our observed low nitrate yield from -pinene and potentially explanatory of low aerosol yield . additional observations of noy balance relevant to the understanding this subsequent radical chemistry are discussed below . the importance of the nitrate radical in initiating rapid soa growth was tested by conducting another limonene experiment ( expt . 8) , where no3 was generated by o3 + no2 ( more typical in the atmosphere ) rather than n2o5 dissociation . in this case , limonene was oxidized by a mixture of o3 and no3 , rather than only the no3 radical . oh scrubber was used , so oh recycling may have also contributed to oxidative chemistry in this case , given the 86% oh yield from ozonolysis of limonene . we observed in this mixed - oxidant case both a 3-fold lower peak number concentration and slower initial volume growth than in the case of no3 alone at nominally the same initial oxidant concentration ( [ n2o5]i = 10 ppb vs [ o3]i = 12 ppb and [ no2]i = 6.3 ppb , in both cases with [ limonene]i = 10 ppb ) . the rate constant of o3 + limonene is 2.1 10 cm molec s , in contrast to no3 + limonene s 1.22 10 cm molec s. in the observed volume growth curves , later in the experiment the total soa volume in the mixed - oxidant case catches up to the no3-only experiment . hence , limonene ozonolysis and nitrate oxidation produce similar eventual soa mass yields at comparable initial concentrations , but no3-initiated chemistry appears more effective at nucleating new particles . this is consistent with the initial nitrate oxidation products being higher mass and lower - volatility than their ozone and oh counterparts under dry conditions , but both reactions producing substantial yields of products of intermediate volatility that begin to condense as aerosol loading increases . the wide range of observed soa yields from no3 oxidation of various bvoc is provocative , but perhaps most surprising is the observation that -pinene reacting with no3 produced no soa . because -pinene is frequently used as a model monoterpene in chamber studies , this exceptional behavior relative to all other monoterpenes tested is particularly important . we seek an explanation in the initial oxidation steps of this reaction , and note that -pinene could be expected to produce an internal nitrate rather than a terminal nitrate , such as would be expected in -pinene , -3-carene , or sabinene ( see si figure s6 ) . it has been shown that terminal functional groups result in lower vapor pressure than internal , but this observation would seem to be a very extreme manifestation of that trend . we note that the no3 + -pinene reaction forms little organonitrate ( table 4 ) , and this organonitrate was not observed to partition to the aerosol phase . most of the no3 returns to the gas phase as no2 , consistent with previous observations that -pinene chemistry instead produces the diketone pinonaldehyde ( si figure s6 , red pathway ) , which is expected to be more volatile than the organonitrate alternatives . an example of observed gas- and aerosol - phase organonitrate production is shown in the si ( figure s7 ) . the nitrogen balance of all high concentration experiments , for which organonitrate measurements were available , are reported in table 4 and described below . bvoc - based molar organic nitrate yield , aerosol fraction of organonitrates , and organonitrate fraction of total aerosol mass are all evaluated at 2 h after chamber experiment initiation ( data are all from the high - concentration experiments , when the td - lif instrument was available ) ; n2o5-based no2 and ans + pns production are evaluated at 30 min after chamber experiment initiation . for -pinene , limonene , and -pinene , no3 decays slowly enough that this n balance analysis can be reliably repeated at 2 h. these are the numbers reported in italics . several parameters are not available for -caryophyllene , because the n2o5 decay was very rapid and not well - characterized . we determined molar organonitrate yields from bvoc ( [ans + pns]/[voc ] in ppb units , using method 1 voc ) , fraction of total organonitrates in the aerosol phase ( [ans + pns]aero/[ans + pns ] in ppb units ) , and the fraction of total aerosol mass that is due to organonitrates ( mans , aero / mo in g m ) , all evaluated at 2 h into the experiment to capture longer - term trends . the calculation of total organonitrates requires subtraction from the total td - lif ans + pns signal the fraction of chamber n2o5 that is detected in this channel ( empirically determined by comparison to crds to be 11% ) , and if dinitrates are present , then they would appear as 2 nitrates . the calculation of total and organonitrate aerosol mass concentrations require ( a ) the conversion of gas phase organonitrates to mass concentration , using assumed values for the average organonitrate molecular weight ; and ( b ) assumptions about average density of smps - measured aerosol and the average organonitrate molecular weight to convert mixing ratios to mass concentrations for comparison to smps - derived total aerosol mass concentration . we assume an aerosol density of 1.4 g cm , and that ans+pns are on average hydroxynitrates of molecular weight 214 g mol ( 286 g mol for -caryophyllene ) , hence at the experiments ambient pressure ( patm = 0.8 atm ) , 1 ppb = 7.0 g m ( 9.4 g m for -caryophyllene ) . in addition , we investigate the observed nitrogen balance in these high - concentration experiments by comparing molar no2 release from n2o5 ( [no2]/[n2o5 ] in ppb units ) and organonitrate formation ( [ans + pns]/[n2o5 ] in ppb units ) to assess closure . for these measurements , we use data 30 min after initiation of the chamber experiment , to ensure that [n2o5 ] is still well - constrained . first , we note that in all experiments except -pinene , organonitrates constitute a substantial fraction of total aerosol mass ( greater than 60% ) , and a large fraction ( 50100% ) of observed organonitrates are in the aerosol phase . this indicates that the organonitrates produced in no3 + monoterpene or sesquiterpene reactions tend to be of low volatility , with the largest aerosol - phase fraction ( lowest volatility ) nitrate arising from the oxidation of the single sesquiterpene tested , -caryophyllene . there is a correlation between organic nitrate molar yields and aerosol mass yields ( see table 3 ) , with -pinene s low organonitrate yield corresponding to no soa production , while -3-carene and limonene have both larger organonitrate yields and soa yields . however , the correlation is not strong ( consistent with previous observations by hallquist et al . ) . for example , -caryophyllene does not fit the trend : despite the largest soa yield , it has the second - lowest organonitrate yield . we note that the -caryophyllene experiment differed from others in terms of initial conditions , with [ bvoc ] [ n2o5 ] , which may explain this discrepancy . in addition , as a sesquiterpene ( c15 ) , -caryophyllene starts at a lower volatility than other precursors , which may also affect the degree to which non - nitrate products might also partition to the aerosol phase . its larger size also may affect vapor - phase wall losses , which are observed to be faster for larger carbon number molecules , potentially depressing apparent soa yield relative to the monoterpenes ( c10 ) . the nitrogen balance provides additional indications of differences among precursors ( a schematic of n balance is shown in si figure s8 ) . no2 release greater than 1 indicates that some of the no3 + bvoc products fragment to rerelease no2 ( the first no2 molar equivalent comes from the initial dissociation of n2o5 ) . this no2 release is greatest for -pinene , which does not produce soa , suggesting that the non - nitrate / fragmentation products are less condensable . in all cases , the total n balance after 30 min reaction time sums to less than two total , the total that would be expected for closure ( as there are two molar equivalents of n in each n2o5 precursor upon which the n balance is based ) . this shortfall could be due to faster wall losses of organonitrates , or to the production of noy species not measured , most notably , hno3 . previous chamber studies of no3 + -pinene and limonene have demonstrated substantial hno3 formation ; in these experiments , -pinene and limonene exhibit the least total n closure . furthermore , as can be seen in the three available time - dependent n balances , the n shortfall is largest at the beginning of the experiments , when [ no3 ] most dramatically exceeds [ bvoc ] , resulting in the greater likelihood of other later - generation no3 reactions such as h abstraction to produce hno3 . in summary , soa yield is greater for the sesquiterpene than monoterpenes , and within the monoterpenes is roughly correlated with the production of low - volatility nitrates . soa formation is hindered by excessive fragmentation and no2 release ( as observed in -pinene ) . the observed soa yields range widely , from 0 to 86% at 10 g m background aerosol , but display consistent trends across experiments at different initial concentrations , suggesting that differences are a function of bvoc precursor molecular structure . in past global modeling efforts , a single yield has been applied for aggregate no3 + terpenes , for example , pye et al . , which gives soa yield at 10 g m of 26% and find that adding in no3 oxidation of terpenes increased global soa production by 3 tg / yr . if we apply our speciated yields to the case of a rocky mountain front range forest , where the mix of bvoc is 1:1:1 -pinene/-pinene/-carene , then we obtain a bvoc - weighted yield of 24%36% ( range based on yields from two voc methods ) , which falls in line with the pye et al . however , in parts of the country where -pinene is dominant among terpenes ( coastal pacific northwest , deep south ) , this aggregate number may overestimate no3-initiated soa production , while areas where other terpenes dominate ( central plains , northeastern u.s . ) , the aggregate may underestimate no3-initiated soa production . an additional factor to note when comparing to the real atmosphere is that the relative importance of no3 oxidation vs o3 varies among these molecules : at 10 ppt no3 and 30 ppb o3 , nitrate reaction rates proceed 2090 times faster than ozonolysis for all the monoterpenes , but for -caryophyllene , only half as fast . thus , -caryophyllene will be relatively less likely to be oxidized by no3 . a true assessment of the role of these reactions requires more detailed regional modeling that can capture the spatial overlap of speciated natural bvoc emissions with anthropogenic nox plumes and resulting oxidation chemistry .

the term " idc - p " was first used in 1973 and later defined as a heterogeneous group of tumors , including urothelial carcinoma , squamous cell carcinoma , and prostatic ductal and acinar carcinomas , extending into normal prostatic ducts and acini.8,9 kovi et al.10 were the first group to intensively study intraductal spread of prostatic carcinoma specifically by examining 139 cases of prostate cancer . they were able to find intraductal spread of prostate cancer in 48% of the cases . they concluded that prostate carcinoma cells can invade adjacent benign ducts , like other types of cancers with mucosal spread of cancer cells ( i.e. , breast , urothelial carcinomas , etc . ) , and supplant the normal epithelial components while preserving the general framework of the affected ducts . mcneal et al.11 built upon this idea when they found that cribriform prostatic carcinomas were commonly associated with an intraductal component . in addition , it was noted that the cribriform pattern was seldom found isolated from invasive carcinoma , at least suggesting that it was unlikely to be a precursor lesion.11 in a subsequent landmark study , mcneal and yemoto1 offered evidence that idc - p was a distinct biological entity with definable morphologic criteria . the morphologic criteria proposed included " complete spanning of the ductal and acinar lumen by several trabeculae of malignant epithelial cells with foci of trabecular fusion."1 this study of 476 radical prostatectomies found that up to 39% of high volume ( 4 - 10 ml ) invasive prostatic carcinomas had an intraductal carcinoma component . they found that these intraductal lesions were frequently adjacent to gleason grade 4 invasive carcinoma . furthermore , these high - grade , large - volume cancers were strongly associated with extracapsular invasion , significant perineural invasion , positive lymph node status , and increased probability of recurrence . the authors concluded that idc - p is not a precursor lesion , but rather an invasive carcinoma spreading into the normal / benign prostatic ducts and acini . this finding was based on two observations : intraductal cribriform lesions were 1 ) rarely found distant from foci of invasive carcinoma , and 2 ) were infrequent in low volume cancers ( < 2 ml ) . soon after mcneal and yemoto 's study,1 wilcox et al.12 embarked on a study of idc - p using criteria established by the prior authors to differentiate idc - p from hgpin . they examined 252 whole - mount radical prostatectomy specimens and found that when idc - p was present , invasive cancers were of higher grade , were more likely to invade seminal vesicles , and had disease progression more often than when idc - p was absent.12 additionally , idc - p was found in 54% of the cases where there was a tumor volume greater than 4 ml . today , our understanding of idc - p as a distinct lesion has evolved into the current morphologic definition of prostate adenocarcinoma cells that span the entire lumen and expand normal prostatic ducts and acini , with at least focal preservation of the basal cell layer . recent studies have refined the diagnostic criteria for idc - p.2,4,13 - 18 cohen et al.2 established a set of histological patterns for idc - p : trabecular ( pattern a ) , cribriform ( pattern b ) , and solid ( pattern c ) . the perimeter compartment consists of peripherally located glandular cells that have malignant features that are identical to severely dysplastic glandular lining cells . the central compartment consists of luminal cells which exhibit varying morphologies that define the three histologic patterns of idc - p.2 however , this finding of two specific cellular compartments is not specific for idc - p and can occasionally be seen in invasive prostate adenocarcinoma . the first histologic pattern , trabecular ( type a ) ( fig . 1a , b ) , exhibits thin cords of cells , approximately 2 cell - layers thick , that span the lumen in the absence of stromal support . these trabeculae intersect one another in a random , but orderly , fashion . in some cases , the trabeculae fail to span the entire lumen and form a focal papillary pattern , which can be difficult to distinguish from hgpin . in the central compartment , 1c ) shows acini formation with punched - out round spaces comprising greater than half of the luminal space . small foci of comedonecrosis can be found occasionally in this subtype . the third pattern , solid ( type c ) ( fig . 2a , b ) , shows a solid proliferation of cells with frequent foci of central comedonecrosis . in this pattern , the central cells attain a level of pleomorphism on par with the perimeter cells to the point where the line between the two compartments is indistinguishable . it should be noted that these different patterns of idc - p correlated with the frequency of high - grade ( gleason 4/5 ) invasive carcinomas in a stepwise fashion , with type c associated with the highest volume and highest grade tumors . in this regard , types a and b could be regarded as low - grade and medium - grade patterns , respectively . these subtypes also showed clinical significance in that the advancing pattern of idc - p correlated with worsening patient prognosis.12 however , it must be emphasized that the presence of idc - p is still a poor prognostic factor , regardless of grading . interestingly , the different patterns also showed differing immunohistochemical staining patterns for muc-2 and ki-67.2 muc-2 staining was seen in most of the type a and b ( micropapillary and cribriform ) patterns but not in type c ( solid ) . positive ki-67 nuclear staining was confined to the perimeter compartment in types a and b but was found in both the central and perimeter compartments in type c. the central and peripheral compartments also showed differential staining patterns . positive prostate - specific antigen ( psa ) staining was limited to the central compartment and did not stain the peripheral compartment while androgen receptor staining was seen in the peripheral compartment and not the central compartment . guo and epstein14 subsequently described four subtypes of idc - p , including solid , dense cribriform , loose cribriform , and micropapillary . they proposed diagnostic criteria of idc - p in prostate biopsy as a proliferation of malignant prostatic glandular cells filling ducts and acini , with some basal cell preservation ( fig . if the first tier criterion is not present , a diagnosis of idc - p can be made if loose cribriform or micropapillary pattern is identified with one of the following : 1 ) prominent nuclear pleomorphism ( nuclear size greater than 6 normal ) or 2 ) non - focal comedonecrosis ( > 1 duct showing comedonecrosis).14 the authors noted that the micropapillary pattern was equivalent to the aforementioned trabecular pattern described by cohen et al.19 again , it must be stressed that idc - p should be differentiated from hgpin because of the poor clinical outcomes in patients with idc - p.13 furthermore , some authors even question the requirement of re - biopsying patients within one year after rendering the diagnosis of hgpin due to the low risk for finding carcinoma in subsequent biopsies.20,21 this distinction can be difficult in some instances as loose cribriform and micropapillary patterns ( as described by guo and epstein14 ) of idc - p can morphologically resemble hgpin . these situations require particular scrutiny and underscore the importance of assessing for either marked nuclear pleomorphism ( > 6 normal ) or non - focal comedonecrosis ( involving > 1 duct ) in these patterns to assist in making the diagnosis of idc - p over hgpin ( table 1).14,22 cohen et al.16 listed major and minor criteria to distinguish idc - p from hgpin . of the five major criteria , the first four are invariably present . these include 1 ) dilated glands greater than 2 times the diameter of normal peripheral zone glands ; 2 ) preservation of the basal cell layer ; 3 ) proliferation of intraluminal malignant cells 4 ) that completely span the lumen ; and 5 ) possible foci of comedonecrosis.16 importantly , while the fifth criterion of comedonecrosis is not always seen , its presence is specific for the diagnosis of idc - p over hgpin . minor criteria include 1 ) intraductal glands branching at right angles with smooth and rounded contours and 2 ) a two - cell population divided into a peripheral layer and central layer . the low grade spectrum of idc - p overlaps with hgpin . even though idc - p with low grade morphology does not meet the diagnostic criteria for idc - p in prostate biopsies , it should not be simply dismissed as hgpin . in lesions that can not be confidently classified , 4 . a recent study found that borderline lesions between hgpin and idc - p were associated with a substantial increased risk ( 55% ) of prostatic carcinoma ( pca ) on subsequent biopsy and these lesions therefore require immediate repeat biopsy.23 there is mounting molecular evidence that idc - p and hgpin are distinct lesions.5 - 7,24 dawkins et al.24 studied twenty radical prostatectomy specimens with idc - p for loss of heterozygosity ( loh ) at known loci on chromosomes strongly associated with prostate cancer and compared the results of hgpin with invasive gleason grade 3 and grade 4 carcinomas . loss of heterozygosity was detected in 60% of idc - p , 29% of gleason grade 4 cancers , 9% of hgpin , and no gleason grade 3 cancers . based on these findings , the authors concluded that idc - p is a distinct pathologic process from hgpin.24 bettendorf et al.6 analyzed hgpin , idc - p , and invasive prostate cancer for loh of tp53 and rb1 ( tumor suppressor genes ) and used comparative genomic hybridization on hgpin and idc - p to further characterize the two types of lesions . loh of both tp53 and rb1 were found frequently in idc - p ( 52% ) and tumor tissue in extraprostatic extension ( 44% ) , and rarely in hgpin ( 19% ) and benign prostatic tissue ( 17% ) . comparative genomic hybridization showed that all of the hgpin lesions lacked chromosomal imbalances in contrast to idc - p where 8/11 cases demonstrated chromosomal imbalances.6 han et al.5 analyzed e - twenty six ( ets ) gene aberrations , the most common of which in prostate cancer is the tmprss2-erg fusion,25 using a break - apart fluorescence in - situ hybridization assay to help establish distinguishing features between hgpin and idc - p . erg gene rearrangement was found in 75% of the cases of idc - p and in none ( 0/16 ) of the cases of hgpin , and the erg gene status was concordant between idc - p and adjacent invasive pca.5 recently , it has been shown that immunohistochemical stains using antibodies against erg correlate strongly with the status of the erg gene fusion , potentially making it an attractive marker for idc - p versus hgpin ( fig . 5).26,27 other researchers have investigated deletions involving the pten locus in idc - p versus hgpin . loss of pten , a tumor suppressor , occurs in up to 70% of invasive prostatic carcinomas and is uncommon in hgpin.28,29 using immunohistochemistry , cytoplasmic loss of pten was identified in 84% of idc - p and was not identified in any cases of hgpin ( 0/39).7 although histologic and molecular evidence strongly suggests that idc - p represents a late - stage intraductal spread of existing , high - grade invasive carcinoma , there are a few cases that involve idc - p without an associated invasive element.4,30 these rare cases suggest that idc - p , in some instances , may act as a precursor lesion in the hgpin pathway of cancer or possibly as a separate de novo pathway . since there have been cases of idc - p alone , with no association whatsoever in radical prostatectomy specimens , studies investigating the morphologic and molecular differences between hgpin and idc - p , with and without invasive carcinoma , are underway to determine whether a subset of idc - p could be a precursor condition . it is clear that the presence of idc - p is associated with high - grade , large - volume cancers with aggressive features , including positive surgical margins and extracapsular extension . several studies have shown that idc - p found at radical prostatectomy in patients treated with neoadjuvant chemotherapy predicts a shorter biochemical recurrence - free survival.31 - 33 furthermore , some preoperative models incorporating idc - p have been shown to improve predictions regarding pathologic stage in radical prostatectomy specimens , and have accurately predicted treatment failure.19 another study looked at the prognostic significance of idc - p in biopsies and radical prostatectomies in patients prior to radiotherapy alone or with androgen deprivation . idc - p was found to be a strong prognostic factor for early biochemical relapse ( < 36 months ) in addition to metastatic failure rate after radiotherapy and clinical progression - free survival.34 it must be noted that idc - p is an uncommon finding . in one prospective study of 1,176 biopsies , the incidence of idc - p was 2.8%.33 however , idc - p clinical significance is well - established , and we recommend including its presence in biopsy and radical prostatectomy reports , regardless of the existence of an invasive component . the main differential diagnoses for idc - p include intraductal spread of urothelial carcinoma , prostatic duct carcinoma and , most importantly , hgpin ( fig . 6 ) . urothelial carcinoma typically shows more nuclear pleomorphism than idc - p ( fig . 7 ) and will not stain with prostate immunohistochemical markers , including psa and prostate - specific acid phosphatase , but positive for high molecular weight cytokeratin , p63 and gata3 ( table 2 ) . prostate duct carcinoma consists of tall , pseudostratified columnar cells with large , elongated nuclei which are in contrast to the cuboidal - to - short columnar cells of idc - p ( table 3 , fig . intraductal growth in prostate duct carcinoma is not uncommon ; however , in contrast to idc - p , there is nearly always an associated invasive component that lacks a basal cell lining . the diagnosis of idc - p over hgpin can be made when there is a solid or dense cribriform ( > 50% cellularity of the lumen ) pattern present . in our opinion , in accordance with the published criteria , it is prudent to realize that there are similarities between certain types of hgpin and idc - p . particularly , the trabecular / micropapillary and loose cribriform patterns of idc - p can overlap with subtypes of hgpin . in these instances , additional diagnostic criteria , including marked nuclear pleomorphism ( > 6 normal ) , non - focal comedonecrosis ( > 1 duct showing comedonecrosis ) , a two - cell population ( peripheral layer and central layer ) , and markedly distended normal ducts / acini ( > 1 mm involving > 6 glands ) , can greatly assist in making the diagnosis of idc - p . additionally , either positive nuclear staining for erg or cytoplasmic loss of pten by immunohistochemistry strongly suggests a diagnosis of idc - p over hgpin . in situations where these lesions may be indistinguishable , the term " atypical intraductal proliferation " may be used with the comment that the differential is between idc - p and hgpin , and that the patient should undergo immediate rebiopsy . lastly , while light microscopic examination may be sufficient for recognition of basal cells , immunohistochemical stains including p63 or high molecular weight cytokeratin may have utility in confirming the intraductal process of idc - p by highlighting basal cells . idc - p is defined as a proliferation of malignant prostate adenocarcinoma cells distending and completely spanning the lumen of normal prostatic ducts and acini , with at least focal preservation of basal cells . idc - p is strongly associated with invasive , high - grade prostate cancers ( gleason grades 4/5 ) with high tumor volumes . recent genetic studies indicate that idc - p represents intraductal spread of invasive carcinoma , rather than a precursor lesion . although rare , it should be reported in prostate biopsies and radical prostatectomies , even in the absence of invasive carcinoma due to its strong association with high - grade , high - volume tumors and predictive and prognostic implications . we recommend the diagnosis and reporting of idc - p in prostate biopsies and radical prostatectomies as seen in table 4.22

modern science through improved sanitation , vaccination , antibiotics and medical attention has eliminated the threat of death from most infectious diseases . but now , too many people are dying relatively young from noncommunicable diseases like heart diseases and cancer . the main contributing factor for this epidemiological transition is change in our lifestyle toward the unhealthy continuum , e.g. , tobacco use , excessive alcohol consumption , unhealthy dietary habits and physical inactivity . this means that death from lifestyle diseases like hypertension , diabetes and cancer are now the primary causes of death . the term lifestyle also reflects an individual 's attitudes , beliefs and , essentially , the way the person is perceived by himself / herself and , at times , also how he / she is perceived by others . this rapidly growing epidemic of noncommunicable diseases is responsible for 60% of the world 's deaths . at least 50% of the deaths in the us each year are due to unhealthy lifestyle . in india also , the situation of lifestyle diseases is quite alarming . the disease profile is changing rapidly . the world health organization ( who ) has identified india as one of the nations that is going to have most of the lifestyle disorders in the near future . nowadays , not only are lifestyle disorders becoming more common but they are also affecting the younger population . hence , the population at risk shifts from 40 + to maybe 30 + or even younger . already considered the diabetes capital of the world , india now appears headed toward gaining another dubious distinction of becoming the lifestyle - related disease capital as well . a study conducted jointly by the all india institute of medical sciences and the max hospital shows that the incidence of hypertension , obesity and heart disease is increasing at an alarming rate , especially in the young , urban population . in most of these lifestyle diseases , the onset is insidious and is usually after the age of 30 years . by the time interventions adolescence is the peak age of onset for serious mental illness like depression and psychosis . physical , emotional , social and sexual change makes adolescents overloaded with stress , which can result in anxiety , withdrawal , aggression , poor coping skills and actual physical illness . several health - compromising behaviors ( e.g. , smoking , alcohol ) as well as health - enhancing behaviors ( e.g. , physical exercise , nutrition ) are adopted in adolescence , and they often persist into adulthood . the who estimates that 70% of premature deaths among adults are due to behavior ( smoking , illicit drug use , reckless driving ) initiated during adolescence . therefore , helping adolescents establish healthy lifestyles and avoid developing health risk behaviors is crucial and should be started before these behaviors are firmly established . the demands on young people are new and unprecedented ; their parents could not have predicted many of the pressures they face . how we help adolescents meet these demands and equip them with the kind of education , skills and outlook they will need in a changing environment will depend on how well we understand their world . there is a definite need to investigate the health behavior of adolescents rather than focus on adults since it will be far more difficult for adults to change their unhealthy habits adopted in their youth . many studies have demonstrated a positive relationship between health - promoting lifestyle and quality of life . mcdaniel found a positive correlation between health - promoting lifestyle and quality of life in 91 older adults , aged 60 - 92 years . it was also demonstrated that there exists a relationship between positive health outcomes and such behaviors as regular exercise , not smoking , maintaining ideal weight , eating a well - balanced diet and managing stress . it was planned to undertake a lifestyle intervention project among the college students of chandigarh . hence , the present pilot study was performed to create an elementary baseline data about the prevalence of lifestyle - associated risk factors among the college students of chandigarh . the present cross - sectional study was conducted in chandigarh during 2009 among university graduates of chandigarh aged 17 - 20 years . an estimated sample size of around 50 students was taken . a class of graduate students was selected randomly from that college and all the students of the class were included after taking written consent for participation . students pursuing their graduation ( regular ) course were eligible for participation in the study . students who were doing their graduation through correspondence ( distant learning ) or during the evening study hours were excluded from the studies . a semi - structured , pretested questionnaire comprising of 28 items regarding activities like diet , physical exercise , spirituality and philanthropy was used in the study . all items are scored on a scale from 1 to 4 ; 1 = never , 2 = sometimes , 3 = often , 4 = routinely , except items of philanthropy that had only two scores . a composite score was obtained , which ranged from 28 to 106 . in analyzing health - promoting behavior , only those who reported often ( score = 3 ) or routinely ( score = 4 ) in each item were considered as health - promoting behavior . to calculate the mean of the lifestyle profile , the scores for all items were summed and then divided by the numbers of items . height , weight , waist and hip circumference of the respondents were measured using standardized equipments and procedures . the response rate for the different questions of the study varied from 80% to 90% . in this survey of 50 students , 30.6% ( n = 15 ) of the respondents were males and 69.3% ( n = 35 ) were females . mean score of lifestyle profile of university graduates was found to be 67.5 12.1 ( maximum attainable score = 106 ) . around one - third ( n = 14 ) of the students were found to be overweight [ table 1 ] . frequent intake of fast food ( n = 12 ) , less consumption of fruits ( n = 26 ) and vegetables ( n = 22 ) and skipping of breakfast ( n = 19 ) were also reported by the students [ table 2 ] . majority ( n = 37 ) of the students exercised as part of their daily routine , but less involvement in yoga ( n = 6 ) and outdoor games ( n = 17 ) was noted [ table 3 ] . majority ( n = 48 ) of the students had faith in god [ table 4 ] . anthropometric measurements of the respondents distribution of students with habits related to diet ( n=49 ) * distribution of students with habits related to exercise ( n=49 ) * spiritual health status of students ( n=49 ) * life of the students in college is a transitional period , offering good opportunities for establishing health - promoting lifestyles . most research on health - promoting behaviors has been undertaken in the us and european countries , where university students are little engaged in health - promoting behaviors , especially healthy diet and physical activity . however , data on health - promoting lifestyles among university students in india are limited . the results from our study provide a glimpse of the lifestyle of north indian college students in chandigarh . however , about one - fourth of the students ( n = 14 ) were overweight , more so in females . however , in a study conducted by rao et al . , more boys ( 72.5% ) were in the overweight category . this may be due to unhealthy dietary habits like frequent intake of fried foods and sweets in comparison with lesser intake of fruits and green leafy vegetables . moreover , students like to lead a carefree life and may find it awkward to show too much concern about their health . this scenario also implies that the bombardment by health - related articles or media advertisements probably do not have much impact on health concern or behaviors of young people . a higher frequency of eating out and having fast food was also reported . they had their meals in the mess / canteen , where the food variety was limited . skipping of meals , especially breakfast , was also observed in nearly one - third ( n = 19 ) of the students . it was a matter of concern since skipping meals is bad for the health of students who need sufficient energy and nutritive diet , as they live an active life . majority of the respondents ( n = 35 ) reported that they exercised as part of their daily routine . this may be due to the fact that adolescents of today are more conscious about their physique and appearance . however , contrary results were found in a study wherein low level of physical activity was found among iranian women . however , yoga was practiced by only six students as compared with exercise ( n = 35 ) , which is similar to the findings of other studies . this may also be indicative of lack of interest of yoga among students and also because yoga requires a special technique . it was also found that only one - third of the students went for outdoor games . apparently , in the modern era of computers , internet and mobile phones provide more choices of entertainment . the present study also revealed that majority ( n = 48 ) of the students had faith in god . most ( n = 36 ) were satisfied with themselves and were clear about the purpose and importance of their life . around two - third ( n = 32 ) of the students had a positive attitude toward life . however , only about one - third ( n = 19 ) made any special efforts to achieve their life goals and around half ( n = 27 ) did not find their day interesting and challenging . this could be probably due to the fact that they were not provided with enough opportunities and had lack of support by family and college authorities . this could be due to the reason that the youth of today are busy in achieving their goals . they do not receive any value inculcation on these aspects , neither in the family nor in their curriculum . the present study provided a glimpse about the prevailing lifestyle of the university graduates of a modern city of north india . the result of the study showed that north indian students had reasonably good orientations toward health behaviors with a mean score of 67.5 . based on the findings of the study , it is recommended that topics related to diet , physical exercise , spiritual growth and philanthropy promotion should be incorporated within the curriculum of students for their all round personality development . the related information , education and communication materials should be disseminated widely in the community . basic health workers like auxillary nurse , midwives and accredited social health activist should be empowered in assessment of risk factors of noncommunicable diseases . the subjects were selected from one college only . all the information collected in the study therefore , it is possible that these may be socially desirable responses , i.e. , the health - promoting behaviors may not be their real or actual behaviors .

functional obstruction and bowel dilatation proximal to the affected segment , first described by harald hirschsprung in 1888 , is a complex disorder resulting from absence of ganglion cells in the bowel wall leading to functional obstruction and bowel dilatation proximal to the affected segment [ 1 , 2 , 3 ] . it is usually diagnosed in infants but hirschsprung 's disease ( hd ) may be diagnosed in the older children presenting with chronic constipation , which is unresponsive to conven - tional treatment . as constipation is a common problem among children and only small minority of patients have an organic cause for their constipation , it is necessary to distinguish between these two conditions . constipation is defined in neonates as failure to pass meconium within the first 48 hours of life and in the older children as the infrequent passage of stool of increased consistency . a number of constipated children are referred for rectal biopsy to exclude a possible hd . the diagnosis of hd is based on a combination of symptoms , radiological study , rectal manometry , and histological features of rectal biopsy . the histological features of hd include the absence of ganglion cells of the myenteric ( auerbach 's ) and submucosal ( meissner 's ) plexuses , and increased number of hypertrophic nerves [ 2 , 3 ] . rectal biopsy with histopathologic examination can reliably exclude hd and it is the gold standard for the definitive diagnosis of aganglionosis . it is said that 12% to 17% of children undergoing rectal biopsy are found to have hd [ 46 ] and so about 80% of patients receive an unnecessary surgical procedure . lewis et al hypothesized that key features in the history , physical examination , and radiographic evaluation would allow us to avoid unnecessary rectal biopsies . in a retrospective study they found that a history of delayed passage of meconium , abdominal distension , vomiting or the results of a contrast enema identified all patients with hd and excluded hd in approximately 36% of patients with idiopathic constipation ; in a child presenting only with constipation and none of the above features , it is not necessary to perform a rectal biopsy . ghosh and griffiths in a retrospective study in 186 children concluded that if the age at onset of constipation is after the neonatal period , rectal biopsy is unnecessary and it is unlikely that the child has hd . the aim of our study was to evaluate rectal biopsies from constipated children of different age groups to see in which age hd is more likely in order to avoid unnecessary rectal biopsy and on the other hand in which age group we need to pay more attention to avoid missing hd . the names and record numbers of children with chronic unrelenting constipation undergoing a rectal biopsy to exclude hd were obtained from the histopathology department of children 's medical center . cases were divided into 4 groups according to their age : a ) neonatal period : the first 4 weeks of life , b ) between 5 to 12 weeks old , c ) 13 weeks to 1 year old , and d ) above 1 year old . a detailed retrospective demographic review , including age of beginning of signs and symptoms , gender , family history of disease , signs and symptoms , paraclinical data and congenital anomalies , was made in all cases . rectal biopsies are preformed at least 1 cm above the pectinate line and types of biopsies were submucosal biopsies and full thickness muscle coat stained with hematoxilin and eosin . in aganglionic biopsies at least 30 - 50 sections were examined to make the diagnosis of hd . aganglionic cases less than 0.5 cm in diameter , presence of squamous epithelium or striated muscle fibers or submucosal biopsies in which the amount of submucosa was less than mucosa were excluded as the signs of inadequacy or inappropriateness . no extra sampling was imposed to individuals and no extra the parents were not charged . with exclusion of inadequate biopsies ( 6% of all biopsies ) , 172 biopsies were available from 168 children with constipation , 127 ( 75% ) had hd and 41 cases had normal biopsies with normal ganglion cells in appearance and distribution . the mean age of constipated patients at the time of biopsy was 39 months and the mean age of patients with proven hd was 18 months . table 1 shows frequency of hd among patients in different age groups based on the time of diagnosis . frequency of hd among patients in different age groups ( at the time of biopsy ) in 93 children with hd , the parents could remember the beginning time of constipation , in 85 ( 91% ) cases it was in neonatal period ( p<0.005 ) although the time of taking biopsy was later in many . in 8 cases the beginning of clinical manifestation was beyond neonatal period . in the remaining children ( 28 cases ) the parents could not remember the time they noticed constipation ( at or beyond neonatal period ) . from 27 cases without hd , 5 cases had constipation from neonatal period . in 22 cases the beginning of clinical manifestation was beyond neonatal period and in the remainder ( 14 children ) the beginning of clinical manifestation was not clear . frequencies of different clinical manifestations in patients with hd are listed in table 2 . frequency of different clinical manifestations of patients with hd congenital anomalies associated with hd were found in 9.6% and included 4 cases with cns malformation , 3 cases with congenital heart disease , 2 cases with anatomical gastrointestinal malformation , 2 cases with genitourinary malformation , 1 case with congenital dislocation of hip , 1 case with polydactyly and one case with down syndrome . barium enema was done in 103 children and there were false positive and false negative results in 7 and 3 cases , respectively , the diagnostic sensitivity was 91.3% . constipation accounts for nearly 3% of visits to a general pediatric office and 25% of visits to a pediatric gastroenterologist[7 , 1 ] . 95% of children with chronic constipation have functionalconstipation while only 5% have an organic cause for their symptoms , and a rectal biopsy , the goldstandard for the diagnosis of hd , is necessary to exclude this condition[1 , 8 ] . some authors are of opinion that if the onset of constipation is beyond neonatal period , the child is unlikely to have hd . the indications for rectal biopsy in children before 6 months of age : ( a ) delayed meconium passage ; ( b ) low intestinal obstruction of unknown cause ; ( c ) severe constipation ; ( d ) chronic abdominal distention and ( e ) failure to thrive [ 9 , 10 , 11 ] . our results show that males were affected 4 times more than females and it is parallel to those of other studies like results reported by amiel and halevy . in a study in korea the most frequent sign of disease in neonates in our study was abdominal distension and delay in meconium passage which is the same as in other studies [ 1 , 2 ] . most cases with hd were diagnosed in neonatal period ( 1.8% of patients without and 30% of patients with hd were neonates , p<0.004 ) . in 85 ( 91% ) cases the beginning of constipation was in neonatal period and in five non - hd cases the constipation was present in newborn infant ( p<0.008 ) . our data supports griffith 's results suggesting that if the onset of constipation occurs after the neonatal period , it is unlikely that the child has hd and therefore a rectal biopsy is unnecessary . also nicola in showed that in sixty percent of patients with hd and 15% of patients without hd the onset of symptoms was in the first week of life . in our study , a large number of suspected children had hd . this may be because our hospital as a referral one is usually attended by cases with prolonged or complicated diseases ; also the reason may be the good selection of patients for taking biopsy since in suspicious cases anorectal manometry and/or a barium enema is undertaken as screening methods before undergoing rectal biopsy . in 9.6% of our cases this is lower than in australian and boston studies which show 16% ( australia ) and 22% ( boston ) of children with associated anomalies [ 13 , 14 ] . these findings plus the fact that 9% of these children had also down 's syndrome , is evidence for the assumption that the etiology of hd may be partially genetic . the diagnostic sensitivity of barium enema in our study was about 91.3% and was close to amsterdam study in 2005 . according to our data and those of griffith and also nicola studies it can be concluded that delay in meconium passage is the most important clinical sign of hd and it should be emphasized in the patients history if noticed . furthermore , radiologic findings in older cases that are unresponsive to medical therapy are other means to select correct patients for biopsy .

malignant tumors , most often originating in the lung and breast , occasionally metastasize to intraocular structures . most intraocular metastases occur in the uveal tract ( iris , ciliary body , and choroid ) . in a study of 520 eyes with uveal metastases , only 8% occurred in the iris among 950 metastatic lesions . in 1 study , the most common complaints were blurred vision , ocular pain , visible iris mass , and photophobia . visual acuity was 20/40 or better in 27 ( 68% ) of the 40 affected eyes . ocular metastasis is extremely rare in esophageal cancer in general , and we are aware of only 1 reported case of metastasis of esophageal cancer to the iris in particular , which was treated effectively with systemic chemotherapy . in this case report , we describe an isolated esophageal metastasis to the iris that was effectively palliated using stereotactic body radiation therapy ( sbrt ) . a 53-year - old man with no significant past medical history presented with a 6-month history of pain when swallowing and regurgitation . esophagogastroduodenoscopy revealed a large , circumferential , distal esophageal mass , with biopsy revealing adenocarcinoma . staging pet / ct did not reveal any regional lymphadenopathy or distant spread of disease . the patient underwent an en bloc esophagectomy , with pathology demonstrating t3n2 ( stage iiib ) disease , and he was subsequently treated with adjuvant chemotherapy . he was well , with no clinical or radiographic evidence of disease recurrence for 4 months , until he began experiencing pain and vision loss in his left eye . ophthalmic exam showed a visual acuity of 20/20 in the right eye and 20/100 in the left . intraocular pressure in both eyes and an examination of the right eye was normal . in the left eye , the pupil was irregular and poorly reactive , but there was no afferent pupillary defect . there was a cystic thickening of the iris stroma with eversion of the temporal pupillary margin ( fig . dilated fundus exam of the left eye showed cystoid macular edema , but no choroidal metastasis . ultrasound biomicroscopy and anterior segment oct suggested that the lesion was only in the iris and did not involve the ciliary body , although it may have extended to the iris root . contrast - enhanced ct of the head and mri of the brain and orbit did not reveal any abnormal mass or suspicious enhancing lesions . in addition , a restaging pet / ct scan did not reveal disease recurrence elsewhere in the body . intraocular biopsy was not performed to avoid the risk of tumor seeding at distant sites via the iris vascularization . he began treatment with intravitreal bevacizumab 1.25 mg for cystoid macular edema and was referred for radiation therapy . the patient was simulated in the supine position and a frameless technique was utilized , which included a custom aquaplast mask with u - frame for immobilization . the previously acquired contrast - enhanced ct and mri were fused to the planning ct simulation , and all previous clinical evaluations were carefully analyzed to help delineate the target volume and normal surrounding critical structures . the gross tumor volume was defined , and then the clinical target volume was determined . due to concerns about eye motion , the clinical target volume was contoured to include as much of the anterior half of the orbit as possible . a planning target volume margin of 2 mm was added . the novalis exactrac patient positioning platform ( brainlab ag , heimstetten , germany ) was used for immobilization . highly conformal radiation therapy , consisting of 8 fixed beams , was used to deliver 35 gy in fourteen 2.5-gy daily fractions using 6 mv photons with a 0.5-cm bolus ( fig . when the patient returned 3 weeks later , he had acute anterior uveitis with 3 + anterior chamber cell and flare . his vision had decreased to 20/400 , and cystoid macular edema worsened . rather than bevacizumab , one month later , which was 2 weeks after completion of sbrt , the patient reported subjective visual improvement . one month later , the iris metastasis had totally regressed and his vision improved to 20/150 . at follow - up 6 months after the last radiation treatment , there was no recurrence of the iris metastasis ( fig . 1 ) , his macular edema had totally resolved , and snellen visual acuity had returned to 20/30 . metastasis to the choroid is most common , while involvement of the iris is rare . the goal of treatment is alleviation of clinical symptoms and preservation of visual acuity , while survival outcomes are dictated by the prognosis of the primary disease . treatment options include chemotherapy or radiotherapy , while resistant cases may require enucleation for relief of severe symptoms . a number of reports have documented effective local control of ocular metastasis with conventional fractionated radiotherapy . the majority of the studies used doses between 30 and 40 gy with 23 gy fractions over a 34 week period , with response rates up to 89% [ 4 , 5 , 6 ] . brachytherapy using intraocular plaques has also been used , providing the advantage of shorter treatment duration but requiring an invasive procedure . sbrt is a non - invasive treatment that can be administered in a much shorter time period than traditional fractionated external beam radiation therapy . although its efficacy and safety profile have been established in nearly every primary malignancy and disease site throughout the body , we are aware of only 1 report detailing its use in metastasis to the eye . . reported on 10 patients with unifocal choroidal metastasis who were treated using sbrt with 1220 gy in a single fraction or 30 gy in 10 fractions . with a median follow - up period of 6.5 months , local control was achieved in all treated lesions , with 5 lesions showing radiographic evidence of tumor regression . intravitreal bevacizumab has been used as an adjuvant therapy for choroidal metastases from various primary tumors and has caused tumor regression in metastases that either failed chemotherapy or developed months after primary chemotherapy was completed [ 8 , 9 ] . the indications for intravitreal bevacizumab were loss of vision or threatened loss of central vision . however , since he received sbrt simultaneously , we can not speculate on the effects of bevacizumab as a primary treatment . the use of bevacizumab is a reasonable option for management of ocular metastases , especially if other effects of the metastases are seen , e.g. macular edema .

methylmalonic acidemia ( mma ) is an inborn error of metabolism caused by deficiency of the mitochondrial enzyme , methylmalonyl - coa mutase ( mut ) . this vitamin b12 dependent enzyme catalyzes the isomerization of l - methylmalonyl - coa into succinyl - coa at the terminal step of propionyl - coa oxidation . isolated mma exhibits genetic heterogeneity and most commonly is caused by mutations in the mut gene , resulting in either complete ( mut ) or partial ( mut ) loss of enzymatic activity , but can also be caused by cbla ( mmaa ) , cblb ( mmab ) , and the much rarer cbld variant 2 ( mmadhc ) complementation classes . the latter forms of mma are the result of enzymopathies that impair the intracellular transport and metabolism of cobalamin . unlike mut mma , most patients with cbla and 50% of cblb forms of mma are responsive to parenteral vitamin b12 , a clinical and biochemical determinant that has been associated with improved survival and milder disease . mma patients exhibit a well - recognized phenotype that features massive elevations of disease - related metabolites in the body fluids and multisystemic complications such as growth impairment , pancreatitis , metabolic stroke of the basal ganglia , intellectual disability and end stage kidney disease . although this disorder has historically had a poor prognosis , with most vitamin b12-nonresponsive patients dying in the first decade of life , earlier diagnosis through newborn screening and more aggressive treatment has afforded increased survival . this highlights the need to understand the natural history of mma and to develop improved therapies . chronic kidney disease is a serious complication of mma , developing in most mut and cblb patients by the age of 8 years . although more commonly associated with vitamin b12 non - responsive forms of mma , patients with mut and cbla subtypes also can develop renal insufficiency and ultimately require kidney transplantation . while the mechanisms of renal injury in mma have not been elucidated , the few reported kidney biopsies have consistently demonstrated tubular injury and interstitial fibrosis with secondary glomerulosclerosis . the primary pathological manifestation of kidney injury in mma may be mediated through mitochondrial dysfunction in the proximal renal tubule , as has been described in a mut mouse model . the most common marker of renal function , serum creatinine , correlates poorly with glomerular function in patients with mma . walter et al . documented that some mma patients had a markedly depressed glomerular filtration rate ( gfr ) in the setting of normal serum concentrations of creatinine . cystatin c based estimates of gfr , which are less dependent upon muscle mass , have not yet been evaluated in the mma population . while the direct measurement of glomerular filtration function with agents such as inulin and iohexol is more accurate than gfr estimates , such testing is not routinely performed in mma patients even though a small study conducted in the late 1990s documented reduced cr - edta based gfr measurements in very young children with mma . although these authors did not describe other biochemical , imaging or enzymatic studies in the patient cohort , their observations highlight the need to develop better disease - associated biomarkers and to assess the utility of non - invasive imaging techniques to monitor and predict kidney growth in affected children . renal size has been proven to be a valuable and useful parameter in evaluating renal growth in normal populations and those with genetic disorders . furthermore , studies in healthy controls and patients with kidney disease have repeatedly documented the correlation between renal length and other markers of renal size such as renal volume , and gfr . using ultrasonographic measurements derived from a large single center cohort of patients with mma , we examined kidney growth and assessed the contribution of selected clinical and laboratory variables to renal size using regression analyses . the predictive equations and kidney size nomograms we have developed should provide a clinical tool to help define parameters associated with disease progression in mma , afford prognostication for long - term management and help assess renoprotective interventions in the future . clinical and laboratory investigations were conducted through nih study 04-hg-0127 clinical and basic investigations of methylmalonic acidemia and related disorders analysis included 84 renal ultrasound studies , each imaging the right and left kidney , from 50 non - transplanted patients aged 2.2 years to 36.3 with enzymatic and mutation confirmed forms of mma . we treated each ultrasound from subjects with consecutive studies as a separate data point instead of averaging each patient 's data because of the significant time interval , typically greater than one year , between visits . patients were excluded after they had undergone a kidney or combined liver - kidney transplantation . the mma patients were compared to a published control group of 209 patients from u.s . medical centers aged 0 to 19 years with indications for ultrasound that included urinary tract infections , enuresis , aniridia , scoliosis , and undescended testicle ; this approach has been used previously . data included height , weight , age as well as concentrations of serum creatinine , serum methylmalonic acid , cystatin c , and urine methylmalonic acid . all laboratory testing was done by standard measures through either the department of laboratory medicine at nih or mayo medical laboratories . patients were subdivided into mut , cbla , and cblb groups based on cellular complementation and molecular testing . body surface area ( bsa ) was calculated using the formula : bsa(m ) = ( [ height(cm ) * weight(kg)]/3600 ) . estimated glomerular filtration rate ( egfr ) for children < 18 years of age was calculated using updated schwartz equations that include serum creatinine , or serum creatinine and cystatin c , respectively . for adults , the ckd - epi creatinine and ckd - epi creatinine cystatin c egfr equations were used . the philips iu22 xmatrix ultrasound system with the c5 - 1 1 - 5 mhz bandwidth transducer and the general electric logiq e9 with the c-1 - 5-d 1 - 5 mhz bandwidth transducer were used to obtain abdominal ultrasound studies for all study participants . the original measurements were made in the context of research , and read by one of three radiologists . pearson 's correlation was used to determine variation between independent variables . published data from age - matched controls were compared with mma study patients using a two - sample t - test of the means assuming unequal variances and by a linear regression model that included the average of the right and left kidneys as the dependent variable and age as the independent variable . in addition to variation with age , we examined the correlation of renal length with height because mma patients can be shorter than healthy individuals . this linear model was compared to a different control group that created a nomogram of right and left renal length versus height using measurements from 325 german children ages 3 days to 16 years without known kidney disease . using renal length as the dependent variable , the following independent variables were included in a multiple regression equation : age , height , weight , body surface area ( bsa ) , body mass index ( bmi ) , serum methylmalonic acid ( mmas ) , urine methylmalonic acid ( mmau ) , serum creatinine , cystatin c , egfr creatinine ( creatinine based ) , egfr creatinine - cystatin c ( creatinine and cystatin c based ) , gender and enzymatic subtype ( complementation class ) . the best model , as determined by the highest correlation coefficient ( r ) , was developed using stepwise regression analyses . individual predictor variables were eliminated if the regression coefficient ( ) had a statistical significance of p > 0.05 using the wald test . beta weights were calculated to assess the unique contribution of independent variables relative to the regression model ( beta weight is the change in renal length when an independent variable increases one standard deviation while other independent variables are held constant ) . stata version 11 ( statacorp lp , college station , tx ) was used for data manipulation and analysis . when two or more variables were closely correlated ( multicollinearity ) , such as mmas and serum creatinine ( pearson coefficient of correlation 0.808 ( p < 0.0001 ) ; see table s1 ) , they were not included in same multiple regression model . multicollinear relationships were observed between the variable group of creatinine , cystatin c and mmas , and when controlling for height , the r values for these predictors were similar ( table s2 ) . height , bsa , and weight also demonstrated multicollinearity when controlling for height , with r values of 0.767 , 0.757 , and 0.712 , respectively . clinical and laboratory investigations were conducted through nih study 04-hg-0127 clinical and basic investigations of methylmalonic acidemia and related disorders analysis included 84 renal ultrasound studies , each imaging the right and left kidney , from 50 non - transplanted patients aged 2.2 years to 36.3 with enzymatic and mutation confirmed forms of mma . we treated each ultrasound from subjects with consecutive studies as a separate data point instead of averaging each patient 's data because of the significant time interval , typically greater than one year , between visits . patients were excluded after they had undergone a kidney or combined liver - kidney transplantation . the mma patients were compared to a published control group of 209 patients from u.s . medical centers aged 0 to 19 years with indications for ultrasound that included urinary tract infections , enuresis , aniridia , scoliosis , and undescended testicle ; this approach has been used previously . data included height , weight , age as well as concentrations of serum creatinine , serum methylmalonic acid , cystatin c , and urine methylmalonic acid . all laboratory testing was done by standard measures through either the department of laboratory medicine at nih or mayo medical laboratories . patients were subdivided into mut , cbla , and cblb groups based on cellular complementation and molecular testing . body surface area ( bsa ) was calculated using the formula : bsa(m ) = ( [ height(cm ) * weight(kg)]/3600 ) . estimated glomerular filtration rate ( egfr ) for children < 18 years of age was calculated using updated schwartz equations that include serum creatinine , or serum creatinine and cystatin c , respectively . for adults , the ckd - epi creatinine and ckd - epi creatinine cystatin c egfr equations were used . the philips iu22 xmatrix ultrasound system with the c5 - 1 1 - 5 mhz bandwidth transducer and the general electric logiq e9 with the c-1 - 5-d 1 - 5 mhz bandwidth transducer were used to obtain abdominal ultrasound studies for all study participants . the original measurements were made in the context of research , and read by one of three radiologists . demographic variables included age , genotype , and gender . the paired t - test was used to compare right and left kidney length . pearson 's correlation was used to determine variation between independent variables . published data from age - matched controls were compared with mma study patients using a two - sample t - test of the means assuming unequal variances and by a linear regression model that included the average of the right and left kidneys as the dependent variable and age as the independent variable . in addition to variation with age , we examined the correlation of renal length with height because mma patients can be shorter than healthy individuals . this linear model was compared to a different control group that created a nomogram of right and left renal length versus height using measurements from 325 german children ages 3 days to 16 years without known kidney disease . using renal length as the dependent variable , the following independent variables were included in a multiple regression equation : age , height , weight , body surface area ( bsa ) , body mass index ( bmi ) , serum methylmalonic acid ( mmas ) , urine methylmalonic acid ( mmau ) , serum creatinine , cystatin c , egfr creatinine ( creatinine based ) , egfr creatinine - cystatin c ( creatinine and cystatin c based ) , gender and enzymatic subtype ( complementation class ) . the best model , as determined by the highest correlation coefficient ( r ) , was developed using stepwise regression analyses . individual predictor variables were eliminated if the regression coefficient ( ) had a statistical significance of p > 0.05 using the wald test . beta weights were calculated to assess the unique contribution of independent variables relative to the regression model ( beta weight is the change in renal length when an independent variable increases one standard deviation while other independent variables are held constant ) . stata version 11 ( statacorp lp , college station , tx ) was used for data manipulation and analysis . when two or more variables were closely correlated ( multicollinearity ) , such as mmas and serum creatinine ( pearson coefficient of correlation 0.808 ( p < 0.0001 ) ; see table s1 ) , they were not included in same multiple regression model . multicollinear relationships were observed between the variable group of creatinine , cystatin c and mmas , and when controlling for height , the r values for these predictors were similar ( table s2 ) . height , bsa , and weight also demonstrated multicollinearity when controlling for height , with r values of 0.767 , 0.757 , and 0.712 , respectively . ultrasound measurements were available for 58 patients with isolated mma who participated in our study . the average age of patients was 11.9 years and the age range was 2.2 years to 36.3 years . after excluding 7 patients who had kidney transplants and one patient with distorted renal anatomy due to severe scoliosis , 50 patients ( 86.2 % ) were evaluated for further study . of these patients , 35 were mut subtype yielding 63 ultrasound studies , 9 cbla subtype ( 14 ultrasound studies ) , and 6 cblb subtype ( 7 studies ) . a single kidney study includes an ultrasound of both right and left kidneys . eighteen patients had sequential ultrasound studies yielding an additional 33 kidney studies . in the entire study group , the mean left kidney length was 0.37 cm greater than the right ( p < 0.001 ) ; similar differences were noted in studies of healthy children . based on these findings , the data were divided into left and right kidneys for subsequent multiple regression analyses . the mut patients had slightly but not significantly smaller kidneys on average compared to the cbla and cblb subtypes , and the enzymatic subtype was insignificant when added into the regression analysis ( controlling for height and cystatin c ) : mut ( p = 0.487 ) , cbla ( p = 0.208 ) , and cblb ( p = 0.485 ) . figure 2(a ) and figure 2(b ) are nomograms showing renal length variation with age and height , respectively . for more severely affected patients , with serum methylmalonic acid levels greater than 2000 micromolar , renal lengths were smaller across the age and height spectrum ( figure 2 ) . when comparing to a control group , mma patients had a significant difference in average renal length ( p < 0.05 ) , as shown in table 1 and figure 1 . the regression equation for the control group was renal length ( cm ) = 6.79 + 0.22 * age ( years ) for children older than 1 year . the linear regression equation for mma subjects was 6.80 + 0.09 * age ( years ) with p < 0.001 for both the constant and age coefficient . in addition to age ( r = 0.40 ) , renal length varied linearly with height ( r = 0.51 ) [ figure 2 ( b ) ] and bsa ( r = 0.52 ) . because the published controls averaged right and left kidney lengths when developing their nomogram , we used an average of the right and left kidneys for comparison . with height as the independent variable and renal length as the dependent variable , mma patients had a diminished slope compared to historical controls , indicating that in mma there is a decreased renal growth rate that is independent of height . for this group of published controls , renal length nomograms were developed for left and right kidneys separately . the control group equations were : left kidney length ( cm ) = 0.051 * height ( cm ) + 1.8 , and right kidney length ( cm ) = 0.050 * height ( cm ) + 1.8 . the mma patient equations were left kidney length ( cm ) = 0.033 * height ( cm ) + 4.0 , and right kidney length ( cm ) = 0.037 * height + 3.0 ( p < 0.001 for coefficients ) . serum creatinine , mmas and mmau were analyzed according to enzymatic subtype and age ( table s3 ) . for the mut and cbla groups , serum creatinine and mmas increased with age compared to mmau , which remained unchanged over time . the number of patients in the cblb group was too small to see trends similar to the mut and cbla groups . table s1 shows that creatinine , mmas and mmau significantly ( p < 0.05 ) correlated to age with pearson 's correlation coefficients of 0.503 , 0.235 , and 0.256 , respectively for all enzymatic subtypes . in more severely affected patients with a serum methylmalonic acid of 2000 micromolar or greater , serum creatinine rises faster with age [ figure s1(a ) ] ; conversely , mmas vs. age has a greater slope when correlated with egfr creatinine greater than 60 ml / min/1.73 m [ figure s1(b ) ] . routine measurement of serum cystatin c began midway through the study and 33 of the 84 ultrasounds having matched serum cystatin c values . cystatin c ( table s1 ) correlated closely with serum creatinine and mmas and had pearson correlation coefficients of 0.790 and 0.768 , respectively . we also analyzed mmas compared to egfr creatinine - cystatin c and found that mmas rises sharply when the egfr creatinine - cystatin c is below 60 ml / min/1.73 m ( figure 3 ) . the most predictive multiple regression model for renal length found cystatin c and height to be the optimal independent variables with beta weights of 0.51 and 0.75 , respectively . figure s2 is a representation of this regression model showing that renal length is inversely proportional to cystatin c when stratified by height . the final predictive equation for left kidney length ( cm ) = 4.9 0.92*cystatin c + 0.036*height ; for the right kidney , predicted length ( cm ) = 3.6 0.92*cystatin c + 0.043*height . table s2 compares mmas , cystatin c , creatinine , and egfr based multiple regression models stratified by different creatinine levels . the regression model using mmas was found to be highly predictive of renal length when serum creatinine was below 1.5 ( table s2 ) . ultrasound measurements were available for 58 patients with isolated mma who participated in our study . the average age of patients was 11.9 years and the age range was 2.2 years to 36.3 years . after excluding 7 patients who had kidney transplants and one patient with distorted renal anatomy due to severe scoliosis , 50 patients ( 86.2 % ) were evaluated for further study . of these patients , 35 were mut subtype yielding 63 ultrasound studies , 9 cbla subtype ( 14 ultrasound studies ) , and 6 cblb subtype ( 7 studies ) . a single kidney study includes an ultrasound of both right and left kidneys . in the entire study group , the mean left kidney length was 0.37 cm greater than the right ( p < 0.001 ) ; similar differences were noted in studies of healthy children . based on these findings , the data were divided into left and right kidneys for subsequent multiple regression analyses . the mut patients had slightly but not significantly smaller kidneys on average compared to the cbla and cblb subtypes , and the enzymatic subtype was insignificant when added into the regression analysis ( controlling for height and cystatin c ) : mut ( p = 0.487 ) , cbla ( p = 0.208 ) , and cblb ( p = 0.485 ) . figure 2(a ) and figure 2(b ) are nomograms showing renal length variation with age and height , respectively . for more severely affected patients , with serum methylmalonic acid levels greater than 2000 micromolar , renal lengths were smaller across the age and height spectrum ( figure 2 ) . when comparing to a control group , mma patients had a significant difference in average renal length ( p < 0.05 ) , as shown in table 1 and figure 1 . the regression equation for the control group was renal length ( cm ) = 6.79 + 0.22 * age ( years ) for children older than 1 year . the linear regression equation for mma subjects was 6.80 + 0.09 * age ( years ) with p < 0.001 for both the constant and age coefficient . in addition to age ( r = 0.40 ) , renal length varied linearly with height ( r = 0.51 ) [ figure 2 ( b ) ] and bsa ( r = 0.52 ) . because the published controls averaged right and left kidney lengths when developing their nomogram , we used an average of the right and left kidneys for comparison . with height as the independent variable and renal length as the dependent variable , mma patients had a diminished slope compared to historical controls , indicating that in mma there is a decreased renal growth rate that is independent of height . for this group of published controls , the control group equations were : left kidney length ( cm ) = 0.051 * height ( cm ) + 1.8 , and right kidney length ( cm ) = 0.050 * height ( cm ) + 1.8 . the mma patient equations were left kidney length ( cm ) = 0.033 * height ( cm ) + 4.0 , and right kidney length ( cm ) = 0.037 * height + 3.0 ( p < 0.001 for coefficients ) . serum creatinine , mmas and mmau were analyzed according to enzymatic subtype and age ( table s3 ) . for the mut and cbla groups , serum creatinine and mmas increased with age compared to mmau , which remained unchanged over time . the number of patients in the cblb group was too small to see trends similar to the mut and cbla groups . table s1 shows that creatinine , mmas and mmau significantly ( p < 0.05 ) correlated to age with pearson 's correlation coefficients of 0.503 , 0.235 , and 0.256 , respectively for all enzymatic subtypes . figure s1 shows serum creatinine , mmas and mmau variations with age . in more severely affected patients with a serum methylmalonic acid of 2000 micromolar or greater , serum creatinine rises faster with age [ figure s1(a ) ] ; conversely , mmas vs. age has a greater slope when correlated with egfr creatinine greater than 60 ml / min/1.73 m [ figure s1(b ) ] . routine measurement of serum cystatin c began midway through the study and 33 of the 84 ultrasounds having matched serum cystatin c values . cystatin c ( table s1 ) correlated closely with serum creatinine and mmas and had pearson correlation coefficients of 0.790 and 0.768 , respectively . we also analyzed mmas compared to egfr creatinine - cystatin c and found that mmas rises sharply when the egfr creatinine - cystatin c is below 60 ml / min/1.73 m ( figure 3 ) . the most predictive multiple regression model for renal length found cystatin c and height to be the optimal independent variables with beta weights of 0.51 and 0.75 , respectively . figure s2 is a representation of this regression model showing that renal length is inversely proportional to cystatin c when stratified by height . the final predictive equation for left kidney length ( cm ) = 4.9 0.92*cystatin c + 0.036*height ; for the right kidney , predicted length ( cm ) = 3.6 0.92*cystatin c + 0.043*height . table s2 compares mmas , cystatin c , creatinine , and egfr based multiple regression models stratified by different creatinine levels . the regression model using mmas was found to be highly predictive of renal length when serum creatinine was below 1.5 ( table s2 ) . chronic kidney disease due to progressive tubulointerstitial injury is emerging as a frequent manifestation in mma and eventually results in the need for dialysis or kidney transplantation : half of our cohort of mma patients of all ages and 100% of our mut patients over age 15 either have been transplanted or have an egfr below 60 ml / min/1.73 m. kidney disease in this population is notoriously difficult to diagnose and monitor early in life , as reliance on serum creatinine - based egfr estimates may over - state the true gfr due to low muscle mass and kidney biopsy , the most accurate test for determining the nature and extent of renal disease , is invasive and is rarely offered to mma patients . once clinically manifest , renal disease in mma patients has a relentless progression to kidney failure as is illustrated by the linear decline of egfr in the patient population , with a faster rate of decline apparent in those with more severe metabolic parameters ( figure 4 ) . the failure to detect chronic kidney disease early is certain to delay the implementation of renoprotective strategies in affected infants and children . whereas blockade of the renin - angiotensin system ( ras ) is the current standard of care for adults with chronic kidney disease , experience with renoprotective measures in the pediatric population has not been as thoroughly studied . for example , there has only been one published article addressing renoprotection with ras blockade in mma despite the formal recognition that such studies should be pursued . the successful small molecule treatments of related metabolic disorders that feature tubulointerstitial disease and progressive kidney failure , such as nephropathic cystinosis and tyrosinemia type i , and striking preclinical efficacy of systemic aav gene delivery to treat the kidney disease of mma in a mouse model provides strong practical and theoretical support for the study of renoprotective interventions in mma patients . to predict renal length in mma subjects , we first created a multivariate model and compared our data to healthy controls in the medical literature using age as the independent variable . renal length was chosen as the dependent variable from other ultrasongraphic measurements such as renal volume and cortical thickness because renal length has the least amount of inter - observer variation and has been established to correlate with gfr . compared to published healthy controls , most age groups in the mma cohort had significantly smaller renal lengths ( table 1 ) . the healthy control group and the mma group both had similar constants ( y - intercept ) , 6.79 and 6.80 respectively , suggesting that kidney size was normal at birth in the mma patients ; however , the slope ( cm / year ) of kidney length increase in the mma group was less than half that of the controls : 0.09 cm / year versus 0.22 cm / year . in comparison to another large study of 778 healthy children less than 18 years of age , the mma kidney growth slope was only a third of the controls : 0.09 cm / year versus 0.3 cm / year . these data provide a marker of the natural history of kidney growth in mma , one that might be used in future studies to examine the effects of renoprotective regimens and perhaps gene therapy on the renal phenotype seen in the patients . we were also interested in the comparison of mma patients to healthy control subjects when height was the independent variable , especially because mma patients have been shown to be shorter than age matched healthy subjects . although most of the standardized nomograms created for renal length use age as the independent variable , one study of healthy children developed a linear model with height as the independent variable . the slope of the nomogram for kidney length vs. height in the mma patients was less than this particular control group , with right and left kidney mma slopes of 0.037 cm ( renal length)/cm ( height ) and 0.033 cm / cm , respectively , and control slopes of 0.050 cm / cm and 0.051 cm / cm , respectively . thus , the comparison of mma patients to yet another set of healthy children via linear modeling when controlling for height also demonstrated that mma patients have slower renal growth , even when controlling for height . using multivariate modeling , we have found that over 70% of the variability ( r ) in kidney length was captured by our model . in order to gauge how our multivariate predictive capability ( r ) compared to those previously reported , we demonstrated a predictive capability of an independent multivariate model for a much larger study of 3031 healthy children under the age of 13 which found over 60% of variability was explained by their model ( r 's of 0.696 and 0.712 for right and left kidney , respectively ) . cystatin c and height were the most predictive independent variables in the multiple regression model . table s2 shows that cystatin c , mmas , or serum creatinine had similar r values when controlling for height and illustrates the significance of these variables as independent predictors of renal length . our results establish that for serum creatinine < 1.5 mg / dl , mmas is a stronger predictor of kidney length in the multivariate model than either cystatin c or egfr creatinine - cystatin c ( table s2 ) . when beta weights are included in the regression analysis , mmas proves to be a highly significant independent variable with a value of 0.42 . the proportional relationship between mmas and smaller renal size in this study combined with a large beta weight for mmas suggests that elevated mmas is strongly associated with renal disease in methylmalonic acidemia when controlling for height by multiple regression . additionally , figure 2(a ) and figure 2(b ) demonstrate smaller renal size versus age and height , respectively , in patients with serum methylmalonic acid greater than 2000 micromolar . the association between mmas and renal failure is also evident in figure 3 where mmas is inversely related to egfr creatinine - cystatin c below 60 ml / min/1.73 m ; additionally , all of our patients with a serum methylmalonic acid level above 1000 micromolar have an egfr creatinine below 60 ml / min/1.73 m. these observations would advocate for the routine measurement of serum methylmalonic acid concentrations in the longitudinal monitoring of patients with mma and highlight the need for future studies to examine serum markers concomitant with a measured gfr . to complement serum creatinine , we obtained cystatin c levels with 33 of the total 84 renal ultrasounds but added this measurement after the protocol began . using the aggregate data , the multivariate model incorporating cystatin c emerged as the most precise model , explaining over 76% of renal length variation ( r = 0.767 ) when controlling for height ( see figure s2 ) . as expected , the regression model using cystatin c was more predictive than the model using gfr estimates calculated from serum creatinine and cystatin c ( table s2 ) , which is most likely due to the muscle mass differences in mma patients and the patients used to derive the egfr equation . our data suggests that serum cystatin c concentration should also be included in the routine monitoring of renal function in mma patients and warrants further study , including validation against direct gfr assessments , especially because recent studies suggest that in the general population , gfr estimates that incorporate both serum creatinine and serum cystatin c appear to be the most accurate . in a population with reduced muscle mass such as mma , the gfr equation using cystatin c or cystatin c and mmas may be preferred . a multicenter , retrospective review of 35 patients suggested that mmau predicted chronic kidney disease as determined from calculations using the schwartz formula ; however , inter- and intra - individual variations were claimed to contribute to overlap in the enzymatic subgroups of this study . in contrast , walter et al . found no correlation between random urinary methylmalonic acid levels and reduction in gfr . we found that the urine methylmalonic acid concentration was not a significant predictor of renal length when controlling for height ( p = 0.471 ) : mmau did not correlate with trends in serum creatinine as can be seen by the statistically insignificant pearson 's correlation in table s1 ( r = 0.0389 ) . given the poor correlation between mmau , and renal length or trends in serum creatinine , our data advocates using mmas as a predictor of renal disease and including this analyte in the routine monitoring of patients with mma . the mut patients had slightly smaller kidneys than the other subgroups ; however , when controlling for cystatin c and height , we did not detect a significant difference between enzymatic subtypes on kidney length . another study found an increase in chronic kidney disease in mut and cblb compared to other enzymatic subtypes causing isolated mma , which would suggest that these individuals might have smaller kidneys . a possible explanation for this discrepancy between the studies may derive from the small number of participants with cblb ; in both our study ( n=6 ) and that of horster et al . in addition to mma subtypes , we did not find gender to be a contributor to renal length in mma patients . boys ' and girls ' body sizes grow at different rates ; however , multiple studies , including ours , have demonstrated that renal length is not significantly different between the sexes . our study has a number of limitations . despite using the largest single center mma patient cohort described to date , the sample size was comparatively small and we were unable to use a discovery and replication design . second , the majority of our data was cross - sectional , which may introduce increased variability in the nomograms compared to data derived from a purely longitudinal design . third , the most severely affected patients may not have been fit for travel to nih , and thus underestimated our effect size . in summary , the rate of renal growth in the mma subjects studied here was one half to one third that observed in controls . renal length is likewise significantly decreased compared to normal controls and predicted by a multiple regression model that uses the readily available clinical variables of height and cystatin c. when serum creatinine values fall in the age normal range , mmas and cystatin c are comparable predictors of renal length , suggesting that both should be included in the routine evaluation in mma patients . in addition to generating a clinically useful nomogram to follow kidney growth in mma , our results capture a marker of the natural history of the renal disease of mma and should assist with the study of therapeutic interventions designed to target kidney disease in this population .

it is usually manifested by neurological syndromes such as tetraparesis , paraparesis or brown - sequard syndrome . sceh presenting with pure motor hemiparesis is extremely rare , and may be mistaken for cerebrovascular stroke . although magnetic resonance imaging ( mri ) is generally considered to be the most reliable diagnostic tool , it may not be available in certain situations . for example , it can take a long time , especially when an extended area of the central nervous system must be examined quickly . we herein report a case of sceh with pure motor hemiparesis in which a cervical computed tomography ( ct ) scan was obtained on admission and led to the rapid and correct diagnosis . this patient was treated without surgery , and her neurological symptoms resolved completely . the appropriate diagnosis and management of sceh is also discussed . an 84-year - old female was brought to the emergency department after the sudden onset of neck pain , followed by right motor weakness . she had no remarkable past medical history and was not regularly taking any medication . on neurologic examination , the motor strength of her right extremities was classified as one out of five , while there was no motor weakness of right facial muscles . routine laboratory and diagnostic studies , including coagulation parameters , were all within normal limits . a brain ct scan showed no hemorrhagic lesion , but a small cavernous angioma in the left postcentral gyrus was present , which was considered irrelevant to the present condition . the paresis improved to some extent , and a cerebral ischemic accident was strongly suspected . the scan revealed a slightly high - density mass posterolateral to the spinal cord extending from c2 to c3 , compatible with an epidural hematoma ( fig . 1 ) . no apparent extravasation or abnormal accumulation of the contrast medium was observed . a cervical mri obtained two days after onset was also compatible with an epidural hematoma ( fig . magnetic resonance images of the cervical spine which were obtained on the third day after the onset . axial ( a ) and sagittal ( b ) t2wis show an extradural mass with hyperintensity that compressed the spinal cord ( arrows ) . her symptoms improved gradually after hospitalization and she began ambulation on the following day with improved motor strength to 4+/5 on the right . a repeated mri on the 7th day showed that the hematoma was in the process of absorption . spinal epidural hematoma is a rare disease ; its incidence is estimated to be only 0.1 per 100,000 patients per year . it has characteristic symptoms , such as paraparesis or tetraparesis with radiating neck or back pain and sensory deficit . to our knowledge , only 3 cases of sceh presenting with pure motor hemiparesis have been reported . all of the reported cases were misdiagnosed as cerebral ischemia and erroneously treated with anticoagulant therapy . the absence of facial weakness or cranial signs suggested that hemiparesis was due to a lesion in the cervical spinal cord . the present case developed sudden hemiparesis after neck pain and was brought to our hospital 40 minutes after the onset . a ct scan of the head and neck was performed within 30 minutes after arrival , which eventually revealed sceh . even though the enhancement of contrast medium is limited to the arterial phase , it often provides valuable diagnostic information . however , brm et al . reported that ct failed to make the diagnosis in one case . a plain ct scan was reported to be helpful in detecting the location of a cervical subdural hematoma . this risk is higher when the thoracic and lumbar region are affect , probably because of air artifacts within the thoracic and peritoneal cavity . therefore , mri has been suggested to be the procedure of choice because of its capabilities for tissue differentiation . nevertheless , when limited to the cervical spine , ct also yields acceptable results . considering the short period of time for the optimal treatment of acute brain ischemia , cervical ct should be chosen to exclude a spinal hematoma prior to administering potentially harmful anti - coagulant therapy , especially if the patient complains of neck pain . the majority of cases have been reported as spontaneous , while coagulation disorders or anticoagulation therapy have also been implied as predisposing conditions . in addition , the previous papers have pointed out other causative backgrounds , such as a vascular malformation , tumor , trauma , lumbar puncture and spinal anesthesia . reported that the posterior internal vertebral venous plexus plays an important role in spinal epidural hematoma , by investigating clinical cases and human cadavers . holtas et al . reported that sceh was located predominantly in the anterior epidural space . in the current case , these findings supported the hypothesis that the posterior internal vertebral venous plexus was the bleeding source . the network of the epidural venous plexus is thought to be especially vulnerable to venous pressure because it lacks valves . it is speculated that increased venous pressure induced by physical activity can result in the rupture of the vessels . the neck pain in the present case began when the patient raised her arms to wash herself . spontaneous resolution of sceh has been reported in only a few cases ; each occurred within the setting of unilateral hemiparesis and dysesthesia . however , if the neurological symptoms are minimal or improving rapidly , conservative management is an alternative choice . sceh may manifest as pure motor paresis with sudden neck pain . even in the mri era , a cervical ct scan is still a reasonable choice for making a quick differential diagnosis of sceh , when considering the therapeutic time window available for the treatment of brain ischemia .