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[ "aging is part of the natural process of human development , and it brings multiple biopsychosocial changes that cause a slow and progressive deterioration of body functions that are essential to life . among these functions that undergo important mostly irreversible changes is hearing.1 hearing loss is considered one of the three most common conditions in the elderly,2 with occurrence of 5 to 20% in 60-year - olds , increasing to 60% in people over 65 years old.1 \n 3 in brazil , 60% of the elderly have some degree of hearing loss.4 \n the term used to describe hearing loss associated with aging is presbycusis , which causes a decrease in hearing sensitivity and a reduction in the ability to understand speech . this occurs due to several degenerative and physiologic changes that affect the inner ear , impairing hearing at high frequencies and affecting communication , especially in noisy environments.1 \n 2 \n 4 \n 5 \n 6 \n 7 other factors associated with the natural degeneration process , such as exposure to loud noise , ototoxic agents , and sequelae of otitis media caused by medical problems and treatments , can contribute to lifelong aggravation of the loss.4 \n 8 \n 9 \n difficult communication associated with hearing loss can lead to social and emotional consequences , especially for the elderly , who have several limitations related to aging that can impact their quality of life , increasing social isolation in addition to causing emotional disorders such as depression.1 \n 4 \n 5 \n 8 \n 10 \n 11 hearing loss associated depression can be even more harmful in the elderly . thus , recommended intervention by hearing aid adaptation contributes to the prevention of emotional disorders such as depression.12 \n depression is defined as a mental disorder that causes feelings of sadness , pessimism , hopelessness , difficulty concentrating , and difficulty making decisions and initiating tasks , among other symptoms.13 despite being very common in the elderly , it can not be attributed solely to aging . studies in so paulo ( brazil)14 and florianpolis ( santa catarina , brazil)15 showed that the factors associated with depression in the elderly are : female , unmarried or widowed , altered cognitive function , dependence , use of many medicines , very old ( over 80 years ) , low education , poor economic status , cognitive impairment , fair or poor self - rated health , functional dependency , and chronic pain . other causes include smoking , comorbidities ( endocrine , vascular , neurologic , oncological ) , and negative changes in relationships with family and friends.15 \n hearing loss may be associated with depression to be a debilitating , chronic disorder.16 \n 17 \n 18 the main goal of an auditory rehabilitation program in elderly patients is to minimize the effects caused by sensory deprivation of hearing and reinstate the patients in their family and society , helping them cope with the disadvantages and limitations caused by hearing loss.3 \n monaural adaptations are commonly performed even in cases of bilateral hearing loss , whereas the most appropriate should be binaural adaptation . the reasons for that may be related to factors such as rejection to the use of two hearing aids , financial issues , aesthetic reasons , problems with manual dexterity , very asymmetric losses , and central processing issues.19 \n given such reality , this work intends to analyze the effects of unilateral adaptation of hearing aids on the symptoms of depression and the social activity constraints of elderly subjects with hearing impairment .", "this is a prospective interventional study of elderly individuals of both sexes with recommended use of hearing aids at a hearing center in porto alegre . inclusion criteria were signature of the free and informed consent ; 60 years or older ; good health ( seniors able to commute to the research location , conduct tests , and answer questionnaires ) ; new hearing aid users with monaural fitting ; and participation in all phases of the research . exclusion criteria were initiation of antidepressant therapy during the study and a history of cognitive and/or neurologic disorders . the study was divided into two phases . in phase 1 , subjects responded to an interview with questions on demographics and health ( general and hearing ) . pure tone audiometry was performed to determine the airway tone thresholds ( frequencies 250 to 8,000 hz ) and bone conduction ( frequencies 500 to 4,000 hz ) in a vocal booth , using an ad-229e interacoustics audiometer ( assens , denmark ) . the presence and degree of hearing loss were classified according to the world health organization by analyzing mean airway tone threshold in the frequencies of 500 , 1,000 , 2,000 , and 4,000 hz.20 \n after that , seniors were asked to answer two questionnaires individually , without the intervention of third parties : the geriatric depression scale ( gds ) and the hearing handicap inventory for elderly short version ( hhie - s ) . the gds , an instrument validated for portuguese and composed of 15 yes - or - no questions , is used to detect depression symptoms in the elderly . thus , scores less than 5 points indicate absence of symptoms , scores of 5 to 10 indicate mild to moderate symptoms , and scores of 11 or more indicate severe symptoms of depression.21 \n after completing the first phase , participants answered a summarized version of the hhie - s . the questionnaire was prepared in 198222 and translated and adapted to brazilian portuguese.23 it contains 10 questions that aim to assess the impact of hearing loss on the emotional and social status of elderly people . all 10 questions provide three choices of answers : for each yes answer , 4 points is assigned ; each sometimes , 2 points ; and each no , 0 points . the total score ranges from 0 to 40 points , and the higher the score , the greater subjects ' self - perception of social activity constraints : 0 to 8 points indicate no social activity constraints ; 10 to 23 points , mild to moderate social activity constraint ; and 24 to 40 points , significant social activity constraints.8 \n 22 \n after testing , the participants were subjected to the selection and unilaterally hearing aid adaptation processes thirty days after purchasing the device , they returned to the hearing center to participate in the second phase of the research . in phase 2 the research project was approved by the research committee and by the ethics and research committee of the institution ( protocol 266.060 ) . patients who agreed to participate signed an informed consent and had their rights guaranteed for confidentiality , nonidentification , and withdrawal of participation . the results were analyzed using the statistical package for social science version 20.0 ( ibm , ny , usa ) . to examine associations of categorical data , the chi - square and student t test for paired samples were used . all statistical tests were nonparametric tests , and always observed exact p value ( not asymptotic ) , which are the best measures to statistically evaluate a study when the sample is restricted . the chi - square test considered the p value of fisher exact test , and for the kappa coefficient of agreement , the exact p value ( not asymptotic ) was observed .", "the sample consisted of 13 elderly patients with hearing loss , ages from 60 to 90 years ( mean 72.85 11.05 years ) , 10 women ( 76.9% ) and 3 men ( 23.1% ) . table 1 shows data on the characterization of sample in terms of age , gender , mean thresholds , and degree of hearing loss . \n table 2 depicts an association analysis between the gds classification of phases 1 and 2 , indicating that there was no significant association between the variables in the periods before and after the adaptation ( p = 0.615 ) . thus , of the 6 patients ( 46.2% ) who initially had mild symptoms of depression , only 2 ( 15.4% ) of them continued to have mild symptoms . all seniors who initially had severe symptoms of depression ( 15.4% ) also started to lose some symptoms of depression in phase 2 . hhie classification analysis in phases 1 and 2 of the study are presented in table 3 . no significant association was found for these variables in the periods before and after the adaptation ( p > 0.999 ) . thus , the only senior who had mild to moderate social activity constraints in the first phase showed reduction in the second phase . after prosthetization , participants who had severe social activity constraints in phase 1 started to show no social activity constraints or mild to moderate social activity constraints . only 1 ( 7.7% ) the lack of association between the classification of the gds and hhie on the two phases is an important point , because it shows that there were different results in both assessments ( before and after hearing aid adaptation)that is , on the sample studied , unilateral prosthetization resulted in benefits for patients . \n table 4 shows the scores obtained by the subjects in the instruments used for evaluation in the two phases of the research . there was significant difference between the gds ( p = 0.031 ) and hhie ( p < abbreviations : gds , geriatric depression scale ; hhie - s , hearing handicap inventory for elderly short version . \n", "the analysis of the survey data showed that the age of the individuals analyzed varied from 60 to 90 years . another study that examined the degeneration of the auditory system in the course of aging had a sample with similar age and results.4 \n we had a greater number of female participants . although it has been found that men often have more problems related to hearing loss than women,24 they also have a lower perception of their disability compared with women,1 \n 25 \n 26 which explains why women seek health services more often26 \n 27 and why there is a greater number of female participants in this study . another fact to be noted is that , in terms of population , there is a greater number of older women in brazil , a fact known as feminization of aging.28 \n there was a prevalence of moderate hearing loss in both the right and the left ears , agreeing with other published studies that refer to this as being the most commonly found degree of hearing impairment in the elderly.2 \n 9 \n 25 \n the data show that many seniors had some degree of depression symptoms before prosthetization ( table 2 ) . after unilateral hearing aid adoption , there was improvement in symptoms for most of them . thus , a hearing aid , even when unilateral , promoted elimination or reduction of symptoms of depression in the sample studied . this result had already been described in the literature,2 but we emphasize that the result was obtained with bilateral prosthesis . thus , the adaptation of a single amplifying device can also generate benefit for elderly patients , which is important for the reduction of one of the most prevalent problems in the aging population , depression . the data show that all participants had some degree of social activity constraints before using a unilateral hearing aid ( table 3 ) . after 30 days of hearing aid use , 12 ( 93.3% ) and 13 ( 100% ) participants showed absence or reduction of social activity constraints , respectively . results show evidence of the positive effects of hearing aid use , even if unilateral , helping to reduce the impact that hearing loss has on social relationships and on the quality of life of the elderly.9 results are consistent with another study that examined the short - term benefits of amplification in new users.29 \n the data also show a significant difference in scores obtained in phases 1 and 2 of the gds ( p = 0.031 ) and hhie - s ( p < 0.001 ) , confirming a reduction or elimination of depression symptoms and social activity constraints in elderly patients , agreeing with other published studies ( table 4).2 \n 8 \n 11 \n 29 \n 30 again , it is important to highlight that in the studied group , unilateral prosthesis resulted in substantial benefits not only regarding hearing but also in other matters essential for the well - being and quality of life of individuals . the findings reinforce the importance of hearing aid use in elderly patients , because many times family and social relationships are affected by the hearing impairment . the adaptation to amplification devices , albeit unilaterally , has proved to be effective in reducing symptoms of depression and social activity constraints . moreover , the length of time between assessments was 30 days , shorter than that considered ideal for auditory acclimatization , and even then , benefits occurred , confirming the importance of auditory rehabilitation in elderly .", "results show that unilateral hearing aid contributed to the elimination or reduction of depression symptoms and of social activity constraints in elderly participants in this research sample group ." ]

[ "ameloblastomas are rare benign odontogenic tumours , most commonly found in a patient s lower jaw . although rarely metastatic , the growth of these lesions can result in defects in the jaw easily destroying surrounding bony tissues . for this reason , the treatment is often excision of the lesion , with wide excision margins , resulting in the absence of a part , or all , of the mandible . the surgical approach for correction of this mandibular defect is often reconstruction of the mandible in view of inserting dental implants at a later date . despite surgical advancements , , bone grafting with non - vascularized bone remains a good option where facilities for soft tissue flaps are not readily accessible . creating an effective wound closure and reducing the incidence of infection is an important stage in the success of the operation , and has been combated here by tissue expanding the submucosa , using osmed pellets , prior to surgery . this is a relatively new technique to the mandibular region and has the potential to revolutionize the success of microsurgeries in this area . in this report , i describe a woman who underwent a repair of her mandibular defect by a combination of innovative techniques , in order to reduce the risk of complications and get her back to an aesthetically pleasing level .", "a 35-year - old lady presented to the craniofacial surgeons looking for a permanent fixture to the bony defect in her lower - right mandible . in 1991 she had her first operation to remove the ameloblastoma and then had it further excised 6 years later due to a reoccurrence . this resulted in an absence of teeth from the canines back and removal of bone down to the inferior alveolar nerve . the patient has since been clear of any further reoccurrences and wears lower dentures for aesthetic reasons . she has no significant past medical history nor family history , nor does she have any known drug allergies . she does not smoke or drink alcohol . on clinical examination , the patient experiences mild parasthesia in the distribution of her right inferior dental nerve , particularly the lateral aspect of the lower lip . on examination , there is clear absence of teeth on the lower - right side from the canines back ( to include the premolar , molar and wisdom teeth ) and presence of healthy oral mucosa with laxity to move the submucosa medially . there is a loss of the vertical height of the posterior body of the mandible of about 50% and the vestigial remnants of the lingual plate are just palpable and visible on plain radiograph and ct . scans show that the superior margin of the resection is well corticated under the inferior dental nerve ( fig . , the patient wishes to get dental implants . the surgical approach taken , prior to the implants , ( b ) ct scan of head imaging of the patient 's mandibular defect pre - surgically . ( a ) radiolocal image of patient 's jaw . ( b ) ct scan of head the patient was initially given the option between a bone graft or vascularized tissue ( free fibular flap ) and informed of the complications a bone graft carries a risk of rejection and vascularized tissue needs an increased amount of post - operative care to ensure that the tissue remains vascularized . the patient was not keen on a free flap and given the fact that the bony deformity was < 5 cm , non - vascularized iliac bone graft was deemed to be sufficient to carry out this operation . the first step was achieving significant tissue expansion ( > 2 cm ) using osmed pellets , guided into the submucosa and kept over the alveolar ridge for several months ( fig . ( c ) osmed pellets before insertion into the submucosa image of patient 's oral cavity during insertion of osmed pellets . ( a ) incisions made in the submucosa . ( c ) osmed pellets before insertion into the submucosa two months later , the patient underwent the main bulk of her operation . the osmed pellets were removed from the subperiosteum ( fig . 3a and b ) and the iliac bone graft was taken , made up of cortical and cancellous bone and placed to one side ( fig . an epidural catheter was placed inside the soft tissue of the graft site and left overnight . it had a continuous fusion of a local anaesthetic to improve post - operative pain and encourage early mobilization . \n ( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone submucosa after adequate tissue expansion . ( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone previously , using ct scans , a titanium construct was made to guide the outline of the bone graft and complete the missing height and width of the mandible . once fitted to the patients jaw , it was packed with the bone graft and screwed down into position ( fig . a slight groove was made on the mandible to allow space for the inferior dental nerve to lay , so that the construct did not compact the nerve and cause functional problems . \n figure 5:titanium construct packed with iliac bone graft and screwed into position on the lower mandible titanium construct packed with iliac bone graft and screwed into position on the lower mandible to complete the surgery , the periosteum was sutured over the construct and then the buccal muscosa was sutured over again to form a double - layered closure ( fig . 6a c).the purpose of this was to prevent infection and rejection , by ensuring that there was an increased distance between the foreign object ( titanium construct ) and the outside environment . the dental implants are scheduled to be inserted several months after this operation . \n figure 6:image of patient 's surgical site . ( c ) image of post - surgical site image of patient 's surgical site .", "bony defects in the maxillofacial area are a huge problem in clinical orthodontics , caused by a wide range of injuries and diseases presenting as either cosmetic or functional defects and in some instances both . it is the importance of accounting for the anatomical , functional and aesthetic aspects that make the reconstruction so challenging . often patients suffering from ameloblastomas undergo resection as treatment and are left with bony defects that require reconstruction . in this case , the intention was to realize an aesthetically pleasing appearance for the patient and it has been shown that bone grafting with subsequent dental implants is a successful method . as in this case , it is recommended that non - vascularized autogenous bone grafting can be used when a two - layer watertight closure is attainable . sources of bone could have been harvested from either local or distant sites but in this case , an extra - oral site ( iliac bone ) was used as a moderate amount of bone was needed . the surgery lasted 5 h and incorporated several novel techniques to improve the outcome of the operation . the use of osmed pellets was innovative , as pellets of this size have only recently been experimented on goats maxilla . as well as allowing for space to put the construct , the pellets allowed for a tension - free closure . otherwise , the result would have been a high - tension closure , leading to rapid breakdown of the graft providing an entrance for infection , a common complication occurring when titanium plates are exposed . advantages of the osmed tissue expanders are that the material is safe with a low complication rate and low risk of infection due to small incision site and minimal trauma . the titanium construct is another relatively novel approach and was invaluable in the success of this surgery , as it guided reconstruction of the mandible , allowing for dental implants to be put in at a later date . at present , an alloplastic material such as titanium is often used for mandibular reconstruction as it is resistant to corrosion , adaptable and biocompatible . although the insertion of dental implants is the end goal for this surgery , the importance of this case report is to highlight the novel techniques involved in making this surgery a success . the use of small osmed pellets in the submucosa of the mouth to cause sufficient tissue expansion and create a tension - free closure , combined with the titanium construct packed with grafted iliac bone to rebuild the jaw was an approach recommended by the doctors for the repair of bony defects of < 5 cm . i suggest prudent management of future cases post - operatively to ensure a continued success of the graft ." ]

[ "glucagonlike peptide1 ( glp1 ) , the most potent incretin hormone , stimulates glucoseinduced insulin secretion and inhibits glucagon secretion , which consequently leads to a decrease in hepatic glucose production and blood glucose levels . in addition to its ability to modulate insulin and glucagon secretion , glp1 inhibits gastric emptying and gastric acid secretion , which suppress appetite and energy intake in obese individuals , and in patients with type 2 diabetes . glp1 is secreted from intestinal l cells in response to ingestion of nutrients , including carbohydrate and lipids . a previous study showed that alphalinolelic acid ( la ) promoted glp1 secretion through the stimulation of gproteincoupled receptor ( gpr ) 120 , which is abundantly expressed in the intestine , in mice . furthermore , a recent study has shown that eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , metabolites of la , stimulate endogenous glp1 secretion in mice . these reports show that 3 polyunsaturated fatty acids ( pufa ) , including la , epa and dha , increase glp1 secretion in mice . however , no evidence that 3 pufa ingestion increases glp1 secretion is available in humans . as compared with americans and europeans , japanese eat more fish , which is a major source of epa and dha . hence , serum epa or dha concentrations are higher in japanese than those in americans or europeans . evidence that fish intake prevents atherosclerosisrelated cardiovascular disease have been accumulating . a recent study has shown a significant inverse correlation between serum epa or dha concentrations and cardiovascular risk . furthermore , some studies showed that fish intake reduced the rates of incidence for diabetes in patients with metabolic syndrome , and reduced the rates of death in patients with type 2 diabetes . however , the direct effects of epa or dha consumption on glycemic control in patients with type 2 diabetes have remained uncertain . dpp4 inhibitor increases endogenous active glp1 levels through inhibition of dpp4 enzyme activity , leading to increased circulating insulin levels and decreased blood glucose levels . the circulating glp1 levels were reported to be lower in patients with type 2 diabetes than in nondiabetic patients . therefore , dpp4 inhibitor might be more effective in increasing circulating glp1 levels in patients with type 2 diabetes if they have low glp1 levels . based on these lines of evidence , we hypothesized that dpp4 inhibitor would be effective in patients with type 2 diabetes with high serum 3 pufa concentrations . in the present study , therefore , we investigated the relationship between serum epa or dha concentrations and a decrease in hemoglobin a1c in patients with type 2 diabetes prescribed dpp4 inhibitors .", "serum epa and dha concentrations were measured in 62 consecutive patients with type 2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) . patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0 mg / dl ) were excluded from the present study . sitagliptin at a daily dose of 50 mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period . the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period . after 24 weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . type 2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus . approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients . serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods . hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society skewed variables , such as epa and dha , are presented as the median ( interquartile range ) , and continuous variables are presented as the mean standard deviation ( sd ) . paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa + dha ) and a decrease in hemoglobin a1c was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c , we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses .", "serum epa and dha concentrations were measured in 62 consecutive patients with type 2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) . patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0 mg / dl ) were excluded from the present study . sitagliptin at a daily dose of 50 mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period . the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period . after 24 weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . type 2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus . approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients .", "serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods . hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society .", "means , medians or frequencies of potential confounding variables were calculated . skewed variables , such as epa and dha , are presented as the median ( interquartile range ) , and continuous variables are presented as the mean standard deviation ( sd ) . paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa + dha ) and a decrease in hemoglobin a1c was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c , we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses .", "characteristics of the 62 patients with type 2 diabetes enrolled in the present study are shown in table 1 . the median ( interquartile range ) serum epa concentration was 62.6 g / ml ( 39.588.0 ) and the median ( interquartile range ) serum dha concentration was 139.1 g / ml ( 109.4165.7 ) . among 62 patients , 16 patients were given dpp4 inhibitor as a monotherapy and 46 patients were given it as an addon therapy ( 34 sulfonylurea , 25 metformin , 8 pioglitazones , 5 alphaglucosidase inhibitor ) . data are n , mean sd or median ( interquartile range ) . mean hemoglobin a1c was significantly decreased from 8.1 1.1% at baseline to 7.2 1.0% at 24 weeks after administration of dpp4 inhibitor . correlation between log epa , log dha or log ( epa + dha ) and other variables are shown in table 2 . log epa and log ( epa + dha ) correlated with a decrease in hemoglobin a1c . log dha tended to correlate with a decrease in hemoglobin a1c , but it did not reach statistical significance . dha , docosahexaenoic acid ; epa , eicosapentaenoic acid . a decrease in hemoglobin a1c correlated with bmi ( r = 0.396 , p = 0.0013 ) , age ( r = 0.275 p = 0.0032 ) , hemoglobin a1c at baseline ( r = 0.490 , p < 0.0001 ) , log epa ( r = 0.285 , p = 0.0246 ) and log ( epa + dha ) ( r = 0.260 , p = 0.0411 ) . there were no significant differences in decrease in hemoglobin a1c between male and female patients ( 1.0 0.8% vs 1.0 0.9% , p = 0.9758 ) . decrease in hemoglobin a1c was greater in patients treated with antidiabetic concomitant drugs compared with that in patients without those ( 1.1 0.9% vs 0.6 0.4% , p = 0.0342 ) . in the multiple regression analysis to examine the effects of variables on the decrease in hemoglobin a1c , we included the following independent variables , which were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses : age , bmi , hemoglobin a1c at baseline , usage of antidiabetic concomitant drugs and log epa . multiple regression analysis showed that bmi , hemoglobin a1c at baseline and log epa were independent determinants of a decrease in hemoglobin a1c ( table 3 ) . presence of antidiabetic concomitant drugs ( no = 0 , yes = 1 ) .", "in the present study , serum epa concentrations correlated with a decrease in hemoglobin a1c . this correlation remained significant after adjustment for age , bmi , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . the median ( interquartile range ) serum epa concentration was 62.6 g / ml ( 39.588.0 ; the mean serum epa concentration was 68.6 34.1 g / ml ) , which was almost consistent with previous reports of the epa concentration in general japanese . serum epa concentrations increase with age in apparently healthy japanese . in the present study , however , there was no significant correlation between log epa and age in patients with type 2 diabetes . dpp4 inhibitor increases endogenous active glp1 levels through prevention of glp1 degradation , leading to an increase in circulating insulin levels and a decrease in blood glucose levels . because of the pharmacological action of dpp4 inhibitor on insulin secretion , dpp4 inhibitor is effective in type 2 diabetic patients with impaired insulin secretion , which is a characteristic of japanese patients with type 2 diabetes . indeed , as compared with american and european patients with type 2 diabetes , japanese patients with type 2 diabetes might be higher responders to dpp4 inhibitor . interestingly , the present study has shown a strong correlation between bmi and decrease in hemoglobin a1c , suggesting that dpp4 inhibitor is more effective in lean patients who generally have impaired insulin secretion , rather than in obese patients with insulin resistance . this is consistent with a recent study reporting the association of bmi with efficacy of the dpp4 inhibitor , sitagliptin . previous studies showed that dpp4 inhibitor was effective as an addon therapy to some antidiabetic drugs , including metformin , pioglitazone and sulfonylurea . the profile of diabetic therapy before dpp4 inhibitor administration was as follows : 16 used diet , 15 received sulfonylurea monotherapy , 12 received metformin monotherapy , eight received sulfonylurea and metformin , five received sulfonylurea and pioglitazone , two received sulfonylurea and alphaglucosidase inhibitor , and four took three drugs . we analyzed the correlations between serum epa concentrations and a decrease in hemoglobin a1c in patients with dpp4 inhibitor monotherapy , dpp4 inhibitor addon therapy to sulfonylurea , to metformin , and to sulfonylurea and metformin . log epa tended to correlate with a decrease in hemoglobin a1c only in patients with dpp4 inhibitor addon therapy to metformin , although it did not reach statistical significance ( r = 0.526 , p = 0.0796 ) . there were no significant correlations between log epa and decrease in hemoglobin a1c in other treatment groups . it seems to be difficult to show these results because of the small sample size in each treatment group . in basic studies have shown that gpr120 functioned as a receptor for unstructured longchain fatty acids , and that the stimulation of gpr120 with la promoted glp1 secretion in vitro and in vivo . furthermore , gpr120 has been shown to be abundantly expressed in the intestine both in humans and mice . . however , the mechanism of glp1 secretion by 3 pufa has been less clear . in the present study , furthermore , serum dha concentrations did not show a significant association with the efficacy of dpp4 inhibitor , although they tended to positively correlate with a decrease in hemoglobin a1c . further studies are required to clarify the mechanism of 3 pufastimulated glp1 secretion . taken together with these previous studies , and the ability of dpp4 inhibitor to increase endogenous active glp1 levels , the present result that serum epa concentrations correlated with the efficacy of dpp4 inhibitor might provide indirect evidence that epa has a potential to promote glp1 secretion in humans . limitations of the present study included a small sample size and a short observation period . therefore , the direct link between serum epa concentration and glp1 or insulin concentrations could not be assessed . furthermore , because ingested fatty acids are generally absorbed within the small intestine , epa might be expected to act on gpr120 inadequately after oral administration . there are no reports of a linear relationship between epa intake and serum epa concentrations . however , serum epa concentrations positively correlated with epa intake assessed by a selfadministered diet history questionnaire in a subgroup of patients with type 2 diabetes ( n = 40 , r = 0.419 , p = 0.0066 ) . therefore , we hope that serum epa concentrations could be a marker of epa intake . to our knowledge , this is the first report of the relationship between serum epa concentrations and the efficacy of dpp4 inhibitor in patients with type 2 diabetes , and suggests new avenues for research into incretin therapies . large interventional studies are required to better assess the contributions of epa to the efficacy of dpp4 inhibitor in patients with type 2 diabetes . in conclusion , dpp4 inhibitor is effective in patients with type 2 diabetes with high serum epa concentrations ." ]

[ "pregnancy course was regular , except for maternal consumption of mebendazol at 5 weeks ' gestation to treat a pinworm infection . maternal history was negative for consumption of any known teratogenic class drug during the whole pregnancy . clinical examination at birth was normal , except for complete congenital anonychia of left middle finger ( fig.1 ) . x - ray was performed , showing an absence of the left middle finger distal phalanx ( fig.2).the rest of the nails on the fingers and toes were normal . parents were not consanguineous and have normal fingernails and toenails . the baby was discharged on the third day of life , and was found healthy and thriving at three - month follow - up visit . final diagnosis was that of a sporadic co. x - ray showing absence of left middle finger distal phalanx .", "congenital onychodystrophy ( co ) , also called as iso kikuchi syndrome , was first described by iso in 1969 1 and later by kikuchi in 1974 2 as a clinical syndrome involving dysplasia / absence of fingernails with underlying bone abnormalities . since these first observations , clinical criteria have been expanded to include a number of additional associated conditions derived from small series , case reports , and retrospective reviews over the next 30 years . co clinical criteria are the following : \n\n unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n\n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n\n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n \n unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n \n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n \n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n genetic loci responsible for the condition are still under investigation : linkage to the known keratin gene clusters on 12q12 and 17q21 has been excluded by krebsova et al . in 2000 ; 7 a putative isolated congenital nail dysplasia locus , designated ndic , has been identified on 17p13 , although the identified region harbors no genes known to be involved in skin or nail abnormalities 7 . several acquired isolated nail disorders which may represent differential diagnoses of co are presented in table1 \n 8 . differently from all these conditions , co presents as a congenital hypoplasia , dysplasia , or absence of one or more fingernails , and is typically accompanied by underlying phalanx bone disease . acquired isolated nail disorders 8 several heterogeneous multisystem pathologies may also come with ungueal abnormalities ; these are summarized in table2 \n 813 . 813 \n triangular nail lunula \n \n fingernails and toenails hypoplasia 8 \n \n absent or hypoplastic patellae \n \n elbow dysplasia , often involving posterior subluxation of the radial head \n \n iliac horns dysplasia \n \n arrested or reduced nail growth \n \n thickened and over - curved nails with absence of cuticle \n differently from the above - mentioned conditions , classic co does not come with involvement of systems other than nails and relative phalanges . kikuchi originally suggested fetal grip as a causative mechanism of nail and phalanx ischemia , leading to dysplasia or even complete resorption of such structures 14 . this theory , however , is not consistent with the recent acquisitions on developmental biology stating that limbs and bones develop early in fetal life , prior to fetus ' ability to exert a significant grip . more recently , several pathogenetic mechanisms have been proposed for this condition : \n\n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n\n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent y shape of the dysplastic distal phalanx , when present;17 \n\n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n\n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n \n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n \n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent y shape of the dysplastic distal phalanx , when present;17 \n \n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n \n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n a literature research showed no proven association between the use of mebendazole during pregnancy and co. conversely , helminth infection during pregnancy is associated with poor cognitive and gross motor outcomes in infants , so that measures to prevent helminth infection during pregnancy should be reinforced;21 anthelminthic therapy is actually recommended in infected pregnant women , and it has been associated to a decreased rate of maternal anemia and low birth weight 22 .", "nevertheless , clinical follow - up is needed in order to exclude other conditions associated with multisystem pathology ; a complete family history may also be warranted to determine sporadic or hereditary transmission of such a condition .", "" ]

[ "glycogen synthase kinase-3 ( gsk-3 ) , an evolutionarily conserved ubiquitous serine threonine kinase consisting of and isoforms , is a multifaceted protein with diverse cellular and neurophysiological functions . the main structural difference between gsk-3 and gsk-3 isoforms lies in the n- and c - terminal regions , while their sequences within the kinase domain are highly homologous . a growing body of evidence indicates that gsk-3 is pro - apoptotic and that its dysfunction may be linked to the pathophysiology of mood disorders , schizophrenia , diabetes , and various neurological / neurodegenerative diseases , among others ( for review , meijer et al . , 2004 ; , 2007 ; chiu and chuang , 2010 ; li and jope , 2010 ) . gsk-3 inhibition has attracted widespread attention as one of the critical therapeutic targets whereby lithium exerts its effects on mood stabilization , neurogenesis , neurotrophicity , neuroprotection , anti - inflammation , and others ( for review , rowe and chuang , 2004 ; rowe et al . , 2007 ; beurel et al . , 2010 ) . pharmacological inhibition or gene knockout / knockdown of this kinase mimics the anti - depressant and anti - manic effects of lithium observed in rodent models ( gould et al . , 2004 ; kaidanovich - beilin et al , 2004 , 2009 ; obrien et al . , 2004 ; rosa et al . , 2008 ; omata et al . , 2011 ) . the activities of gsk-3 are negatively regulated by phosphorylation of gsk-3 at ser21 and gsk-3 at ser9 . gsk-3 can be inhibited by lithium through direct binding to the atp - dependent magnesium - sensitive catalytic site of the enzyme ( klein and melton , 1996 ; stambolic et al . , 1996 ) , and/or indirectly through enhanced serine phosphorylation of gsk-3 isoforms by multiple mechanisms ( figure 1 ) . lithium has been shown to enhance gsk-3 serine phosphorylation by activation of protein kinase a ( pka ; jope , 1999 ; liang et al . , 2008 ) , or phosphatidylinositol 3-kinase ( pi3-kinase)-dependent akt ( chalecka - franaszek and chuang , 1999 ) and protein kinase c- ( kirshenboim et al . , 2004 it has also been reported that lithium can disrupt the -arrestin-2pp2a akt complex that dephosphorylates / inactivates akt , thereby enhancing gsk-3 serine phosphorylation ( beaulieu et al . , 2005 ) . moreover , it has been proposed that lithium can interrupt auto - regulation of gsk-3 via disinhibition of the inhibitory action of inhibitor-2 complex on protein phosphatase-1 ( pp-1 ; zhang et al . , 2003 ) . this article reviews the findings supporting the role of gsk-3 inhibition in mediating lithium s neuroprotective effects against excitotoxicity in both cultured neurons and animal models of ischemic stroke . lithium , a competitive inhibitor of magnesium , directly inhibits atp magnesium - dependent catalytic activity of gsk-3 . lithium can indirectly increase this serine phosphorylation of gsk-3 through pi3-kinase - mediated phosphorylation / activation of akt , pi3-kinase - mediated activation of pkc , and camp - dependent activation of pka . lithium can also increase the serine phosphorylation of gsk-3 by disrupting the -arrestin-2 ( arr2)pp2a akt complex that dephosphorylates and inactivates akt . in addition , by disinhibiting the inhibitory action of inhibitor-2 ( i-2 ) on protein phosphatase - l ( pp-1 ) that dephosphorylates gsk-3 at serine residues , lithium s direct inhibition of gsk-3 interrupts this auto - regulation of gsk-3 and further decreases gsk-3 activity . lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections .", "we have designed isoform - specific small interfering rnas ( sirnas ) to distinguish the functional and regulatory differences between the two gsk-3 isoforms in rat cerebral cortical neuronal cultures ( liang and chuang , 2007 ) . transfection with sirna for gsk-3 or gsk-3 or with dominant - negative mutants specific for either isoform produced almost complete protection against glutamate - induced , n - methyl - d - aspartate ( nmda ) receptor - mediated excitotoxicity . the sirna - induced neuroprotection was associated with enhanced n - terminal phosphorylation in both gsk-3 isoforms . moreover , transfection with or isoform - specific dominant - negative mutants of gsk-3 mimicked lithium - induced neuroprotection against glutamate excitotoxicity . these results strongly suggest that both gsk-3 and are involved in glutamate - induced neuronal death and that both isoforms are the initial targets of lithium - elicited neuroprotection . gsk-3 has also been implicated in neuronal development , maturation / differentiation , and aging in the mammalian cns ( spittaels et al . , 2002 ; kim et al . substrates phosphorylated by gsk-3 include metabolic , signaling , and structural proteins as well as transcription factors . it is known that inhibition of gsk-3 results in activation , and sometimes suppression , of an array of transcription factors ( for review , grimes and jope , 2001 ; jope and roh , 2006 ; chiu and chuang , 2010 ) . among the long list of transcription factors regulated by gsk-3 are cyclic amp response element binding protein ( creb ) , nuclear factor-b ( nf-b ) , activating protein-1 ( ap-1 ) , heat - shock factor-1 ( hsf-1 ) , -catenin , t - cell factor ( tcf)/lymphoid enhancer factor ( lef ) , and p53 . dysfunction of gsk-3-mediated phosphorylation of transcription factors is believed to relate with the pathophysiology of various pathological conditions ( for review , chiu and chuang , 2010 ) . we found that gsk-3 silencing activated camp response element ( cre)- and nf-b - responsive transcription more robustly than gsk-3 silencing ( liang and chuang , 2006 ) . dna array further identified two novel gsk-3-regulated transcription factors , early growth response-1 ( egr-1 ) and smad3/4 , both of which play important roles in growth , differentiation , survival , and plasticity of brain cells ( harada et al . , 2001 ; derynck and zhang , 2003 ; lee and kim , 2004 ; droguett et al . , 2010 ) . specifically , the binding activity of egr-1 was down - regulated by sirna for gsk-3 , but was up - regulated by sirna for gsk-3 ( liang and chuang , 2006 ) . by using sirnas or dominant - negative mutants specific to gsk-3 isoforms , inhibition of gsk-3 the differential roles of gsk-3 isoforms are further supported by the opposite effects of gsk-3 and sirnas on the protein levels of plasminogen activator inhibitor type-1 ( pai-1 ) , a smad3/4-regulated gene product . these results demonstrate that selective silencing or inhibition of the two gsk-3 isoforms could produce different and sometimes opposite effects on the regulation of certain transcription factors including novel gsk-3 targets ( liang and chuang , 2006 ) . for example , the disruption of gsk-3 in mice is embryonic lethal , despite the normal expression of gsk-3 , indicating that the presence of isoform can not compensate for the loss of isoform ( hoeflich et al . , 2000 ) . transfection and sirna studies suggested that gsk-3 inhibition decreased the processing of -amyloid ( a ) precursor protein to form a140 and a142 , while gsk-3 appeared to have a lesser role ( phiel et al . , 2003 ) . in addition , gsk-3 , but not gsk-3 , is required for interferon--induced activation of signal transducer and activator of transcription-3 ( beurel and jope , 2009 ) . together , these findings underscore important similarities and differences between the roles of gsk-3 isoforms and in cell survival as well as transcription , and suggest that the development of isoform - specific inhibitors may be essential for therapeutic intervention of gsk-3-related neuropathological conditions . we have also explored lithium s effects on smad3/4-dependent transcriptional activity and the underlying mechanisms . smad3/4 is a down - stream mediator of the signaling pathway triggered by transforming growth factor- ( tgf- ) , and plays a prominent role in regulating the expression of proteins involved in neuronal survival , differentiation , and synaptic plasticity ( for review , gomes et al . , 2005 ) . treating cultured cortical neurons with therapeutically relevant concentrations of lithium significantly decreased smad3/4-dependent transactivation and protein levels of pai-1 , a tgf--responsive smad3/4-dependent gene product ( liang et al . , 2008 ) . of particular relevance to the therapeutic efficacy of lithium , pai-1 has been implicated in the etiology and progression of neurodegenerative diseases and mood disorders ( for review , pawlak et al . , 2003 ; lithium s effects on smad3/4 likely result from cross - talk of signaling pathways between camp / pka and pi3-kinase / akt / gsk-3. we have shown that lithium - induced smad3/4 suppression involved gsk-3 inhibition through the activation of pka and cell survival factor akt followed by the phosphorylation of gsk-3 at ser9 and creb at ser133 ( liang et al . , 2008 ) . creb binding protein ( cbp ) and p300 are known to be co - activators of creb . our data further demonstrated that over - expression of p300 , but not cbp , completely antagonized lithium - induced reduction of pai-1 promoter activity . a series of experimental data support the notion that , in smad3/4 signaling , the inhibitory effects of lithium are due to complex formation of activated creb and p300 , which results in limited interactions of p300 with the transcription factors / smad complexes . this in turn prevents efficient smad3/4-dependent transcription of smad3/4-dependent genes such as pai-1 and p21 ( figure 2 ) . transcriptional activations triggered by stimulation of cell surface tgf- and bdnf receptors are mediated by smad3/4- and pi3-kinase / akt - dependent pathways , respectively . lithium treatment - induced inhibition of gsk-3 , directly and indirectly via camp - dependent activation of pka as well as bdnf - stimulated activation of pi3-kinase / akt pathways , potentiates bdnf - induced phosphorylation / activation of creb . this in turn increases cre - mediated transactivation and expression of survival factors such as bdnf and bcl-2 . enhanced gene transcription triggered by bdnf , via sequestration of transcriptional co - activator p300 , suppresses smad3/4-dependent transactivation and subsequently decreases the expression of tgf--responsive genes , pai-1 , and p21 . lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections . dashed lines represent pathways with reduced activity as a result of lithium treatment .", "lithium - induced neuroprotection against glutamate excitotoxicity was first noted in rodent primary neuronal cultures of cerebellar granule cells ( cgcs ) , cerebral cortical neurons , and hippocampal neurons ( nonaka et al . , 1998 ) . this experimental paradigm was selected because glutamate - related excitotoxicity has been implicated in many neurodegenerative diseases including stroke ( for review , chuang , 2004 ; chiu and chuang , 2010 ) . our pioneering studies have shown that glutamate - induced , nmda receptor - mediated excitotoxicity was robustly reduced by extended lithium chloride pretreatment ( 57 days ) in cultured rat cgcs and cortical neurons , partly via inhibition of nmda receptor - mediated calcium influx ( nonaka et al . , 1998 ; hashimoto et al . , 2002a ) . moreover , these effects of lithium were likely due to the attenuation of constitutive phosphorylation at tyr1472 of the nr2b subunit of nmda receptors , possibly as a result of inhibiting src tyrosine kinase ( hashimoto et al . , 2002a , 2003 ) . although glutamate - induced excitotoxicity in cultured cortical neurons was blocked by treatment with either lithium or mk-801 ( an nmda receptor antagonist ) , the src kinase inhibitor su6656 only partially diminished this toxicity ( hashimoto et al . , 2003 ) , suggesting that other components are involved . in cgcs , lithium - induced neuroprotection against glutamate excitotoxicity was associated with up - regulation of the anti - apoptotic protein bcl-2 , down - regulation of the pro - apoptotic proteins p53 and bax , and suppressed release of cytochrome c from mitochondria ( chen and chuang , 1999 ) , whereas the involvement of gsk-3 in the regulation of nmda signaling by lithium treatment is currently unclear and requires further investigations . cyclin - dependent kinase 5 ( cdk5 ) also regulates signaling mediated by nmda receptors , either directly through phosphorylation of the nr2b subunit or indirectly through phosphorylation of psd-95 ( morabito et al . , 2004 ; zhang et al . , 2008 when it binds to p25 ( the product of calpain - mediated cleavage of p35 ) , cdk5 becomes pro - apoptotic and its activity is dysregulated ( lee et al . , p25 accumulation was observed in neurons in response to glutamate or oxidative stress , and also in the brains of several animal models of neurodegenerative diseases . sustained activation of cdk5 in neurons has been implicated in many neurodegenerative diseases ( cruz and tsai , 2004 ; dhariwala and rajadhyaksha , 2008 ) . in cultured rat cgcs , lithium pretreatment prevented colchicine - induced apoptosis and associated increase in cdk5 expression and fragmentation of p35 into p25 ( jorda et al . . additionally , pretreatment with lithium also attenuated intracellular calcium increase , calpain activity , cdk5 activation , and cellular death in primary cultured hippocampal neurons and rat striatum following the treatment of 3-nitropropionic acid ( crespo - biel et al . , 2009 ) , a succinate dehydrogenase inhibitor ( for review , brouillet et al . , 1999 ) . therefore , lithium - induced inhibition of calpain and cdk5 activation may also contribute to protection against glutamate excitotoxicity . prior to changes in gene expression , lithium rapidly and transiently activated the cell survival pi3-kinase and its down - stream target , akt-1 , through phosphorylation at ser473 , thereby reversing glutamate - induced inactivation of this signaling pathway in cgcs ( chalecka - franaszek and chuang , 1999 ) . activated akt is known to affect several anti - apoptotic targets including bcl-2 associated death promoter ( bad ) , creb , members of the forkhead family , and procaspase-9 ( for review , neri et al . , 2002 ; nicholson and anderson , 2002 ; huang and reichardt , 2003 ) . in addition , lithium also triggered ser21 phosphorylation of the isoform of gsk-3 ( and hence resulted in inhibition ) , and this effect was prevented by a pi3-kinase inhibitor ( chalecka - franaszek and chuang , 1999 ) . another signaling pathway affected by lithium is the mitogen - activated protein ( map ) kinase pathway . one of the down - stream targets of map kinase is creb , a transcription factor that is involved in learning and memory , and promotes the expression of bcl-2 as well as brain - derived neurotrophic factor ( bdnf ; for review , finkbeiner , 2000 ) . in cgcs , toxic concentrations of glutamate - induced an nmda receptor - dependent decrease in creb phosphorylation at ser133 and creb - driven transcriptional activity ( kopnisky et al . , 2003 ) . concurrent with its neuroprotective effects , long - term ( but not acute ) lithium treatment suppressed glutamate - induced dephosphorylation of creb . we also found that glutamate rapidly activated c - jun - n - terminal kinase ( jnk ) and p38 kinase in cgcs , resulting in a robust increase in ap-1 binding ( chen et al . , 2003a ) . these two kinases are also activated by a variety of apoptotic insults ( for review , mielke and herdegen , 2000 ) , and ap-1 has been known to be activated by different stress factors as well . experiments using lithium and curcumin , a selective ap-1 inhibitor , suggest that nmda receptor - mediated apoptotic death requires concerted action of jnk and p38 to enhance ap-1 binding , and that lithium s neuroprotection is mediated , at least in part , by suppressing the jnk and p38 kinase pathways . as one of the major neurotrophins , bdnf is essential for cortical development , synaptic plasticity , and neural survival , and is likely a key mediator of the clinical efficacy of anti - depressants and anxiolytic drugs ( for review , woo and lu , 2006 ) . the notion that bdnf plays a key role in neuronal survival is supported by our observation that bdnf and neurotrophin-4 ( nt-4 ) , but not nt-3 , completely protected immature cgcs from apoptosis induced by cytosine arabinoside ( leeds et al . , 2005 ) . it was first reported that chronic treatment with lithium increased the expression of bdnf in the rat brain ( fukumoto et al . , 2001 ) , and we have documented that bdnf protein levels were increased in cortical neurons following lithium treatment ( hashimoto et al . , 2002b ) . we hypothesized that this bdnf up - regulation and subsequent activation of its receptor trkb might play a critical role in mediating the neuroprotective effects of lithium . in confirmation of this hypothesis , we found that lithium s neuroprotection against glutamate excitotoxicity was blocked by a trkb inhibitor , k252a , or by a neutralizing antibody against bdnf , and was mimicked by exogenous bdnf in rat cortical neurons . in addition , lithium increased intracellular levels of bdnf and this was followed by activation of trkb . furthermore , lithium - induced neuroprotection was prevented in cortical neurons from heterozygous ( + / ) or homozygous ( / ) bdnf knockout mice ( hashimoto et al . , 2002b ) . rodent bdnf has a complex genomic structure that makes it an ideal target for multiple and complex regulation . we found that treatment of rat cortical neurons with therapeutic concentrations of lithium ( e.g. , 1 mm ) caused a significant increase in the levels of bdnf exon iv - containing mrna , while levels of exon i , ii , or vi - containing mrna remained unchanged ( yasuda et al . , 2009 ) . it is known that exon iv - containing bdnf transcripts are expressed in response to kcl - induced depolarization in rat cortical neurons ( tao et al . , 2002 ) . this transcriptional activation requires utilization of the promoter region 80 bp up - stream from the transcription initiation site of exon iv - containing three calcium responsive elements ( cares ; chen et al . , 2003b ) . we generated various bdnf promoter iv deletion constructs to investigate whether lithium treatment causes an increase in bdnf promoter iv activity , and , if so , which region of promoter iv confers the sensitivity to this drug . we identified that the drug - induced up - regulation of exon iv - containing bdnf transcript was associated with a significant increase in the activity of bdnf promoter iv and total bdnf protein . to our surprise , the lithium - responsive element(s ) in promoter iv resides in a region up - stream from the cares responsible for depolarization - induced bdnf induction ( 170 to 704 bp ) . moreover , activation of bdnf promoter iv occurred in cortical neurons depolarized with kcl and depletion of these three cares failed to abolish lithium - induced activation . importantly , we found that lithium - induced activation of promoter iv was mimicked by pharmacological inhibitors of gsk-3 ( sb216763 , sb415286 , inhibitor i , and inhibitor vii ) or by transfection with specific sirna for gsk-3 or gsk-3. additionally , their dominant - negative mutants also mimicked lithium - induced activation of promoter iv . these results demonstrate that gsk-3 is the initial target of lithium to selectively activate bdnf promoter iv and that bdnf induction by lithium involves a novel responsive region in promoter iv of the bdnf gene . lithium - induced , gsk-3-dependent bdnf promoter iv activation could be a part of the molecular mechanisms underlying its neuroprotective effects and as such , possibly accounts for the therapeutic actions in bipolar patients . it should be noted that in addition to lithium , other gsk-3 inhibitors have been shown to almost completely block glutamate - induced excitotoxicity in rat cortical neuronal cultures ( liang and chuang , 2007 ) . these include atp - competitive inhibitors , sb216763 and sb415286 , and atp - non - competitive inhibitors , inhibitor i and vii . as mentioned in the preceding section , glutamate - induced death of cortical neurons was mitigated by silencing of gsk-3 and/or , or both isoforms , or inhibition of gsk-3 activity via transfection with dominant - negative mutants of gsk-3/ isoforms ( liang and chuang , 2007 ) . studies from other laboratories also supported the roles of gsk-3 inhibition in protecting neurons from glutamate neurotoxicity . for example , stimulation of nmda receptors in cultured rat hippocampal or cortical neurons activated gsk-3 by pp-1-mediated serine dephosphorylation of gsk-3 ( szatmari et al . , 2005 ) . treatment of primary rat cortical neurons with -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa ) , lithium or sb216763 blocked glutamate - induced caspase-3 activation and excitotoxicity , and the protective effects of ampa required pi3-kinase akt - dependent serine phosphorylation of gsk-3 ( nishimoto et al . , 2008 ) . further , in organotypic cultures of chick embryo spinal cord , lithium prevented kainate - induced excitotoxic death of motoneurons by targeting gsk-3 , and this neuroprotection was associated with cytopathological changes ( caldero et al . , 2010 ) .", "stroke is the third leading cause of death in the united states and a major global cause of serious long - term disability in adults . ischemic strokes represent approximately 87% of all cases , while the rest are hemorrhagic strokes ( roger et al . , 2011 ) . in addition to physical deficits , stroke victims also suffer from vascular depression and dementia , both of which are difficult to treat with conventional medicine . it is becoming clear that there is a substantial increase in extracellular glutamate in the brain following cerebral ischemia , and that a significant portion of ischemia - induced brain damage is mediated by over - stimulation of nmda receptors . shortly after ischemia , the interruption of cerebral blood flow depletes oxygen and glucose and subsequently prevents atp production . inadequate atp supply will cause the malfunction of atp - dependent ion pumps and alter the ion concentration gradient across the neuronal membranes . the resulting failure to transport glutamate leads to an accumulation of glutamate in the extracellular space and over - stimulates nmda receptors , which leads to a toxic influx of calcium and in turn drives the activation of damaging calcium - mediated intracellular enzymes . this cascade of events ultimately results in mitochondrial failure , production of reactive oxygen species , neuroinflammation , and cell necrosis and apoptosis ( allen and bayraktutan , 2009 ; deb et al . , 2010 ) . gsk-3 has been strongly implicated in the neuronal cell death caused by cerebral ischemic insult . one study in rats subjected to transient middle cerebral artery occlusion ( mcao ) demonstrated a rapid increase in the expression of cytoplasmic and nuclear gsk-3 protein in ipsilateral lamina i , ii , v , and vi in young rat brains , whereas in lamina v and vi in old rat brains ( sasaki et al . although the phosphorylation status of gsk-3 was not mentioned , these findings implicate a role of gsk-3 in cerebral ischemic injury . it is well known that gsk-3 can be phosphorylated at serine and tyrosine residues in which ser9 phosphorylation renders it inactive , while tyr216 phosphorylation is necessary for its functional activity ( hughes et al . , 1993 ) . discrepancies exist in the literature regarding changes of ser9 and tyr216 phosphorylation levels following cerebral ischemia . the phosphorylation levels of gsk-3 at ser9 and akt at ser473 were reported to be markedly enhanced in the vulnerable hippocampal ca1 region , but not in the ischemia - resistant ca3 region in rats subjected to transient global cerebral ischemia , while there was no change in levels of tyr216 phosphorylation or total gsk-3 ( endo et al . , 2006 ) . levels of gsk-3 ser9 phosphorylation were also increased shortly after permanent focal cerebral ischemia and decreased to basal levels or even lower 24 h after ischemic onset ( sasaki et al . , 2006 ; gao et al . , 2008 however , it has also been reported that transient focal cerebral ischemia in rats caused an increase in gsk-3 tyr216 phosphorylation in degenerating cortical neurons with no alteration in ser9 phosphorylation ( bhat et al . , 2000 ) . this discrepancy may stem , in part , from the difference of ischemic severity and ischemic models across various studies . it appears that transient focal cerebral ischemia tends to activate gsk-3 and subsequently to induce apoptotic cell death . in contrast , gsk-3 is inactivated shortly after permanent focal cerebral ischemia or global cerebral ischemia , which in turn may promote survival of vulnerable neurons . ischemia both gsk-3 and gsk-3 mediated the expression of a lethal protein , neuronal pentraxin 1 ( russell et al . , 2011 ) . in light of these findings the beneficial effects of lithium in rodent cerebral ischemic models demonstrated by us and others support this notion . in an initial study , long - term lithium pretreatment at therapeutically relevant doses decreased brain infarct volume , reduced apoptotic cell death and improved behavioral performance after permanent cerebral ischemia - induced by mcao ( nonaka and chuang , 1998 ; xu et al . , 2003 ) . in a subsequent study , we demonstrated that subcutaneous injection of rats with lithium at therapeutic doses ( e.g. , 0.5 and 1.0 meq / kg ) after the onset of transient mcao markedly decreased infarct volume , reduced tunel - positive dna damage , and suppressed neurological deficits measured by sensory , motor , and reflex tests ( ren et al . , 2003 ) . the time window for these beneficial effects was at least 3 h after the onset of ischemia . heat - shock protein 70 ( hsp70 ) , a well - established cytoprotective factor against apoptosis , was induced in the ischemic penumbra where neuronal recovery takes place . post - insult treatment with lithium increased the dna binding activity of hsf-1 to the heat - shock element , superinducing hsp70 which inhibits brain ischemia - induced apoptosis ( ren et al . , 2003 ) . lithium - elicited gsk-3 inhibition is likely associated with hsf-1 activation and hsp70 induction ( bijur and jope , 2000 ) . notably , post - insult lithium treatment mitigated apoptosis and brain damage by preventing gsk-3 and erk dephosphorylation , suppressing calpain and caspase-3 activation , and inhibiting mitochondrial release of cytochrome c and apoptosis - inducing factor in a neonatal hypoxic these findings suggest that lithium - induced gsk-3 inhibition contributes to its anti - apoptotic effects under ischemic conditions . in addition , it was found that lithium pretreatment largely suppressed ischemia - induced exploratory behavioral changes and memory impairments in gerbils after global cerebral ischemia ( bian et al . , 2007 ) . these behavioral benefits were associated with an increase in the number of viable cells and a decrease in apoptotic cells in the ca1 hippocampal area of ischemic gerbils . moreover , lithium - induced neuroprotection in the ischemic brain was accompanied by down - regulation of pro - apoptotic p53 in the ca1 , and up - regulation of anti - apoptotic bcl-2 and hsp70 , both of which are targets of gsk-3 . it is likely that lithium protection against ischemia - induced injury involves multiple mechanisms . in the rat hippocampus , lithium was reported to inhibit ischemia - induced nmda receptor hyperactivation by inhibiting nmda subunit 2a tyrosine phosphorylation and its interactions with src and fyn through psd-95 ( ma and zhang , 2003 ) . lithium also attenuated hypoxia - induced serine dephosphorylation of gsk-3 and in the mouse brain ( roh et al . , 2005 ) . additionally , in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation , lithium showed neuroprotection in conjunction with hsp27 activation ( cimarosti et al . , 2001 ) . post - ischemic inflammation is a dynamic process involving a complicated set of interactions between inflammatory cells and molecules ( iadecola and alexander , 2001 ) . a recent study documented the anti - inflammatory effects of lithium in a neonatal rat hypoxic ischemic model . post - insult lithium treatment significantly reduced total tissue loss following hypoxia ischemia , and this beneficial effect of lithium was associated with inhibiting microglia activation and attenuating levels of pro - inflammatory cytokines or chemokines , such as interleukin-1 and chemokine ligand 2 ( li et al . , 2011 ) . hsp70 over - expression can inactivate the key inflammatory transcription factor nf-b by stabilizing the nf-b - ib complex , and thereby preventing nuclear translocation of activated nf-b subunits in a mouse mcao model ( zheng et al . , 2008 ) . besides anti - inflammation , lithium also increased proliferation and differentiation of hippocampal neural progenitor cells in both non - ischemic and ischemic brains without altering the relative levels of neuronal and astrocytic differentiation , and this effect lasted at least 7 weeks after hypoxia ischemia in neonatal rats ( li et al . , in line with this finding , chronic lithium pretreatment was found to increase the generation and survival of newborn cells in the hippocampal dentate gyrus , and did not affect the neuronal or astrocytic differentiation of these newborn cells in a transient four - vessel occlusion model ( yan et al . erk1/2 phosphorylation following ischemia was enhanced by lithium treatment , while erk1/2 inhibitor u0126 prevented the effects of lithium in increasing brdu - positive cells and improving spatial learning and memory ( yan et al . , 2007 ) . in fact , chronic lithium treatment has been demonstrated to increase activity in the mek / erk pathway in vivo , and lithium s neuroprotection has been suggested to depend on the induction of this signaling pathway ( einat et al . in addition , it was reported that activation of erk associates with and phosphorylates gsk-3 at the thr43 residue , which primes this kinase for its subsequent phosphorylation at ser9 by p90rsk , resulting in inactivation of gsk-3 and up - regulation of -catenin ( ding et al . , 2005 ) . therefore , lithium might affect gsk-3 phosphorylation through the mek / erk pathway , which in turn inhibits this kinase via rsk . gsk-3 is also negatively regulated by the wnt/-catenin signaling pathway and , accordingly , activating the canonical wnt pathway has been shown to contribute to adult hippocampal neural progenitor cell proliferation triggered by lithium treatment ( wexler et al . , 2008 ) . in a collaborative study , the neurohemodynamic aspects of recovery induced by delayed chronic lithium treatment were assessed using functional magnetic resonance imaging ( mri ; kim et al . , 2008 ) . rats were subjected to transient mcao and then injected with lithium ( licl , 1 meq / kg , s.c . ) this delayed lithium injection was followed by daily injections , and on day 15 , an mri scan was performed to monitor changes in blood oxygen level dependence ( bold ) and functional cerebral blood volume ( fcbv ) responses using electric stimulation of forelimbs . the mean activated volume ratio and total activation magnitude ratio between ipsilateral and contralateral cortices for both bold and fcbv were significantly higher in the lithium - treated than in the saline - treated rats . the lithium - induced increase in fcbv in the peri - infarct regions suggests a possible vascular transformation . indeed , the size and distribution of immunohistochemical staining of cd31 , a microvasculature marker , were enhanced by lithium treatment in the peri - infarct regions . co - localized with cd31 , the tissue staining of matrix metalloproteinase-9 ( mmp-9 ) was also much more pronounced following lithium treatment , suggesting mmp-9-dependent neurovascular remodeling in the recovering brain area . moreover , treatment of cultured rat brain endothelial cells with lithium in a follow - up study was also found to increase the protein levels of vascular endothelial growth factor ( vegf ) via a mechanism involving the pi3-kinase and gsk-3 signaling pathways ( guo et al . , since vegf has been linked to angiogenesis , neurogenesis , and neuroprotection ( for review , fan and yang , 2007 ) , vegf over - expression may contribute to lithium s ability to promote neurovascular remodeling and to induce functional recovery after ischemic stroke . ample evidence supports the therapeutic potential of mesenchymal stem cells ( mscs ) in several human diseases including stroke . however , it is increasingly recognized that the effectiveness of msc transplantation is limited by their poor migration toward disease target sites such as ischemic brain regions . in a recent study , we investigated whether treatment of mscs with lithium and another mood stabilizing drug , valproic acid ( vpa ) , would enhance cell migration ( tsai et al . , 2010 ) . we found that treatment of mscs with lithium ( 2.5 mm for 1 day ) selectively elevated the transcript and protein levels of mmp-9 and its enzymatic activity . these effects were mimicked by pharmacological inhibition or gene silencing of gsk-3. lithium treatment also potentiated stromal cell - derived factor-1 ( sdf-1)-dependent msc migration across the extracellular matrix , which was suppressed by two mmp-9 inhibitors , doxycycline and gm6001 . short - term ( 3 h ) exposure of mscs to a relatively high concentration ( 2.5 mm ) of vpa markedly increased the transcript and protein levels of cxc chemokine receptor 4 ( cxcr4 ) . vpa - induced cxcr4 expression required its ability to inhibit histone deacetylases ( hdacs ) , including the hdac1 isoform , and involved histone hyperacetylation at the cxcr4 gene promoter . vpa treatment enhanced sdf-1-mediated msc migration , which was completely blocked by amd3100 , a cxcr4 antagonist . notably , combining lithium and vpa treatment further increased msc migration , and the additive enhancement of migration was completely blocked by the co - presence of amd3100 and gm6001 . our results suggest that lithium and vpa stimulate msc migration through distinct targets and mediators : gsk-3mmp-9 and hdac cxcr4 , respectively ( tsai et al . , 2010 ) . in a follow - up in vivo study , mscs were primed with lithium and/or vpa and then injected into the tail vein of transient mcao rats 24 h after ischemic onset . priming with lithium or vpa increased the number of mscs homing to the cerebral infarcted regions such as the cortex and striatum 2 weeks after transplantation , and co - priming with lithium and vpa further enhanced this migratory effect ( tsai et al . , 2011 ) . mcao rats receiving lithium- and/or vpa - primed mscs showed improved functional recovery , reduced infarct volume , and enhanced angiogenesis in the infarcted penumbra regions . these beneficial effects of lithium and vpa priming were reversed by pharmacological inhibition of mmp-9 and cxcr4 , respectively , suggesting that these effects were likely mediated by lithium - induced mmp-9 up - regulation and vpa - induced cxcr4 over - expression . together , these findings raise the potential utility of using mscs primed with inhibitors of gsk-3 and hdac to enhance the migration and homing capacity for transplantation into stroke victims . in addition to lithium , other pharmacological gsk-3 inhibitors have been shown to exert neuroprotective effects against cerebral ischemia by various groups . a specific gsk-3 inhibitor , chir025 , was demonstrated to protect cultured hippocampal neurons from glutamate excitotoxicity and to attenuate death of cortical neurons following oxygen glucose deprivation , an in vitro model of cerebral ischemia ( kelly et al . , 2004 ) . moreover , chir025 reduced infarct size in focal cerebral ischemic rats , but did not affect tunel - positive neurons or caspase-3/9 activities , although bcl-2 expression was increased . gsk-3 enzymatic activity was markedly elevated after transient mcao in rats , and this gsk-3 activation was blocked by jugular vein injection of gsk-3 inhibitor viii ( koh et al . , 2008 ) . pre- or post- ( up to 2 h ) mcao injection with inhibitor viii also reduced blood glucose levels , infarct size , caspase-3 activity , and water content in the ipsilateral brain hemisphere . furthermore , ischemia - induced inflammation - related signals such as cox-2 over - expression and neutrophil infiltration were alleviated by this gsk-3 inhibitor . prophylactic or therapeutic administration of a gsk-3 inhibitor tdzd-8 reduced infarct volume and cerebral injury in the rat hippocampus after transient ischemia ( collino et al . , 2008 ) . this was accompanied by suppression of ischemia - induced oxidative stress , apoptosis , and neuroinflammation . delayed treatment with compound i , a gsk-3 and cdk inhibitor , decreased tunel - positive cells in the ipsilateral hippocampus and striatum of adult ( but not juvenile ) mice subjected to hypoxic these neuroprotective effects of compound i were associated with long - lasting functional recovery . finally , gsk-3 inhibition by sb216763 counteracted oxygen glucose deprivation - induced mitochondrial biogenesis impairment and reduced mitochondrial reactive oxygen species generation in primary cortical neurons ( valerio et al . , 2011 ) . when systematically administrated to permanent mcao mice , sb216763 decreased infarct volume and restored the loss of mitochondrial dna , thus supporting a novel role of gsk-3 inhibitors in stimulating the renewal of functional mitochondria following ischemic stroke .", "a growing body of evidence supports that lithium , a mood stabilizer used to treat bipolar disorder , has neuroprotective properties in both cellular and in vivo experimental settings . one of the major targets of lithium is gsk-3 , a serine / threonine kinase implicated in the pathogenesis of diverse cns disorders . lithium inhibits gsk-3 activity by direct binding to the enzyme or indirectly by enhancing serine phosphorylation of both and isoforms through multiple mechanisms . lithium has been used as a prototype drug to seek evidence for the involvement of gsk-3 inhibition in lithium - induced protection against excitotoxicity in cultured neurons and animal models of cerebral ischemic stroke . lithium at therapeutically relevant concentrations robustly protected primary brain neurons from glutamate - induced , nmda receptor - mediated excitotoxicity . the neuroprotective effects of lithium were associated with gsk-3 inhibition , and were mimicked by other pharmacological gsk-3 inhibitors , by silencing gsk-3 and/or isoforms , or by expression of isoform - specific dominant - negative mutants . these results support the roles of gsk-3 inhibition in lithium - elicited protection against excitotoxicity . lithium rapidly activated the cell survival pi3-kinase akt signaling pathway to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation as well as apoptosis . lithium also caused an increase in the expression of cytoprotective bcl-2 and suppressed glutamate - induced up - regulation of pro - apoptotic p53 and bax , resulting in blocking cytochrome c release from mitochondria . bdnf promoter iv was selectively activated by gsk-3 inhibition using lithium or other drugs or through gene silencing / inactivation of either isoform . this effect on however , there is a gap in the understanding of how gsk-3 inhibition causes an increase in bdnf promoter activity . in addition , lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and suppression of p38/jnk and ap-1 activation , thus reducing apoptosis . this effect appears to stem from inhibition of src / fyn kinase to suppress nr2b tyr1472 phosphorylation of the receptor . it remains to be explored as to whether this lithium - induced action on nmda receptors is related to gsk-3 inhibition . the potential roles of these other targets in mediating the neuroprotective effects of this drug also deserve future investigation . it is well known that glutamate overflow and nmda receptor hyper - stimulation are early events following cerebral ischemia . in rodent ischemic models , pre- or post - insult treatment with therapeutic doses of lithium decreased infarct volume , caspase-3 activity and apoptotic cells in the injured brain . the beneficial time window of lithium is at least 3 h after the ischemic onset . up - regulation of hsp70 and bcl-2 as well as down - regulation of p53 likely contributed to the protective effects of lithium in the ischemic conditions , thus supporting similar underlying neuroprotective mechanisms in the excitotoxic cellular models and animal models of ischemic stroke . limited data suggested that lithium might also display anti - inflammatory effects by inhibiting ischemia - induced microglia activation and pro - inflammatory factors release . delayed and chronic injections of lithium improved functional mri responses such as increases in bold and fcbv . indeed , lithium was found to induce two pro - angiogenic factors , mmp-9 and vegf in a gsk-3-dependent manner . lithium has also been reported to stimulate erk1/2 activity and to enhance proliferation of hippocampal neural progenitor cells and memory performance after ischemia . finally , lithium promoted migration of mscs in vitro by up - regulation of mmp-9 through gsk-3 inhibition and this migratory effect was potentiated by co - treatment with vpa , another mood stabilizer . notably , transplantation of lithium vpa co - primed mscs into ischemic rats markedly increased msc migration to the injured brain regions , decreased infarct size and improved the neurological performance . lithium - induced stem cell migration , neurogenesis , and angiogenesis all likely contribute to functional recovery . figure 3 illustrates proposed molecular events leading to lithium - induced beneficial effects following cerebral ischemia . it should be noted that several other gsk-3 inhibitors have also been reported to exert beneficial effects in rodent ischemic models and their actions were accompanied by suppression of ischemia - increased gsk-3 activity . accordingly , gsk-3 inhibitors have therapeutic potential to treat stroke and other excitotoxicity - related neurodegenerative diseases . lithium has been used in bipolar patients over 60 years and its clinical profiles are well understood . therefore , lithium is a prime candidate for use in clinical trials of new therapies for stroke victims . the neuroprotective effects of lithium against cerebral ischemia are proposed to result from its interactions with cell survival and apoptotic machinery . a significant portion of brain damage following cerebral ischemia is caused by an increase in extracellular glutamate and subsequent over - stimulation of nmda receptor - mediated toxic increase in intracellular calcium . this signaling pathway plays a critical role in mediating glutamate - induced caspase activation and apoptosis . lithium at therapeutically relevant concentrations inhibits nmda receptor - mediated calcium influx , which in turn decreases subsequent activation of jnk , p38 kinase , and transcription factor ap-1 . inhibition of intracellular calcium increase also attenuates the activity of calpain and calpain - mediated activation of pro - apoptotic cdk5/p25 kinase . on the other hand , lithium can directly and indirectly reduce the activity of constitutively activated gsk-3 by multiple mechanisms , leading to disinhibition of several transcription factors , such as creb and hsf-1 , and resulting in induction of major cytoprotective proteins such as bdnf , vegf , mmp-9 , hsp70 , and bcl-2 . a decrease in gsk-3 activity further reduces the activity of pro - apoptotic protein p53 and its downregulating effect on bcl-2 . bdnf , via activating its cell surface receptor and the down - stream erk and pi3-kinase / akt pathways , induces neuroprotective effects in part by inhibiting gsk-3 and stimulating creb . induction of bdnf is an early and essential step for neuroprotection and is involved in lithium - induced neurogenesis . in addition , superinduction of hsp70 by lithium treatment not only inhibits brain ischemia - induced apoptosis , but also contributes to the anti - inflammatory effects of lithium through inactivation of nf-b . counteraction of gsk-3 inhibition of vegf and mmp-9 by lithium enhances angiogenesis and neurovascular remodeling . taken together , these effects of lithium in reducing apoptosis , suppressing inflammation , enhancing angiogenesis and neurogenesis , contribute to behavioral improvement and functional recovery after ischemia . lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ]

[ "studies have shown the incidence of injury from squash to range from 35.5 to 80.9 per 100,000 players . trauma to the head ( including the eyes ) is the most common cause of squas - related hospital presentation , comprising 48.7% of all emergency department presentations . craniofacial and spinal fractures typically result from high - energy blunt force to the skeleton , as seen in motor vehicle accidents . this is the first case reported of a patient sustaining such extensive head , neck and spinal injuries from a squash - related injury .", "a 55-year - old man was brought in by ambulance after colliding with the corner of the wall and floor of a squash court , without the use of protective eye - glasses or mouth - guard . he was chasing a ball toward the corner of the court when he fell and hit the junction of the wall and floor with his head . on examination in the emergency department , the patient had a glasgow coma scale ( gcs ) of 14 , with confusion to time and place . initially , the patient reported diplopia at primary position but examination by the surgical team elicited normal eye movements with nil complaint of diplopia . there was significant boggyness to palpation of the forehead with a palpable defect of the frontal bone . the cervical spine was slightly tender over / c1 - 2 and c5 - 6 . upper limb examination was limited by pain maximal over the distal radii but bilateral arm weakness was noted with nil sensory dysfunction . primary and secondary surveys did not elicit any further findings . a non - contrast computed tomography ( ct ) imaging of the facial bones [ figure 1 ] showed a le fort type ii fracture , with comminuted fractures of the roofs of the orbit bilaterally and a depressed fracture of the left orbital floor . there was also fracture of the right maxilla at the junction of the maxilla and zygomatic arch , extending towards but not involve the right orbital floor . on the left , there was fracture of the anterior part of the left zygomatic arch which extended into the anterolateral wall of the left maxillary sinus . a large , depressed fracture of the frontal bone was also evident , extending superiorly from the superior lateral wall of the right orbit and extending across the frontal bone and inferiorly toward the mid - portion of the roof of the left orbit . the fracture involved the inner and outer tables of the frontal bone and was depressed by at least 3.5 mm . there was also a comminuted fracture of the nasal bone , which was not significantly displaced . anterior and anterolateral views of three - dimensional ct reconstructions demonstrating lefort type ii , frontal , orbital and zygomatic fractures ct brain showed tiny subarachnoid or intraparenchymal haemorrhages but there was no evidence of extensive intra- or extra - axial haemorrhage . imaging of the cervical spine [ figure 2 ] showed an undisplaced fracture of the posterior arch of c1 on the right side and a type 3 dens fracture with posterior displacement of 4 mm but no contact with the spinal cord . anterolateral view 3 dimensional ct reconstruction demonstrating type 3 dens fracture with posterior displacement and fracture of c7 spinous process subsequent ct of the thoracic spine showed a comminuted t6 vertebral body fracture with loss of vertebral height and extension into the left costovertebral joint . there was a small bony fragment projecting posteriorly into the spinal canal and making contact with the cord , but there was nil evidence of compression . associated with this finding were two to three hyperdense foci within the spinal cord likely to represent small contusions . the above findings were confirmed on magnetic resonance imaging ( mri ) of the spine , which also demonstrated a small cortical buckle at the t6 level without significant cord impingement . the patient underwent craniotomy , obliteration of the frontal sinus and open reduction internal fixation ( orif ) of the frontal bone . orif of the maxilla bilaterally , orif of the infra - orbital rim and supra - orbital rim bilaterally and inter - maxillary screw fixation was also performed . the patient was admitted to the neurosurgical intensive care unit post - operatively and underwent cervical fusion with odontoid screw fixation of the c2 fracture one week after surgical correction of the head injuries . post - operative ct scans demonstrated complete bony union in the c2 fracture site [ figure 3 ] and uncomplicated fixation of the facial bone fractures [ figure 4 ] . post - operative lateral x - ray of the cervical spine in extension demonstrating union of dens fracture with overall alignment in - tact post - operative coronal ct demonstrating surgical correction of multiple craniofacial fracture sites", "our patient suffered injuries to multiple sites in the cranium , facial bones and the vertebrae that have previously not reported in the literature . the thinnest and weakest segment of the orbit is the floor , which is the most common site of fracture . our patient suffered a le fort type ii fracture , which is pyramid shaped and passes through the posterior alveolar ridge , lateral walls of maxillary sinuses , inferior orbital rim and nasal bones . he also suffered a depressed frontal bone fracture involving the inner and outer tables , an extremely rare complication of sport seen in only 1.3% of sports related craniofacial fractures . spinal injury is associated with sports in approximately 8.7% of cases , usually in relation to contact sports such as wrestling . our patient had a fracture of the posterior arch of c1 on the right side and a type 3 dens fracture , which required cervical fusion . there was also fracture of the c7 cervical process , from forceful flexion of the cervical spine .", "we report the first case of extensive head and spinal injury resulting from collision with the wall of a squash court . the injuries ranged from the frontal bone to the thoracic vertebra and are usually seen in high velocity trauma ." ]

[ "lung cancer is a leading cause of cancer mortality in the usa and other developed countries.1 the two major forms of the disease are non - small cell lung cancer and small cell lung cancer , which account for 85% and 15% of all lung cancers , respectively.2 despite advances in preliminary detection and standard treatment with combination chemotherapy and radiotherapy , the prognosis for patients with lung cancer has not improved significantly in the last 20 years,3,4 and survival rates have changed little over the past 2 decades . currently , an enormous amount of research is aimed at understanding the molecular and cellular biology of lung cancer ; however , much more work is needed to better understand the correlation between gene regulation and lung cancer . growing evidence shows that aberrant hypermethylation in 5-cpg islands in the promoter regions is a major mechanism for silencing tumor suppressor or other cancer - associated genes in many kinds of human cancer.58 loss of function in cancer suppressor genes may hinder inhibition of growth of cancer cells , which leads to malignant transcription and translation during replication of dna . a number of genes , including the cyclin - dependent kinase inhibitor ( p16 ) , the tumor suppressor gene ras association domain family protein 1a , kelch - like ech - associating protein 1 , the dna repair gene mgmt , and the cystic fibrosis transmembrane conductance regulator are demonstratively methylated in lung cancer.912 cdh1 , a member of the transmembrane glycoprotein family , also known as cadherin-1 , cam 120/80 , epithelial cadherin ( e - cadherin ) , or uvomorulin , is encoded by the cdh1 gene ( 16q22.1).13 cdh1 is a calcium - dependent cell - cell adhesion glycoprotein containing three domains , ie , five extracellular cadherin repeats , a transmembrane region , and a highly conserved cytoplasmic tail.14 \n cdh1 as a tumor suppressor gene plays an essential role in maintaining cell adhesion and adherent junctions in normal tissues . cdh1 expression is frequently absent in a variety of epithelial tumors , and loss of normal intercellular junctions results in promotion of cancer invasion and metastasis and is correlated with several types of cancers.1517 however , the association between and clinicopathological significance of cdh1 promoter hypermethylation and lung cancer remains unclear . in this study , we systematically investigate studies of cdh1 promoter hypermethylation and lung cancer , and validate the association between cdh1 promoter hypermethylation and lung cancer using meta - analysis methods . in addition , we summarize these findings and discuss tumor suppressor function , as well as the clinicopathological significance of cdh1 in lung cancer .", "a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions . the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] . the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data . titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria . details and additional information were identified and collected from the full text of these articles . a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data . studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers , two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls . methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19 the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) . pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer . cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters . otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) .", "a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions . the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] . the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data . titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria . details and additional information were identified and collected from the full text of these articles .", "a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data . studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers , only the most recent and complete paper was selected to avoid overlap .", "two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls . methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19", "the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) . pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer . cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters . otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) .", "the process followed to select the papers used in this report is shown in figure 1 . ninety papers were identified by electronic database searching and 20 further papers by manual searching . eighty - five papers were excluded for being duplicate publications , being an irrelevant title or abstract , or being a relevant title and abstract but not published in the english language . after retrieval of the full - text articles , eleven papers were excluded for not having a large enough study population or for being reviews . thirteen further studies were screened out ( involving 866 cases and 757 controls ) based on the inclusion and exclusion criteria in the pooled analysis . the characteristics of the studies are shown in table 1.2436 six papers were used to study the correlation between cdh1 methylation and sex status . five papers provided smoking history data , allowing an investigation of the influence of smoking on cdh1 methylation . three studies investigated the effect of clinical stage , ie , stage i , ii , iii , or iv . one paper discussed lymph node metastasis and another discussed degree of differentiation . analyzing tissue samples from 657 patients and 593 controls , the mean frequency of cdh1 methylation was 32% ( range 8.33% to 66.32% ) in tumor tissue and 9% ( range 0.00%27.78% ) in tissue from controls . this result indicates that the occurrence of cdh1 methylation is higher in tumor tissue than in normal tissue . using the fixed model , meta - analysis showed that 657 cases and 593 controls from 12 studies were pooled or as shown in figure 2 ( or 3.89 , 95% ci 2.875.27 , p<0.00001 ) . these findings indicate that cdh1 methylation is a key molecular event in tumor tissue but not in normal tissue . the results also show heterogeneity across the included studies ( i \n 2 is 61% , ie , more than 50% ) . given this significant heterogeneity , a subgroup analysis was performed to investigate sex status , smoking history , pathological type , clinical stage , differentiation degree , and lymph node metastasis to observe the relationship between cdh1 methylation and clinical characteristics ( see figure 3 ) . however , there was no correlation between cdh1 promoter methylation and any of these factors ( sex status , or 0.78 , 95% ci 0.411.50 , p=0.46 ; smoking history , or 0.97 , 95% ci 0.531.79 , p=0.93 ; pathological type , or 0.97 , 95% ci 0.591.60 , p=0.91 ; clinical stage , or 1.48 , 95% ci 0.812.68 , p=0.2 ; lymph node metastasis , or 0.68 , 95% ci 0.133.63 , p=0.65 ; and differentiation degree , or 1.01 , 95% ci 0.343.02 , p=0.99 ) . the or ranged from 0.78 to 3.89 , which was not a significant change , suggesting that the results of our meta - analysis were not significantly unstable . the funnel plot shown in figure 4 is partially symmetric , indicating low publication bias regarding cdh1 methylation in lung cancer .", "hypermethylation of tumor suppressor genes and hypomethylation of oncogenes are two essential components of the molecular mechanism involved in the epigenomic regulation of initiation and progression of cancer . as a tumor suppressor gene , cdh1 maintains cell - cell adhesion and keeps epithelial cells arranged in normal arrangement and layer . in vitro studies demonstrate that loss of expression or function of cdh1 can activate transcription factors associated with epithelial - mesenchymal transition , leading to metastasis of cancer cells.37 \n cdh1 methylation has been detected in several types of carcinoma , including breast cancer , gastric cancer , and lung cancer.3840 a comprehensive evaluation of markers of methylation in lung cancer is needed to better understand the relationship between cdh1 methylation and lung cancer . although a large number of studies have demonstrated a possible relationship between cdh1 methylation and lung cancer , a meta - analysis can summarize the relevant studies and compare different subgroup characteristics . in this meta - analysis we analyzed data from 13 studies that included 657 tumor tissue samples and 593 control samples . the results show that the cdh1 methylation level in the cancer group was significantly higher than in the control group . the pooled or using the fixed - effect model was 3.89 ( 95% ci 2.875.27 versus the control group ) . cdh1 methylation plays a key role in the induction of lung cancer due to silencing of the tumor suppressor gene cdh1 . this conclusion is consistent with that of a previous study.36 since changes in cdh1 promoter hypermethylation are reversible , drug treatment promoting demethylation may be useful for delaying carcinogenesis and progression . treatment with 5-aza-2-deoxycytidine showed that migration of a549 cells decreased markedly upon restoration of cdh1.41 these preclinical studies show the therapeutic potential of restoration of tumor suppressor expression via epigenetic modulation . we further determined the clinicopathological significance of cdh1 promoter hypermethylation in patients with lung cancer . for smoking history , the summary or was 0.97 ( 95% ci 0.531.79 ) in the 65 cases and 27 controls . smoking may target other specific genes for methylation or mutation , for example , methylenetetrahydrofolate reductase.33 other subgroup meta - analysis was performed , including for sex status ( or 0.78 , 95% ci 0.411.50 ) , pathological type ( or 0.97 , 95% ci 0.591.60 ) , clinical stage ( or 1.48 , 95% ci 0.812.68 ) , lymph node metastasis ( or 0.68 , 95% ci 0.133.63 ) , and degree of differentiation ( or 1.01 , 95% ci 0.343.02 ) . cdh1 methylation determined by clinical staging shows a slightly more significant association than the other subgroups . interestingly , cdh1 methylation was detected much more frequently in stages iii and iv than in stages i and ii . however , cdh1 methylation itself does not correlate with pathological type , sex status , lymph node metastasis , or degree of differentiation . possible reasons for this finding might be the widely heterogeneous results for the subgroups or the lack of cases and controls in the subgroups . other potentially significant factors may need to be investigated , such as patient age , tumor size , and biopsy sample control.42", "cdh1 methylation might be a biomarker of lung cancer , with potential value in predicting the prognosis of the disease , and warrants further studies involving more clinical cases for meta - analysis in the future . in addition , the potential variables on cdh1 methylation from different group database are still not clear due to the limitation of the statistical power of meta - analysis ." ]

[ "nerve conduction studies ( ncs ) are an objective , quantitative , and reproducible measure of peripheral nerve function and are widely used in the diagnosis of neuropathies [ 1 , 2 ] . they can be used to monitor neuropathic disease progression and the efficacy of interventions in clinical trials . however , non - uniform electrophysiologic test procedures degrade reproducibility . potential sources of variation include the use of different emg instruments at different test sessions or sites , inconsistent placement of recording and stimulating electrodes , use of non - standardized distance measurements , use of sub - maximal electrical stimuli , poor skin preparation resulting in high skin impedance , and failure to maintain limb temperature within an acceptable range or to compensate for temperature . all these factors may compromise the repeatability of ncs measurement and lead to erroneous diagnostic conclusions . aside from true physiological changes , factors that influence repeatability of ncs measurements are broadly grouped into two categories : inter - tester variability and intra - tester variability . inter - tester variability refers to variability of a test parameter measured on a single individual when repeat test measurements are made by two or more examiners . intra - tester variability refers to variability of a test parameter when repeat test measurements are made by a single examiner . in the absence of physiological changes , both inter - tester and intra - tester variability are influenced by electrodiagnostic examination technique . automated ncs instruments may improve ncs repeatability by utilizing prefabricated electrode arrays and automating evoked waveform analysis . the objective of this study is to quantify ncs repeatability utilizing the nc - stat ( neurometrix , inc . , waltham , massachusetts ) , an automated ncs system [ 810 ] . we hypothesized that use of the nc - stat system would yield highly reproducible ncs results .", "all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy . the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) . each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . , waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation . the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves . tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used . each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks . the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor . the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli . supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) . snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count . time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via docking station ( a telecommunication port for secure data transfer via telephone line ) . it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via docking station ( a telecommunication port for secure data transfer via telephone line ) . the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease . concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal . the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease . the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist . the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal . the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line . responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . at the conclusion of ncs for a given subject , motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters . supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz . for recording snaps , 615 individual waveforms were averaged depending upon the signal - to - noise ratio . up to 10 f - wave responses ( with 12 traces as a maximum ) up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n 2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle . bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . dotted line is the calculated mean f - wave latency for the f - wave set . sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle . bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . dotted line is the calculated mean f - wave latency for the f - wave set . all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies , distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as + in the lower left panel of figure 2 ) . dml values from four cmap waveforms were averaged and reported ( after temperature correction ) . cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap . f - wave onset latency was identified for each trace with an identified f - wave response ( traces with + , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) . the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) . the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) . all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) . the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant . statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] . the pearson product - moment correlation ( cc ) was used to assess the association between ncs parameters obtained 7 days apart . intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests . coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv ) the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size , 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others , were also obtained for selected parameters . the mean and standard deviation of the difference between the test and retest results paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean .", "all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy . the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) .", "each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . , waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation . the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves . tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used . each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks . the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor . the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli . supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) . snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count . time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via docking station ( a telecommunication port for secure data transfer via telephone line ) . it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via docking station ( a telecommunication port for secure data transfer via telephone line ) . the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease . concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal . the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease . the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist . the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal . the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line . responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . both the active and inactive electrodes were located just distal to the medial malleolus . a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . at the conclusion of ncs for a given subject , test data were uploaded to a central database . motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters . supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz . 615 individual waveforms were averaged depending upon the signal - to - noise ratio . up to 10 f - wave responses ( with 12 traces as a maximum ) were recorded for the median and ulnar nerves . up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n 2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle . bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . dotted line is the calculated mean f - wave latency for the f - wave set . sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle . bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . dotted line is the calculated mean f - wave latency for the f - wave set .", "all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies , distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as + in the lower left panel of figure 2 ) . dml values from four cmap waveforms were averaged and reported ( after temperature correction ) . cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap . f - wave onset latency was identified for each trace with an identified f - wave response ( traces with + , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) . the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) . the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) . all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) . the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant .", "statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] . the pearson product - moment correlation ( cc ) intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests . coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv ) the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size , 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others , the mean and standard deviation of the difference between the test and retest results were also reported . paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean .", "their ages ranged from 24 to 52 years ( mean 37.1 , sd 8.6 years ) . height varied from 157 to 180 cm ( mean 173 , sd 7.9 cm ) . total of 15 subjects ( 5 females ) volunteered for the lower extremity repeatability study and nine of them had also enrolled in the upper extremity study . their age range was 2247 years ( mean 32.6 , sd 8.2 years ) and height range was 157180 cm ( mean 172 , sd 8.2 cm ) . the rivs for mean fwl at 5th and 95th percentiles were [ 11.4% , 9.4% ] , [ 5.1% , 3.3% ] , [ 4.9% , 4.5% ] , and [ 2.8% , 4.0% ] , respectively , for median , ulnar , peroneal , and tibial nerves . at p = 0.05 level , paired t - tests indicated that the means of all test and retest ncs parameters were the same . pearson ccs for all latency parameters were greater than 0.80 with the exception of median nerve mean fwl ( cc = 0.69 ) . all upper extremity amplitude parameters had ccs greater than 0.85 . for lower extremity nerves , all latency results were based on temperature corrected latencies . without temperature compensation , latency parameters exhibited lower repeatability . for example , the cc values for ulnar nerve dml , mean fwl , and dsl without temperature correction were 0.72 , 0.93 , and 0.72 , respectively . standard deviation of the difference between test and retest results would have been 0.31 , 1.04 , and 0.34 . table 1statistical measures of nerve conduction study parameter repeatabilityriv percentiles(day 8day 1)nerveccicccov10th ( % ) 90th ( % ) meanstdevpaired t - testdistal motor latency ( dml)median0.8720.8700.0329.55.10.0550.1940.289ulnar0.8890.8320.0458.311.00.0260.1900.605peroneal0.8640.8450.06115.48.10.1240.4530.307tibial0.8500.8240.0386.88.80.0160.2390.799mean f - wave latency ( fwl)median0.6920.6790.0316.64.40.0371.9700.943ulnar0.9490.9210.0173.91.10.2860.9150.246peroneal0.8960.9010.0174.73.40.1631.5630.693tibial0.9430.9400.0131.83.50.2781.1450.363distal sensory latency ( dsl)median0.8870.8790.0287.24.00.0560.1890.270ulnar0.8150.7970.0375.79.00.0120.2070.825compound muscle action potential ( cmap ) amplitudemedian0.8790.8620.10919.219.90.0310.1980.550ulnar0.9810.9740.0455.710.70.2880.6090.088peroneal0.3920.3310.29830.899.50.8491.9370.112tibial0.7140.7280.14830.925.40.1651.6010.695sensory nerve action potential ( snap ) amplitudemedian0.9540.9540.05811.87.80.6738.4020.761ulnar0.9900.9820.0424.410.91.4855.0630.275cc : pearson product moment correlation to assess the association between test - retest results . riv : relative interval variation defines the difference between test - retest as a percentage of average . paired t - test results indicate the means of test and retest are not statistically different . dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference . dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference . fwl is the arithmetic mean of individual f - wave latencies in an f - wave set . statistical measures of nerve conduction study parameter repeatability cc : pearson product moment correlation to assess the association between test - retest results . riv : relative interval variation defines the difference between test - retest as a percentage of average . paired t - test results indicate the means of test and retest are not statistically different . dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference . dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference . fwl is the arithmetic mean of individual f - wave latencies in an f - wave set .", "several studies ( summarized in table 2 ) have examined ncs parameter repeatability [ 2 , 4 , 5 ] . in , the repeatability of ncs was evaluated for 60 sites participating in a clinical trial , with ncs oversight performed by an experienced , insightful and knowledgeable core lab . our study yielded similar results , though our study demonstrated a higher cov for peroneal cmap amplitude ( 9% vs. 29.8% ) . in , 132 healthy subjects were retested at a time interval of 14 weeks and icc and riv were used to measure the test - retest repeatability . in comparison with that study , the riv ( 5th and 95th percentile ) for tibial nerve f - wave latency was tighter in our study ( [ 2.8% , 4.0% ] vs. [ 4.6% , 5.7% ] ) while the relationship was reversed for median nerve fwave latency results ( [ 11.4% , 9.4% ] vs. [ 6.7% , 6.7% ] ) . decreased repeatability of median nerve f - wave latency may be attributed to the lower amplitude signal - to - noise ratio as median nerve f - waves were acquired via a volume - conduction recording technique . indeed , latency repeatability was much higher for ulnar f - waves recorded directly over the muscle . the icc for ulnar f - wave latency was 0.92 in our study , higher than the icc of 0.59 reported in a similar study of 49 healthy adults . salerno and colleagues studied upper extremity sensory test - retest repeatability based on 158 active workers who were tested by a neurologist and a physiatrist . our results fell into the upper range of their repeatability results as measured by cc and icc . \n table 2repeatability results reported in the literaturesnerveccicccovdistal motor latency ( dml)median0.8260.04/0.08 > \n peroneal0.05/0.08tibial0.6170.08minimum f - wave latency ( fwl)ulnar0.59tibial0.921distal sensory latency ( dsl)median0.820.920.820.920.04ulnar0.370.640.320.63compound muscle action potential ( cmap ) amplitudemedian0.7770.07/0.11peroneal0.09tibial0.846sensory nerve action potential ( snap ) amplitudemedian0.830.880.834/0.810.880.08ulnar0.680.850.680.85 132 healthy subjects retested in 14 weeks . results for control subjects from 60 sites with core lab support in a clinical trial . 33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined . repeatability results reported in the literatures 132 healthy subjects retested in 14 weeks . results for control subjects from 60 sites with core lab support in a clinical trial . 33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined . it is possible that the small recording electrodes ( surface areas 2 cm ) used in our study degraded repeatability , since tjon - a - tsien and colleagues noted that when electrode size was changed from 0.78 to 7.65 cm , the cov of cmap amplitude improved from 11 to 7% ( lower cov is associated with higher repeatability ) . dml repeatability was less affected by electrode size in both our study and the study of . however , control of skin temperature is often difficult to achieve , especially in different testing environments . in the present study , skin temperature adjacent to the recording site was acquired automatically using an embedded probe and latencies were adjusted to the reference temperature . as expected , temperature correction improved ncs repeatability . for example , the cc increased from 0.72 ( without temperature correction ) to 0.89 ( with correction ) for both motor and sensory latencies of the ulnar nerves . temperature correction also reduced the standard deviation of the test - retest latency difference by as much as 39% ( ulnar dml ) . the principal limitation of the current study was the small number of subjects evaluated relative to several prior investigations . the short test - retest interval of 1 week reduced the likelihood of intervening physiological changes . it would be valuable to quantify repeatability of the automated ncs instrument in neuropathic nerves . at the time of this study , temperature correction may improve the repeatability of the amplitude parameters , especially for sensory responses . the reproducibility of individual ncs parameters followed the same pattern as those observed in traditional settings . f - wave latency had the greatest repeatability , followed by dml , dsl , and snap amplitude . the cmap amplitude was the least reproducible parameter , with peroneal cmap amplitude repeatability the lowest among all nerves . a larger recording electrode size should improve its repeatability . f - wave latency repeatability for median nerve was lower due to the lower signal - to - noise ratio inherent in volume - conduction recordings . as a result , over - muscle recording would be expected to enhance f - wave repeatability , which was the case for the ulnar nerve . further studies with longer test - retest intervals and a higher subject count would be valuable in confirming the high reproducibility of ncs parameters measured in this study . extending the study to pathologic nerves and validating the equivalent or superior performance of automated ncs instrument should increase the adoption rate of the automated ncs technology in patient management" ]

[ "this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials . eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above . these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial . in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment . other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel . all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy . this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) . patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy . they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed . the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims . nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events . the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 . scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale . a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale , a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 . a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status . similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not . time - to - cancer progression was defined as the time from registration to documentation of cancer progression . survival time was defined as the time from study registration to death due to any cause . a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles . no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups . a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events . because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment . all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) .", "this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials .", "eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above . these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial . in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment . other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel .", "all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy . this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) . patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy . they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed .", "the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims . nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events . the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 . scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale . a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale , a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 . a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status . similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not . time - to - cancer progression was defined as the time from registration to documentation of cancer progression . survival time was defined as the time from study registration to death due to any cause . a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles . no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups . a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events . because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment . all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) .", "a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study . one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses . forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively . lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight . in addition , 73% reported that when they have an important decision to make , they always have someone who can take them to their medical appointments , and 71% reported that they always have someone from whom they can seek help if they have medication side effects . the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) . it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) . additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) . although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) . there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) .", "a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study . one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses . forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively .", "lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight . in addition , 73% reported that when they have an important decision to make , they always have someone to talk with . always have someone who can take them to their medical appointments , and 71% reported that they always have someone from whom they can seek help if they have medication side effects . the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) . it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) . additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) . although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) . there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) .", "the present study was designed to assess and evaluate the role of social support in a group of geriatric non - small cell lung cancer patients who were enrolled in two different cancer therapeutic clinical trials . this study found that this cohort of geriatric patients had high levels of social support , as suggested by the fact that 81% reported that they always had someone who could take them to medical appointments , and 71% reported that they always had someone from whom they could seek help if they had medication side effects . however , social support was not associated with improved survival or diminished side effects from chemotherapy . moreover , no differences were observed between men and women in terms of baseline social support . thus , although previous studies have reported that social support carries a positive impact on clinical outcomes and although gender differences in extent of social support have been hypothesized , the present study did not observe such findings . why might the results of this study be divergent from the others that preceded it ? first , relying on a clinical trial to answer such questions provides a solid infrastructure that ensures reliable outcome data . the fact that all clinical trial participants were followed prospectively in such a thorough and systematic fashion makes it unlikely that patients particularly those who have poor social support and who are thereby especially vulnerable to being lost to follow up would , in fact , be lost to follow up . such artificially compromised outcomes were side - stepped by building the study around two clinical trials , as was done here . thus , the present study s meticulous follow up may have allowed for a more definitive and accurate assessment of the predictive capabilities or lack thereof of social support . second , and more importantly , one might argue that patients willingness to participate in a clinical trial is associated with a high level of social support and that this selection bias may have contributed to the lack of differences between groups . the fact that 82% of patients reported that they always had someone to bring them to medical appointments speaks to the possibility that this group as a whole has a high level of social support . similarly , the median social support score of 72 in this study appears greater than the 63 score derived from a community - based study , an observation that also suggests cancer clinical trial participants have high levels of social support.19 in short , although the findings from this study suggest that social support does not influence clinical outcomes , these findings might pertain to only a culled group of geriatric clinical trial participants . future studies may choose to focus further on the extent of social support among geriatric patients who participate in clinical trials , and further studies may also choose to study further whether social support is a predictive factor for the patients who can and do enroll in cancer clinical trials . two final points merit further discussion . first , although the current study did not find that social support was predictive of outcomes , there are a variety of other potentially clinically relevant factors that had not been captured and adjusted for . these include socioeconomic status , ongoing tobacco use , use of nutritional supplements , as well as several other factors . other investigators may choose to further study the issue of social support in the context of these other factors . second , although this study found that social support is not associated with clinical outcomes among elderly lung cancer patients , there remains no question that these patients have major needs that are likely exacerbated by a lack of social support . patients with metastatic non - small cell lung cancer suffer from a poor prognosis , as seen from both studies presented here where the prognosis was poor , and previous studies have demonstrated that elderly patients suffer from a wide constellation of cancer - related symptoms , including depression , guilt , debility , pain , and dyspnea.1013 although the present study did not observe an association between social support and clinical outcomes , there nonetheless remains a strong need for maximizing social support to help with some of the cancer - related challenges these patients face ." ]

[ "port - wine stains ( pwss ) are congenital vascular malformations comprised of ectatic dermal capillaries and affecting 0.3 - 1% of newborn infants . acquired pwss are an uncommon entity developing later in life , but morphologically and histologically similar to the congenital lesions . we hereby report a case of 41-year - old male patient who acquired pws about a year ago .", "a 41-year - old male presented to the outpatient department of dermatology of our tertiary care hospital with complaint of asymptomatic reddish patches involving the lower part of right leg for 10 months . the patch started from near the ankle and gradually spread to involve whole of the medial aspect of right leg over a period of 10 months . we could not elicit any history of antecedent mechanical or thermal trauma , drug intake , topical application or excessive ultraviolet exposure . dermatological examination revealed a diffuse partially blanchable erythema extending from the right ankle and involving chiefly the medial aspect of right leg up to the knee [ figure 1 ] . there were also a few discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg [ figure 2 ] . a diffuse partially blanchable erythema extending from the right ankle and involving the medial aspect of right leg up to the knee discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg on the basis of the clinical examination , a provisional diagnosis of acquired pws was made and the patient investigated . the histopathological examination revealed multiple dilated , variably sized capillaries lined by a single layer of endothelial cells throughout the dermis showing lobular arrangement at places [ figure 3 ] . color doppler of the limb did not reveal any abnormality of the underlying arteries and veins . ( a ) multiple dilated variably sized capillaries in the dermis ( h and e , 40 , arrows ) . ( b ) high power showing groups of capillaries lined by a single layer of endothelial cells ( h and e , 250 , arrow ) laser therapy was discussed with the patient , but he declined treatment for the lesion .", "pwss are congenital vascular malformations usually presenting at birth in the form of pink - red to purple macules , which become darker , raised , and nodular as the person ages . the most accepted hypothesis is a defective embryological maturation of the sympathetic fibers , resulting in a loss of normal sympathetic control of the cutaneous vessels which leads to ectasia . various factors have been proposed for the occurrence of acquired pwss . of these , trauma has been found to be the most important causative factor in majority of the cases . trauma - induced pws was first described by fegeler in 1949 and hence called fegeler syndrome . in a review of 59 patients with acquired pws , trauma was found to be a causative factor in 17 ( 29% ) cases . it has been proposed that injury may result in loss of a previously effective sympathetic regulation of the cutaneous blood flow , leading to development of the lesions . reports of acquired port - wine stain few cases of acquired pws have also been proposed secondary to oral medications like isotretinoin , oral contraceptive pills , simvastatin , and metformin . isotretinoin causes skin fragility and frictional trauma , while simvastain and metformin have been shown to promote angiogenesis by upregulation of vascular endothelial growth factor . isolated cases of acquired pws following frostbite injury , obstruction of the peritoneovenous shunt , herpes zoster infection , cerebral arteriovenous malformation , spinal root compression , and solid brain tumor have also been reported . no definite cause was found for the development of lesion in our patient . in such cases , a likely possibility of having a latent vascular anomaly which manifests clinically after unnoticed trauma an unusual finding in our patient was the presence of a lobular arrangement of dilated vessels in the dermis which has not been observed in previous cases of congenital or acquired pws . interestingly , there are multiple reports of development of pyogenic granulomas ( pg ) within pws which also shows a lobular pattern of dilated vessels . it has been proposed that the pg probably results from an underlying arteriovenous anastamosis frequently associated with the pws . though , there was no clinically appreciable pyogenic granuloma like lesion , the distinct histological features seen in our patient might be attributed to a similar pathogenetic mechanism . as regards therapy , it includes treatment with krypton , argon , argon - pumped , tunable dye , neodymium : yttrium - aluminum garnet , copper vapor , and pulsed dye lasers . acquired pws tends to have a quicker and better response to pulsed dye therapy than congenital pws . the case report demonstrates a rare occurrence of a vascular malformation in an adult patient . although no definite cause could be appreciated for the development of lesion in our patient , further reports and future insights into the pathogenesis of such cases would broaden our knowledge regarding this infrequently reported phenomenon . this is the first report of an acquired port - wine stain from india and previous reports of such cases have also been reviewed ." ]

[ "ionizing radiation is a well - known carcinogen in humans , and breast is one of the most sensitive organs to radiogenic cancer . the rate of breast cancer in postwar japan was among the lowest in the world , but breast cancer contributed a disproportionately large fraction of the radiation - related cancer burden in atomic bomb survivors [ 2 , 3 ] . the data from the hiroshima and nagasaki survivors provides strong evidence for increased breast cancer following single acute doses of 20 cgy and linearity with increasing dose [ 36 ] . also , an increase in the incidence of breast cancer has been observed in areas affected by the chernobyl accident , which resulted in radioactive contamination of large areas of belarus and ukraine . a twofold increase in risk was observed when comparing the most ( > 40 msv cumulative dose ) and least contaminated regions . interestingly , the increase appeared 10 years after exposure and was most prominent in women exposed at younger age . more than 50,000 women in the united states have been treated with chest radiation ( 20 gy ) for a pediatric or young adult cancer . children treated from cancer with radiotherapy have a 2.9 relative risk of subsequent malignancy compared to those who were not [ 8 , 9 ] . a systematic review of 14 studies concluded that risk of breast cancer increased as early as 8 years following chest radiation and did not plateau with increasing length of follow - up . studies estimating low - dose radiation - induced cancer risk from diagnostic x - rays and ct scans have found a small but significant increased lifetime risk [ 11 , 12 ] . while the benefits of diagnostic x - ray and ct scans outweigh potential individual lifetime risk , their use should be justified and alternatives considered . we know remarkably little of molecular mechanisms that may be protective or risky for breast cancer after exposure to low - dose ionizing radiation ( ldir ) . identification of transcriptomic changes induced by ldir in mammary tissue will be valuable to elucidate the molecular mechanisms associated with radiation - induced breast cancer . long noncoding rnas ( lncrnas ) , which initially were thought of as transcriptional noise , are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic , transcriptional , and posttranscriptional regulation of gene expression [ 13 , 14 ] . the lncrnas have a weaker evolutionary constraint and lower levels of expression compared to the protein - coding transcripts [ 15 , 16 ] but exhibit more tissue specific expression than the protein - coding genes . recently , a number of studies have shown that lncrna expression can be deregulated in human cancers [ 17 , 18 ] . as the functions of individual lncrnas in cancer are beginning to be elucidated , they are being categorized and referred to as either tumor suppressor or oncogenic lncrnas , in the same way as traditional protein - coding cancer genes . however , the relevance of lncrnas in the cell and tissue response to ionizing radiation has not yet been characterized . in this study , we used agilent sureprint g3 microarrays to profile lncrna and mrna from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10 cgy of x - radiation . we identified lncrna and mrna expression signatures for each time point after irradiation in comparison to sham . of the total 1338 lncrnas identified to be differentially expressed after ldir in either balb / c or spret / eij , 1337 had a significantly correlated expression pattern with at least one mrna that was also differentially expressed after ldir . our results indicate lncrnas may exert a partial or key role in the regulation of coding rna expression induced by radiation .", "balb / c and spret / eij mice were purchased from jackson laboratory , housed four per cage under a 12 hr light and 12 hr dark cycle , and fed with lab diet 5008 chow and water ad libitum . the mice were irradiated whole body at 8 - 9 weeks of age to a single dose of 10 cgy using a precision x - ray inc rad320 320 kvp x - ray machine , operated at 300 kv , 2 ma . mammary tissues were collected for gene expression profile at 2 , 4 , and 8 weeks after irradiation . all animal experiments were performed at lawrence berkeley national laboratory and the study was carried out in strict accordance with the guide for the care and use of laboratory animals of the national institutes of health . the animal use protocol was approved by the animal welfare and research committee of the lawrence berkeley national laboratory . agilent sureprint g3 mouse ge 8x60k microarrays were used according to the manufacturer 's protocol ( arrays contained 39,430 entrez gene rnas and 16,251 lncrnas ) . genes that were differentially expressed between sham and irradiated were identified by the unpaired student 's t - test with a p value cut - off of 0.05 ( p value < 0.001 for baseline strain comparison ) and a fold - change criteria of more than 1.5 . for each of the 8 experimental treatment groups , the average expression values of the 3 biological replicates significantly correlated pairs of mrna and lncrna were calculated using a standard permutation test . in brief , for each potential mrna and lncrna pair , the 8 mrna values were randomly rearranged and a correlation coefficient was calculated between the 8 mrnas and 8 lncrnas values . the p value reported in this work represents the percentage of permutations leading to a higher correlation than the original correlation between the 8 mrnas and 8 lncrnas values . in other words , the lower the p value is , the more likely the mrna and lncrna pair is not randomly associated . gene lists were annotated with biological functions using ingenuity pathway analysis ( ipa ) , kegg pathway analysis ( http://bioinfo.vanderbilt.edu/webgestalt/ ) and david go gene ontology ( http://david.abcc.ncifcrf.gov/ ; p 0.05 ) . annotations for the various shapes used in the ipa networks in figures 13 are shown in figure s3 in supplementary material available online at http://dx.doi.org/10.1155/2015/461038 .", "to identify potential lncrnas and mrnas that may determine susceptibility to radiation - induced breast cancer , we profiled two inbred strains of mice with differing genetic susceptibilities : balb / c mice as more sensitive and spret / eij as more resistant . balb / c mice carry two dna - pkcs polymorphisms with reduced catalytic subunit activity and defective nonhomologous - end - joining of double strand breaks . we identified 195 lncrnas as upregulated and 95 lncrnas as downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(a ) ; table s1 ) . additionally , 582 mrnas were upregulated and 402 mrnas were downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(b ) ; table s1 ) . gene ontology analyses of differentially expressed genes between balb / c and spret / eij showed significant enrichment for metabolic processes ( p = 5.9e 06 ) , ion binding ( p = 3.00e 08 ) , and chemokine signaling ( p = 0.02 ) ( figure 1(c ) ; table s2 ) . our analyses identified significant strain differences in gene expression between balb / c and spret / eij mammary tissues . we found significant differences in the expression of a number of chemokines including cxcl10 , ccl6 , and ccl25 , which were expressed at higher levels in mammary tissues of the more sensitive and susceptible balb / c mice and which have previously been associated with breast cancer progression when over expressed . to identify lncrna and mrna expression changes induced by low - dose ionizing radiation ( ldir ) , we profiled lncrna and mrna expression from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10 cgy of x - radiation . for balb / c mice , a total of 357 , 480 , and 335 lncrnas and 550 , 911 , and 389 coding rnas were identified to be differentially expressed at weeks 2 , 4 , and 8 after ir in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) , respectively ( figure 2(a ) ; table s1 ) . for spret / eij , a total of 327 lncrnas and 424 mrnas were identified as differentially expressed at week 4 after irradiation in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) ( figure 3(a ) ; table s1 ) . few coding - rnas and lncrnas were found to be differentially expressed at different time points ( figure s1(a ) and s1(b ) ) and between balb / c and spret / eij ( figure s1(c ) ) . to determine the biological functions associated with the ldir response , we excluded genes whose levels fluctuate in the mouse mammary gland across the estrous cycle . we recently mapped transcript - level changes across the estrous cycle in the murine mammary gland using rna sequencing and defined a comprehensive estrous variable gene signature of 3893 genes whose levels fluctuate in mammary glands of balb / c mice . comparison of our mapped ldir genes in mammary glands of balb / c and spret / eij mice with the estrous signature revealed an approximate 20% overlap in balb / c ( figure 2(b ) ) and 9% overlap in spret / eij mice ( figure 3(b ) ) . nonoverlapping and differentially expressed ldir genes for each of the time points were then computationally mapped to biological functions , pathways , upstream regulators , and networks . these analyses suggested that the ldir response signatures in mammary glands of balb / c mice transitions between time points and is distinct from the ldir response in spret / eij mice . two weeks after ldir exposure pathways and biological functions significantly enriched in mammary glands of balb / c mice compared to sham irradiated mice included chemokine signaling ( p = 0.01 ) , ccr3 signaling in eosinophils ( p = 0.05 ) , cellular movement ( 3.92e 04 < p < 3.41e 02 ) , and cell death and survival ( 1.29e 03 gene interaction networks were enriched for tissue and endocrine system injury ( figure 2(c ) top panel ) and significant predicted upstream regulators ( table s3 ) include gli2 ( p = 5.29e 04 ) and satb1 ( p = 1.98e 03 ) . similar to the two - week ldir response , gli2 was predicted to be an upstream regulator ( p = 4.52e 03 ; table s3 ) . gata3 and stat6 were among other significant upstream regulators associated with the four - week low - dose response ( table s3 ) . we furthermore observed that the mammary gland of balb / c mice four weeks after ldir was enriched for inflammatory response genes ( 1.84e 05 < p < 1.45e 02 ) , cell - cell signaling ( 2.79e 05 < p < 1.60e 02 ) , while gene interaction networks were enriched for lipid metabolism ( figure 2(c ) middle panel ) . interestingly , similar responses were observed in mammary glands of spret mice at 4 weeks after ldir including inflammatory response functions ( 1.69e 03 < p < 4.49e 02 ) , cell - cell signaling ( 5.54e 04 < p < 4.49e 02 ) , suggesting that the functional response is similar across strains and is independent of the gene transcript response . at 8 weeks after ldir we observed downregulation of genes involved in mammary gland development including progesterone receptor , prolactin , amphiregulin , and wnt4 ( figure 2(c ) bottom panel ; table s3 ) . to identify lncrnas potentially regulating the expression of coding rnas in response to radiation , correlation coefficients on expression data were calculated for each coding rna and lncrna that were identified as differentially expressed after ldir . a permutation - based algorithm was then used to determine which correlations were statistically significant ( p < 0.05 ; table s4 ) . we observed that nearly all ldir modulated lncrnas were correlated with at least one of the ldir modulated coding mrnas ( figure 4(a ) ) . to determine whether these correlations were driven by estrous variations , we only considered genes whose expression levels were not overlapping with our previously determined estrous signature ( figure 2(b ) ) . again , we observed that nearly all differentially expressed lncrnas were correlated with at least one differentially expressed mrna suggesting that estrous cycling does not affect the strong correlation between lncrna and mrna expression after ldir . to test the robustness of these correlations , we compared the number of lncrnas associated with at least one mrna at three different p values ( p < 5e 02 , p < 5e 03 , and p < 5e 04 ) . at p < 5e 02 or p < 5e 03 , nearly all ( 97100% ) differentially expressed lncrnas were found to be correlated with at least one mrna ( table s5 ) . at p < 5e 04 , corresponding to a correlation coefficient > 0.9 , we still observed a significant fraction ( 6381% ; table s5 ) of lncrnas correlated with mrnas . representative correlation networks of lncrnas are shown in figure 4(b ) ( p < 5e 03 ) for each of the timepoints . we furthermore observed that the same lncrna correlates with different gene sets across different time points ( figure s2 ) . taken together these data show that ldir induces coordinated changes in lncrna and mrnas and suggests a critical role for lncrnas in mediating the low - dose radiation response .", "in this study , we demonstrate that genetic background strongly influences the expression of lncrnas and their response to low - dose radiation by transcriptomic analysis of mouse mammary glands using microarrays that contain both lncrnas and coding rnas . we have identified a number of lncrnas that are significantly changed after exposure to ldir at three different timepoints after radiation exposure . moreover , the changes in the expression of lncrnas are significantly correlated with the expression of coding rnas , suggesting that lncrnas may coordinate the tissue response to radiation via regulation of coding mrnas . however , the specific regulatory mechanism of this control requires further investigation , and knock - out and overexpression of the lncrna genes in mice and other model systems should be performed to increase our understanding of the regulatory mechanisms in response to ldir ." ]

[ "\n women with psychiatric diseases are treated with antipsychotic medication(s ) with limited evidence to support the safety of their use during pregnancy.olanzapine , a second - generation antipsychotic , is a us food and drug administration pregnancy category c drug with no unequivocal evidence of harm to the fetus.we report the first case of tracheo - esophageal fistula as a possible teratogenic effect of olanzapine exposure . \n", "women with psychiatric diseases may become pregnant and are treated with antipsychotics without any proven evidence of safety . due to ethical issues , pregnant women are rarely included in clinical trials , leading to a dearth of data available on the safety of antipsychotic drugs in this population . there have been reports of congenital anomalies in newborns of mothers exposed to antipsychotic medications during pregnancy [ 14 ] . olanzapine , a second - generation antipsychotic , is a us food and drug administration ( fda ) pregnancy category c drug [ 3 , 5 ] with no unequivocal evidence of harm to the fetus . a literature search revealed that maternal exposure to olanzapine appears to be associated with lumbar meningomyelocele , dysplastic kidney , hip dysplasia , atrioventricular canal defect , club foot , microcephaly , ventricular septal defect , absent fingers , craniosynostosis , cleft lip , encephalocele , aqueductal stenosis , etc . the possibility of further teratogenic effects of olanzapine can not be ruled out . in this case report , we report the first case of tracheo - esophageal fistula ( tef ) as a possible teratogenic effect of olanzapine exposure .", "we report the case of a 29-year - old married woman with bipolar disorder with mixed episodes who had been treated with olanzapine in the psychiatric outpatient department of our institute ( lady hardinge medical college , new delhi , india ) for the last 7 years who delivered a full - term female baby with tef in september 2013 . one week after her marriage in february 2008 , the patient developed psychotic symptoms such as excessive talking , episodes of crying and laughing , and forgetting things frequently . following initial unsuccessful local treatment , she was taken to the psychiatry department of a tertiary care hospital . after initial management of acute symptoms there , she continued her treatment from our institute , where she was prescribed olanzapine ( oleanz 10 mg tablet orally once daily ) . in december 2008 , she had a miscarriage at 4 months gestation , following which she stopped taking the medication of her own choice . a careful history / enquiry from the parents failed to reveal any data available regarding congenital malformation of the aborted fetus . in 2009 , she conceived again ( it is noteworthy that the patient was not taking olanzapine during this pregnancy ) and delivered a full - term healthy female child in january 2010 . eight months after delivery of this baby the patient had a recurrence of psychotic symptoms for which she was prescribed the same treatment . in december 2012 , she conceived a third time but due to persistent psychiatric symptoms , olanzapine was continued throughout the pregnancy by the treating psychiatrist , who considered there to be a favorable benefit : risk ratio for the patient at that time . she received regular antenatal care and was compliant with all prescriptions and instructions , including prophylactic folic acid and two doses of tetanus toxoid . there was no history of maternal alcohol intake , smoking , or exposure to other potential teratogenic drugs or exogenous hormones throughout the pregnancy . all baseline hematological ( hemoglobin 13.8 g / dl , total leukocyte count 6000/l , platelets 2.3 lac/l , etc . ) and biochemical investigations ( random blood sugar 98 mg / dl , blood urea 27 mg / dl , etc . ) tests for trisomy 21 and 18 ( nuchal translucency scan and dual marker tests ) and rubella were negative . , she delivered a full - term female baby by vaginal delivery . immediately after delivery , the baby experienced respiratory distress and excessive salivation , for which she was admitted to the neonatal intensive care unit . on examination , a red rubber catheter could not be negotiated beyond 10 cm in the esophagus . other concomitant anomalies including vertebral , anal , renal , limb ( components of vacterl [ vertebral defects , anal atresia , cardiac defects , tracheo - esophageal fistula , renal anomalies , and limb abnormalities ] ) , eye , ear , and nasal defects were ruled out . the tef was repaired using esophageal esophageal anastomosis with a drainage tube in the fifth intercostal space . on the second post - operative day for this reason , cervical esophagotomy was performed and a gastrostomy was inserted for feeding purposes . the baby was discharged in a stable condition with advice for full breast milk feeding through gastrostomy , some required medications ( multivitamin syrup , calcium , anti - emetic and antibiotics ) for an appropriate duration , and regular vaccination . however , although the baby was well until this point , she was re - hospitalized at 11 months of age because of diarrhea and unfortunately died on 19 august 2014 , probably because of widespread infection ( no definitive record of the last hospitalization is available and a post - mortem was not performed).fig . 1chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula \n chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula", "a number of drugs are clearly known to be associated with teratogenicity , such as thalidomide and isotretinoin , but the majority of drugs do not have conclusive data concerning their effect on fetal development and growth . due to strict ethical constraints , at - risk populations ( including pregnant women ) are not included in most clinical trials , limiting the availability of data on the use of drugs ( including antipsychotics ) in these populations , and thus spontaneous reporting of suspected adverse drug effects constitutes an essential tool for generating data on the safety of drugs , especially in the population of pregnant women . according to a retrospective analysis of data from the lilly safety database , of the 610 prospectively identified pregnancies exposed to olanzapine with an available outcome , there were 401 ( 66% ) normal births , 60 ( 9.8% ) premature births , 57 ( 9.3% ) spontaneous abortions , 49 ( 8% ) perinatal complications , 27 ( 4.4% ) congenital anomalies ( cleft lip , encephalocele , and aqueductal stenosis ) , and 16 ( 2.6% ) other outcomes ( post - perinatal condition , ectopic pregnancy , post - term birth , and still birth ) . other reported congenital anomalies with olanzapine included meningocele , ankyloblepharon , hip dysplasia , acheiria , atrioventricular canal defect , and unilateral club foot [ 1 , 68 ] . a link between teratogenicity and antipsychotics is also supported by the fact that these drugs readily cross the placental barrier [ 1 , 4 , 9 ] , with 23.8% placental passage for quetiapine and 72.1% for olanzapine [ 2 , 4 , 9 ] . newport et al . measured placental passage of medication from mother to fetus by measuring levels in the umbilical cord serum and found that olanzapine has the highest rate of placental passage , compared with haloperidol , risperidone , and quetiapine [ 3 , 9 ] . the overall incidence of esophageal atresia / tef ranges from one in every 2500 to 4500 live births . however , a careful literature search revealed a paucity of data on the incidence of tef in children born to women with psychiatric disease and the problem of confounding by indication can not be ruled out . it has been proposed that antipsychotics interfere with the action of calmodulin , which has an important role in organogenesis . esophageal atresia and abnormalities of pharyngeal glands have also been proposed as being related to neural crest cells . the drugs ( olanzapine in this case ) associated with such anomalies may be hypothesized to cause neural crest cell damage during early fetal development as a causative factor . although the neural crest cell damage - related esophageal abnormalities in many cases are also associated with cardiovascular anomalies , these were absent in the present case . we tried to establish the causality between the adverse outcome ( tef ) and use of olanzapine during pregnancy according to the who - umc ( world health organization uppsala monitoring centre ) adverse drug reaction causality assessment scale and the naranjo causality assessment scale . causality is probable ( score + 5 ) as per the naranjo scale , whereas it is one of the important reasons for lower scores of causality is the non - applicability of many questions in these scales . the current case report of tef associated with antenatal olanzapine exposure demonstrates the need for large clinical studies to generate more conclusive data concerning use of this drug during pregnancy .", "on the basis of the present case and the literature , it is suggested that antenatal olanzapine exposure may be related to the development of tef in newborns . however , the present case report can only be considered as preliminary evidence and further observational prospective studies of women with antenatal exposure to olanzapine should be conducted to explore the strength of association between tef and olanzapine . knowledge of the teratogenic potential of a drug is essential to prevent / reduce the morbidity and mortality associated with a congenital anomaly ( tef in this case ) . such post - marketing reports are effective tools to disclose the adverse effects of drugs , which are difficult to detect in clinical trials and may factually change the safety status of a drug .", "", "", "written informed consent was obtained from the infant s father for publication of this case report ." ]

[ "\n\t\t\t\t olfactory neuroblastoma ( onb ) is a rare malignant tumor arising from the olfactory epithelium of the nasal cavity and the paranasal sinuses . the anatomic origin in the superior nasal cavity leads to nonspecific symptoms that make early diagnosis difficult . direct extension of the tumor into the anterior cranial fossa , either at presentation or at disease recurrence , is common . onb is mostly a locally aggressive tumor that can spread to lymph nodes but metastatic involvement of bone , bone marrow , and other organs was also reported . the most commonly used staging system proposed by kadish is currently based on computer tomography and magnetic resonance imaging . in stage a , the tumor is limited to the nasal cavity , in stage b the tumor extends to paranasal sinus , and in stage c the tumor is extending over the nasal cavity and sinus and/or metastasized distantly . a histological grading system based on microscopic findings proposed by hyams at 1988 showed a clear correlation of survival with histological differentiation of disease . however most authors believe that craniofacial resection followed by rt offers the gold standard treatment in this disease . despite aggressive therapies , local , regional relapse , and distant metastasis occur frequently and often after extended periods of follow - up . data for this case series were acquired retrospectively from the files of division of medical oncology , cerrahpasa medical faculty , istanbul university . additional information regarding the clinical course and outcome was collected from the patients ' charts and phone calls to the patients , their relatives , and their general practitioners . in this study , we have reviewed 19 cases of olfactory neuroblastomas treated over 24-year period and analyzed the clinical features , treatment outcomes , and prognostic factors .", "the data of nineteen patients of olfactory neuroblastomas treated and followed up between 1986 and 2010 at division of medical oncology , cerrahpasa medical faculty , istanbul university , were analyzed retrospectively . patient characteristics , initial symptoms , tumor extent , histologic features , primary therapy , tumor recurrence , and treatments of recurrent disease were determined . patients were staged according to the kadish staging system . a single pathologist with expertise in head and neck neoplasms reviewed the histological specimens . local ethics committee 's approval and patients ' or their next of kin 's informed consent were taken before the study .", "treatment response was assessed by clinical examinations and computed tomography ( ct ) or magnetic resonance imaging ( mri ) . survival rates were calculated from diagnosis to the death of the patient using the kaplan - meier life table method .", "the madian age at diagnosis was 46 ( range : 19 to 68 years ) . tumor staging was kadish stage a in 3 , stage b in 5 , and stage c in 11 patients . four patients had metastasis of cervical lymph nodes , two brains , one bone marrow , one bone and lung metastases , and one bone , mediastinal lymph node , and liver metastases at the time of diagnosis . tumor localization , clinical symptoms , and presentations at the time of diagnosis are listed in table 1 . median time from onset of first symptoms until diagnosis was 8.8 months ( range : 1.829 months ) . the common presenting symptoms were nasal obstruction ( 53% ) , epistaxis ( 31% ) , rhinorrhea , and facial pain ( 26% ) . patient treatments consisted of surgical resection , radiation therapy , and chemotherapy ( ctx ) ( table 3 ) . initial treatments included surgery alone in three patients , radiotherapy ( rt ) with or without ctx in five , surgery plus postoperative rt in seven and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , and finally ctx alone in one . in 14 patients , four of these patients had also radical neck dissection due to cervical lymph nodes involvement . nasal tumor resection in 2 patients , maxillectomy plus orbital exenteration in 1 , and external ethmoidectomy ( ee ) in 1 were also used as surgical treatments . of these 14 patients who underwent surgical tumor resection , 4 were treated with surgery alone , 5 with adjuvant radiation therapy after surgery , 2 with adjuvant ctx after surgery , and the remaining 3 with radiation therapy and ctx after surgery . of the remaining 5 patients , four in advanced stage and one in early stage refused surgical treatment . radiation therapy was administered to two of the patients , radiation therapy followed by ctx to the other two , and ctx alone to one patient . the average radiation dose was 57.9 gy and the ctx was chosen from cisplatin - based regimens . as a part of the primary treatment modality , 12 patients underwent radiation therapy , so did the five relapsed patients . as a part of primary treatment modality 8 patients received ctx , so did the three relapsed patients . fifteen patients died of tumor progression during the follow - up , of whom 9 were at an advanced stage ( stage c ) by the time of diagnosis and 6 at an early stage ( stage a / b ) . kadish stage c patients had a significantly poorer outcome compared to the stage a / b patients ( 2-year survival rate : 71 versus 25% , resp . histopathologically high - grade patients ( grade 3/4 ) had a significantly poorer outcome when compared to the lower grade ( grade 1/2 ) patients ( 2-year survival rate : 16 versus 50% , resp . primary tumor extensions to orbital area had a significantly poorer outcome compared to the tumors with no extension to orbital area ( 2-year survival rate : 22 versus 60% , resp . there was no statistically significant difference in survival rate between male and female patients ( 2-year survival rate : 40 versus 44% , resp . patients with age < 45 years had no poorer outcome compared to patients with age > 45 years ( 2-year survival rate : 41 versus 43% ; p = .059 ) . brain and bone marrow involvements showed poorer outcome compared to those without such involvement ( median survival rate : 9.1 versus 28.8 months , resp . ; survival rate was not significantly different among pateints of kadish c when stratified according to initial treatment ( p = .8 ) . locoregional recurrence developed in 6 and distant metastasis in 6 patients . during the follow - up period , these groups exhibited distant metastasis to the brain , spinal cord , bone , bone marrow , meninges , and lungs . as a treatment for recurrence and metastasis , reoperation of primary site plus radiation therapy in 4 patients and radiation therapy alone in 4 patients were offered .", "onb is a rare malignant tumor that comprises 4,65% of malignant nasal and paranasal tumors . some 1000 cases have been reported in the english literature since it was first introduced in 1924 . the incidence rate is bimodal , with peaks in the second and third decades of life and in the sixth and seventh decades of life and equal among females and males . as for our study , the age at diagnosis was most common in the fourth decade and female - to - male ratio was equal . the average time between the appearance of the first symptom and the diagnosis was reported as 6 months and this period was similar also in our group . the most common symptoms at the initial diagnosis were defined as unilateral nasal obstruction and epistaxis in the literature . this tumor may be misdiagnosed as an undifferentiated small cell carcinoma , melanoma , rhabdomyosarcoma , or other small blue cell neoplasms . hyam proposed a grading system with grades 14 based on the presence or absence of seven different histopathological parameters . these are growth , architecture , mitotic activity , necrosis , nuclear pleomorphism , rosette formation , and fibrillary stroma . . staining paterns for neuron - spesific enolase , chromogranin , synaptophysin , s-100 , and epithelial markers can be helpful . there is evidence that histological grade of onb influences biologic behavior , particularly because it relates to disease progression , local recurrence , and metastasis . for the well - differentiated tumors a slower disease progression and less tendency for local recurrence have been described . in our study , the 2-year survival rate of this grade i / ii - iii / iv disease was 16 versus 50% , respectively . more aggressive treatment , such as surgery , radiation , and intensive ctx , may be useful for patients with grade iii / iv disease . a meta - analysis showed that the median overall survival at 5 years is 45% in 390 patients . the distribution of onb from 21 studies according to kadish staging system was 12% at stage a , 27% at stage b , and 61% at stage c. the mean 5-year survival is 75% for stage a , 68% for stage b , and 41% for stage c . the conclusions of the university of virginia stated that kadish stage is predictive of disease - related mortality . however , in the series of the mayo clinic it was found that kadish stage did not affect the outcome . danish clinicopathological study found that the staging system of kadish was able to stratify into prognostically significant groups . in our study , the 5-year survival rate of these tumors was 26% and median survival time was 23 months . the distribution of onb according to kadish staging system was 16% at stage a , 26% stage b , and 58% stage c. however , in our study , the 5-year survival rates of these stage groups were inferior compared to those reported in other studies . the cause of this poor result was presentation of 5 patients with distant metastases ( 26% ; 2 brain , 1 bone marrow , 2 other sites ) , 4 patients ( 21% ) with cervical lymph node metastasis , 12 patients ( 63% ) with high - grade histology at the time of diagnosis , and inability to evaluate the surgical margins of the resected tumors . the survival rate of node positive patients is 29% compared with 64% of node negative patients . distant metastases have been described in the pancreas , the liver , mediastinum , bone marrow , lungs , leptomeninges , skin , and breasts [ 11 , 12 ] . cervical nodal metastases can develop in 17 to 33% of patients and distant metastases in 10 to 40% of patients over the course of disease metastatic disease at presentation occurs in 10 to 50% of patients , depending on the study [ 6 , 13 , 14 ] . we found the local recurrence rate to be 31.5% and distant metastasis 31.5% after initial surgery . during the follow - up periods these groups exhibited distant metastases to the brain , spinal cord , bone , bone marrow , meninges , and lungs . patients who had brain and bone marrow metastases had poorer prognosis than others having metastatic deposits of other sites . involvement of the orbit is a serious prognostic factor , but when the orbital periosteum is affected without penetration into the orbital structures themselves , the eye may be preserved by resection and grafting of the periosteum without an adverse effect on survival as compared with orbital clearance . irrespective to the most sophisticated imaging , involvement of the dura and orbital periosteum can accurately be determined only at surgery . in our study , this group 's ( 8 patients ) survival rate is poorer than those with no involvement of the orbita ( p = .042 ) . the institute of laryngology and otology , university college london , published a study of 42 patients treated over 23 years . overall and disease - free survival rates were 77 and 61% at 5 years , and 53 and 42% at 10 years , respectively . late recurrences were seen . based on this experience , combined therapy with craniofacial resection and radiation therapy is recommended by this group . in a retrospective study of 47 patients treated at multiple centers in germany from 1979 to 2001 , the 5-year overall survival and event - free survival rate were 64% and 50% , respectively . patients who received multimodality therapy had a significantly better event - free survival rate compared with those who did not receive multimodality therapy ( 74 versus 41% ) . the authors recommend a combination therapy including ctx , surgery , and postoperative radiation therapy for kadish stage c patients . in our study , initial treatment included surgery alone in three patients , rt with or without ctx in five , surgery plus postoperative rt in seven , and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , only ctx in one patient . however , in our study choice of treatment modality did not show a statistically significant difference in survival rate . the reasons for that might be including low number of patients in the study , being 5 distant site metastasis , 4 cervical lymph node metastasis at the time of diagnosis , only one orbital exentration ( although there were 8 orbital involvement ) , and 12 patients exhibiting high grade histology and inability to evaluate surgical margins in resected tumors . in conclusion , advanced tumor stage , the initial histopathologic grade of iii / iv , and involvement of orbita , brain , or bone marrow metastasis were the statistically significant poor prognostic factors , whereas age , sex , and treatment modality were not prognostic factors ." ]

[ "unrepaired dsbs are usually lethal for dividing cells due to the subsequent loss of the acentric fragment and the unstable break - end of the centric fragment . dsbs can be caused enzymatically , by ionizing irradiation , or by other s phase - independent clastogens . dsbs with only one break - end may appear when a replication fork meets a single - strand break or a repair - mediated parental single - strand discontinuity . mostly however , the break is extended by a 5-specific exonuclease activity generating 3-overhanging ends . as soon as overhanging ends meet at short stretches of complementary bases ( single - strand annealing , ssa ) , the distal free ends are resected , and the break becomes ligated , resulting in a ( micro-)deletion ( figure 1a ) . alternatively , the break - ends may invade undamaged homologous template strands and a limited dna synthesis across the break region is followed by re - ligation of both strands ( synthesis - dependent strand annealing , sdsa ) . the result is genetically not detectable when the template was the undamaged sister chromatid ; it appears as a gene conversion if an allele of the homologous chromosome served as a template ( figure 1c ) . if for break - end elongation an ectopic or an extra - chromosomal ( partially ) homologous sequence is used , the result may be linked with an insertion into the break ( figure 1b and puchta , 2005 ) . when the holiday junction ( caused by break - end invasion into a template double strand ) is resolved in connection with an exchange of the flanking region ( figure 1c ) , then , depending on the template double helix involved , a sister chromatid exchange , a crossing over , or a reciprocal translocation results . pathways of dsb misrepair via single - strand annealing(ssa ) or via synthesis - dependent strand annealing ( sdsa ) . ( a ) deletion via exonucleolytic 5-end resection , ssa at complementary overhang sequences , resection of the non - aligned ends , and ligation of break - ends . ( b ) insertion into a dsb by break - end invasion and elongationalong an ectopic and partially homologous ( vertical bars ) template.(c ) re - synthesis of break - ends after invasion into a homologous template double - strand without ( gene conversion ) or with exchange of flanking regions due to appropriate resolution of holiday junctions ( greenarrow heads ) . based on genetic experiments on budding yeast , a further mechanism , break - induced replication ( bir ; , 1996 , 2005 ; morrow et al . , 1997 ; haber , 1999 ; for review haber , 2006 ; mceachern and haber , 2006 ; llorente et al . , 2008 ) has been postulated . with the exception of a direct ligation of clean break - ends , all dsb repair events are linked with a limited replication step , bir is claimed to extend replication from the proximal break - end up to the end of the template chromatid , using a ( homologous ) undamaged double helix as template . mechanistically , bir appears as a non - reciprocal translocation event ( bosco and haber , 1998 ) which can be considered as a type of gene conversion extending from the breakpoint up to the telomere . simultaneously , the original distal fragment ( if occurring ) of the broken double helix is lost ( figure 2 ) . schematic models of replication and chromosome labeling patterns after bir at proximal dsb ends in s and g2 . ( a ) bir through conservative replication of a one ended dsb during s phase . the dsb appears when the replication fork arrives at a single - strand break ( arrow head ) . conservative replication occurs via recurrent strand invasion ( or via unidirectional fork migration ) without resolution of the holiday junction(s ) using the parental double strand as a template . ( b ) bir during g2 phase , through conservative replication at the proximal end of a dsb ( arrow head ) via recurrent strand invasion and/or via unidirectional fork migration without resolution of the holiday junction(s ) using the undamaged sister double helix as a template . ( c ) bir during g2 phase through semiconservative replication achieved by resolution of the holiday junction ( green arrow head ) after invasion of the elongating break - end into the template double strand . full lines unlabeled ; broken lines labeled by edu . the distal fragment of the broken double helix in ( b , c ) gets lost . when a homologous or a heterologous chromatid instead of the sister chromatid is involved in bir , a non - reciprocal translocation between homologous or heterologous chromosomes is mimicked . microhomology - mediated bir involving heterologous chromosomes leads to duplication of the template region and deletion of the region distal the original dsb . the problem of bir before or after regular replication in species with large chromosomes is that breakage occurring distantly to the arm end requires replication of the involved region ( larger than a chromatin unit of jointly regulated replication comprising 1 mb , zink et al . , 1998 ) more than once between two nuclear divisions , thus overriding the licensing mechanism which allows only one round of replication between two divisions . bir occurring during s phase ( figure 2a and hastings et al . , 2009 ) requires a recurrent 3-end invasion into a still unreplicated template chromatid ( most likely the sister chromatid ) , or a continuous migration of the replication fork toward the arm end . resolution of the holiday junction results in a reciprocal exchange of the distal regions between the chromatids involved . bir during s phase without resolution of the holiday junction circumvents the dsb , yielding a conservative replication pattern with regard to the broken chromatid distal to the breakpoint . to test whether bir may occur within chromosomes of higher plants after dsb induction , we treated field bean root tip meristems with the s phase - independent clastogen bleomycin during s or g2 phase and checked for the expected chromatid labeling by incorporation of the base analog ethynyldeoxyuridine ( edu , see kotogany et al . , 2010 ) also semiconservative bir at terminal chromatid regions ( figure 2c ) was not observed after breakage in the presence of the base analog during g2 . our results suggest that bir either does not occur or is so infrequent that its role within the concert of dsb repair mechanisms can at best be a minor one in organisms with a large dna content ( > 1 mb ) per chromosome arm .", "the field bean , vicia faba l. , karyotype acb with six large and individually distinguishable chromosome pairs ( dbel et al . , 1978 ) is much more comfortable for aberration scoring than for instance a. thaliana with smaller chromosomes and less mitoses per root meristem . seeds were germinated for 3 days on wet filter paper at room temperature in the dark . about 12 cm long primary roots were incubated for 18 h in aerated hoagland solution , ph 5.5 , containing 1.25 mm hydroxyurea which synchronizes cells by blocking the cell cycle in early s phase . for mutagen treatment during s phase , the roots were incubated for 1.5 h in hoagland solution containing 10 g / ml bleomycin with or without 20 m edu ( invitrogen ) followed by incubation in hoagland solution for 4.5 h and then 0.05% colchicine for 2.5 h ( to arrest metaphases ) before fixation in ethanol : acetic acid ( 3:1 ) overnight . for scoring of chromatid aberrations , roots were washed for 10 min in distilled water , hydrolyzed for 11 min in 1 n hcl at 60c , stained for 3040 min in feulgen solution and squashed in a drop of 45% acetic acid . only complete metaphase cells were scored for chromatid aberrations : chromatid and isochromatid breaks ( one or both sister chromatids with terminal deletion ) , interstitial deletions , duplication deletion ( the deleted part of one chromatid is inserted into a break of the sister chromatid ) , and reciprocal chromatid translocations . alternatively , the root tips were fixed in 4% formaldehyde for 20 min for isolation of metaphase chromosomes ( schubert et al . , 1993 ) and subsequent detection of edu incorporation using the click.it edu imaging kit from invitrogen according to manufacturer 's instruction . microscopic analysis of chromosomes was performed with an epifluorescence microscope ( zeiss axiophot ) using a 100/1.45 zeiss alpha - plan - fluar objective and a sony ( dxc-950p ) camera . images of fluorescent chromosomes were captured using filter sets f36 - 513 for dapi and f36 - 750 for alexa fluor 594 ( ahf analysentechnik , germany ) . dapi and alexa fluor 594 azide images were merged using adobe photoshop 6.0 software ( adobe systems , san jose , ca , usa ) . for mutagen treatment during g2 phase , primary roots were incubated in hoagland solution with hydroxyurea as above , then in hoagland solution for 4.5 h and subsequently in hoagland solution with bleomycin , with edu , or both for another 1.5 h followed by 2.5 h in colchicine before fixation , staining , and evaluation .", "the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10 g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) . the same bleomycin concentration applied for 1.5 h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations . treatment with edu ( 20 m , 1.5 h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase . bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) . although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin . chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) . t = reciprocal translocation , i = isochromatid break , d = interstitial deletion , b = chromatid break , see figure 3a . when root tips were incubated in 20 m edu 8.57 h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) . no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids . only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid . type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . * ( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) . none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) . thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) . since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone . ( a ) chromatid - type aberrations after bleomycin treatment . left cell : isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots . middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) . the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) . right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . ( b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase . ( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 . the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) . when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5 h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment . no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) . also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed . single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes . after simultaneous exposure to edu and to bleomycin 42.5 h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment . however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions .", "the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10 g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) . the same bleomycin concentration applied for 1.5 h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations . treatment with edu ( 20 m , 1.5 h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase . bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) . although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin . chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) . t = reciprocal translocation , i = isochromatid break , d = interstitial deletion , b = chromatid break , see figure 3a .", "when root tips were incubated in 20 m edu 8.57 h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) . no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids . only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid . type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . * ( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) . none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) . thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) . since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone . isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots . middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) . the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) . right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . ( b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase . ( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 . the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) . when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5 h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment . no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) . also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed . single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes . after simultaneous exposure to edu and to bleomycin 42.5 h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment . however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions .", "if bir occurs in higher plants in the same way as postulated for yeast ( i.e. , as replication extending from the break position up to the corresponding chromosome arm end ) conservative replication in s or g2 ( figures 2a , b ) or semiconservative replication in g2 ( figure 2c ) should be detectable . of the chromatid aberrations observed after bleomycin and bleomycin plus edu treatment during s and g2 , reciprocal translocations ( 25 ) , isochromatid breaks ( 36 ) , and interstitial deletions ( 7 ) require two dsbs each , while simple chromatid breaks ( 87 ) representing terminal deletions , go back to a single dsb . therefore , within 600 g2 and 750 s phase cells , bleomycin caused 223 dsbs which underwent misrepair . thus , on average at least one dsb per six cells was induced . considering that a fraction of dsbs might have been repaired via pathways not resulting in microscopically detectable chromatid - type aberrations ( for instance by sister chromatid exchange , intrachromosomal ssa , or gene conversion ) , the actual number of dsbs may be higher . however , in a number of chromosomes corresponding to 308 s and g2 cells , treated simultaneously with the chromosome - breaking agent bleomycin and the base analog edu , not a single chromosome showed a labeling pattern indicative of bir . in budding yeast , more than half of diploid cells repair ho - endonuclease - induced dsbs by bir using the homologous chromosome as template ( bosco and haber , 1998 ) ; meaning more than half of the dsbs are repaired via bir if the endonuclease ho causes one dsb per diploid yeast cell . had bir occurred with a similar efficiency in the field bean , we should have observed > 223 instead of no chromosomes with asymmetric terminal edu signal . our result does not exclude the occurrence of bir in plants , however , the frequency of such events is below 0.45% ( less than 1 out of 223 ) of the dsbs causing chromosome structural aberrations , i.e. , more than 100-times lower than in yeast . unidirectional replication forks that override distal replication origins have to migrate up to the arm ends , or , alternatively , the requirement of recurrent invasion of 3-ends into other double helices , are presumptions for which experimental evidence is lacking so far . long range replication in addition to the regular round of replication between two nuclear divisions , represent a further unproven assumptions . to our knowledge no experimental evidence as to the occurrence of bir in animals bas been shown ( zhang et al . , 2009 ) . taken together these arguments imply that the existence of a bir pathway in higher eukaryotes is unlikely . even when such events occur , their frequency is much lower than in yeast arguing against their importance as a pathway for dsb repair at least in higher plants such as the field bean . because the 12 megabase pair ( mb ) genome size of saccharomyces cerevisiae is distributed over 32 chromosome arms , the risk that bir interferes with replication licensing at the replicon level is much smaller than for species with orders of magnitude larger genomes with several mb per arm . also complex chromosome rearrangements ( e.g. , difilippantonio et al . , 2002 ; zhang et al . , 2009 ) can more easily be explained by other well - documented pathways ( zhang et al . , 2009 ; schubert and lysak , 2011 ) rather than by bir via recurrent invasion of a dsb - induced 3-end into different template double helices . the more so as experimental approaches even in yeast can not differentiate between bir and gene conversion events involving g2 cross over , followed by appropriate chromatid segregation nevertheless , the increased frequency of interstitial asymmetric labeling patterns of chromosomes exposed to bleomycin and edu during s or g2 might indicate that conserved replication , although not reaching arm ends , could be linked with dsb repair to result in microscopically detectable stretches of gene conversion extending hundreds of kilobases ( possibly , up to the junction with the next replication unit , but not covering dna stretches of > 1 mb up to the arm ends ) . such extended gene conversion - like repair might be considered as bir , if the postulation up to the chromosome terminus is excluded . similar events could potentially have been responsible for the zebra structure of the long arm of chromosome z5a observed in a backcross progeny between elymus trachycaulus gould ex shinners and triticum aestivum l. ( zhang et al . , 2008 ) . the preferential appearance of local asymmetric labeling within heterochromatic regions of the field bean suggests that for late replicating repetitive dna the units of replication regulation are larger or more variable than for euchromatin .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ]

[ "is an effort to manipulate the metabolism of an organism worthy and reasonable , \t knowing \n that this cellular process has been continuously modified and refined through \n evolution and natural selection for adapting , in the most convenient manner , to \n the ongoing environmental conditions ? the \n answer to this question seems obvious when three broad areas of research and \n development are identified in which manipulation of metabolic pathways is \n relevant : ( a ) drug design to treat diseases , ( b ) genetic engineering of \n organisms of biotechnological interest , and ( c ) genetic syndromes therapy . historically , drug design was \n the first area in which modification of metabolism was tried : the primary goal \n of drug administration is the inhibition of essential metabolic pathways , for \n example , in a parasite or a tumor cell . thus , any metabolic pathway can be a potential \n therapeutic target . in the absence of a solid \n theoretical background that may build a strategy for the rational design of \n drugs , the pharmaceutical industry has applied the knowledge of inorganic and \n organic chemistry for the arbitrary and rather randomized modification of \n metabolic intermediaries by replacing hydrogen atoms in a model molecule with \n any other element or compound . the era of rational drug \n design probably started in the 50s when hans krebs proposed that , after having \n an exact description of a metabolic pathway , the pacemaker enzyme or \n rate - limiting step had to be identified . this approach certainly decreased the amount \n of intermediaries to be chemically modified , focusing only on the substrates , \n products , and allosteric effectors of the rate - limiting step , instead of dispersing \n efforts on all the metabolic pathway intermediates . the experimental approaches used in the \n identification of the pacemaker , key enzymes , bottlenecks . limiting steps , or \n regulatory enzymes [ 1 , 2 ] were \n inspection of the metabolic pathway architecture : due to cell \n economy and for reaching the highest efficiency , pathway control must \n reside in the enzymes localized at the beginning of a pathway or after a \n branch ( teleological approach);determination of nonequilibrium reactions : those reactions in which \n the quotient between the mass action ratio ( ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n approach);identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n enzyme of the pathway is the one that has the lowest rate ( kinetic \n approach);enzymes with sigmoidal kinetics : steps that are susceptible to \n alteration in their kinetic properties by compounds different from \n substrates and products and which may coordinate the entire metabolism \n ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n malonyl - coa);crossover theorem . comparing the intermediary concentrations \n between a basal and an active steady - state pathway flux , the rate - limiting \n step in the basal condition will be that for which its substrate \n concentration diminishes and its product concentration increases when the \n system changes from the basal to the active state or vice versa ( crossover \n point on a histogram of each intermediary versus its normalized variation in concentration);the shape of the metabolic flux inhibition curve : a sigmoidal curve \n on a plot of inhibitor concentration versus flux shows that the sensitive \n step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n activity inhibition and pathway flux inhibition because there is an \n excess of enzyme . on the other \n hand , a hyperbolic curve indicates that the enzyme susceptible to the \n inhibitor controls the flux . \n inspection of the metabolic pathway architecture : due to cell \n economy and for reaching the highest efficiency , pathway control must \n reside in the enzymes localized at the beginning of a pathway or after a \n branch ( teleological approach ) ; determination of nonequilibrium reactions : those reactions in which \n the quotient between the mass action ratio ( ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n approach ) ; identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n enzyme of the pathway is the one that has the lowest rate ( kinetic \n approach ) ; enzymes with sigmoidal kinetics : steps that are susceptible to \n alteration in their kinetic properties by compounds different from \n substrates and products and which may coordinate the entire metabolism \n ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n malonyl - coa ) ; crossover theorem . comparing the intermediary concentrations \n between a basal and an active steady - state pathway flux , the rate - limiting \n step in the basal condition will be that for which its substrate \n concentration diminishes and its product concentration increases when the \n system changes from the basal to the active state or vice versa ( crossover \n point on a histogram of each intermediary versus its normalized variation in concentration ) ; the shape of the metabolic flux inhibition curve : a sigmoidal curve \n on a plot of inhibitor concentration versus flux shows that the sensitive \n step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n activity inhibition and pathway flux inhibition because there is an \n excess of enzyme . on the other \n hand , a hyperbolic curve indicates that the enzyme susceptible to the \n inhibitor controls the flux .", "once a site in a metabolic \n pathway has been identified with at least one of the criteria described above \n as the rate - limiting step , researchers have frequently concluded that such \n enzyme or transporter is the only limiting step of the metabolic flux and \n extend this conclusion to all cell types and to all conditions . for example , inspection of the glycolytic pathway ( teleological approach ) suggests that \n hexokinase ( hk ) and phosphofructokinase-1 ( pfk-1 ) ( which are at the beginning \n and after a branch of the pathway ) are the key steps of glycolysis . however , all studies on glycolysis in the 60s , \n 70s , and 80s were performed by taking into account only the intracellular \n reactions from hk to ldh ( i.e. , without including the glucose transport \n reaction through the plasma membrane ) and by considering glycolysis as a linear \n pathway without branches . to this regard , \n it is recalled that the glucose transporter ( glut ) includes a family of \n proteins and genes that are susceptible of regulation . thus , if the extracellular glucose is \n considered as the initial glycolytic substrate , then another potential key step \n would be glut . hence , if all the \n branches of the pathway are considered ( figure 1 ) , then according to the \n teleological approach there will be additional potential rate - limiting sites . application of the thermodynamic and kinetic approaches to glycolysis reveals that hk , \n pfk-1 , and pyruvate kinase ( pyk ) are the rate - limiting steps because in the \n living cell they catalyze reactions that are far away from equilibrium ( /keq = 1010 ) , and they are also the slowest \n enzymes in the pathway by at least one order of magnitude ( they have the lowest vmax values ) . the use of the enzyme cooperativity approach has established that the regulatory \n steps of glycolysis are ( i ) pfk-1 and pyk because they are allosteric enzymes \n and ( ii ) hk because it is inhibited by its products ( g6p and adp , or amp as an adp - analogue ) . \n the application of the crossover theorem \n ( approach no . v ) to glycolysis has shown a consistent variation in the pfk-1 substrate \n ( f6p ) and product ( f1,6bp ) . up to now , \n there are few studies on control of glycolysis using the shape of the inhibitor \n titrating curve ( approach no . vi ) , due to the lack of specific inhibitors for \n any of the three presumed key steps . an exception is iodoacetate which is indeed \n a potent inhibitor of gapdh , but also of other highly reactive \n cysteine - containing enzymes [ 35 ] . by \n using iodoacetate as specific inhibitor , both gapdh activity and flux showed \n identical titration curves , leading to the conclusion that gapdh was the \n rate - limiting step of glycolysis in streptococcus lactis and s. cremoris ( see , \n however , section 3.2 ; glycolysis in lactobacteria below ) . all together , these results constitute \n the main reason why many intermediary metabolism researchers , including the \n authors of biochemistry text books , have proposed hk , pfk-1 , and pyk as the \n rate - limiting steps of glycolysis . in \n consequence , to vary the glycolytic flux , one of these enzymes has to be \n modified . although the above - described experimental approaches are qualitative , full control has \n been automatically assigned to the key steps because the concept of the rate - limiting \n step assumes that there is only one single enzyme controlling the metabolic \n pathway flux ( and the concentration of the final product of the pathway ) and , \n in consequence , assigns values of zero to the control exerted by the other \n enzymes and transporters . however , as analyzed for glycolysis , researchers have commonly identified more than one \n limiting step . in the case of oxidative \n phosphorylation ( oxphos ) , in the 70s and 80s some researchers considered \n cytochrome c oxidase as the rate - limiting step , whereas others preferred the \n atp / adp translocator or the krebs cycle ca - sensitive \n dehydrogenases ( for a review , see ) . rephrasing the initial question , which could be the aim of manipulating a metabolic \n pathway such as glycolysis , knowing its universal distribution in the living \n organisms ? from a clinical standpoint , the inhibition of glycolysis is relevant for the treatment of human parasitic \n or pathological diseases such as cancer . the glycolytic reactions are almost identical in all organisms ; in \n addition , the enzymes catalyzing these reactions are highly conserved throughout \n the evolutionary scale ( their amino acid sequences are highly similar ) . in mammals , the genes of the 12 glycolytic \n enzymes are scattered throughout the genome , generally in different chromosomes , \n whereas in bacteria many of the glycolytic enzymes are clustered in operons . \n however , there are organisms ( like some \n human parasites ) that contain enzymes with remarkable differences in their \n biochemical properties ( substrate selectivity , catalytic capacity , stability , \n and oligomeric structure ) , or in genetic expression regulation in comparison to \n the human enzymes , which could be considered as drug targets . furthermore , some glycolytic products are of commercial interest such as ethanol for wine , \n beer , and other alcoholic beverages ; co2 for bread manufacturing ; and \n lactic acid and other organic acids for cheese production . thus , from a biotechnological standpoint , it \n is convenient to accelerate the pathway flux to diminish the processing time and \n it is also desirable to increase the concentration of the metabolite to obtain \n robust commercial products . here , it is important to emphasize that the metabolic pathways are designed to attain \n changes in flux with minimal disturbances in the intermediary concentrations . for example , the glycolytic flux in skeletal \n muscle can increase from rest to an active state by 100 fold , without large \n changes in metabolites . then , it is physiologically more common to change a metabolic flux and the production of \n the final metabolite in the pathway than varying the intermediary concentrations \n . however , we will see that , by \n using a suitable approach of metabolic control analysis , it is possible to \n design strategies to manipulate not only fluxes but also metabolic intermediary \n concentrations .", "when the yeast saccharomyces \n cerevisiae is exposed to high glucose ( > 2% ; 0.11 m ) , the genes of all \n glycolytic enzymes are induced ( pdc and eno increase their expression by 20 fold ; \n pgk , pyk , and adh , 310 times ; and the \n others , 2 fold in average ) [ 811 ] . however , \n when the methodological development of genetic engineering allowed modulating the \n expression of enzymes within cells , researchers turned to the rate - limiting \n step concept to manipulate a metabolic pathway to increase flux and/or its \n intermediates , hypothesizing that the overexpression of only one , or of a few \n key glycolytic genes , should increase the flux . historically , heinisch in germany was the first author to \n obtain a 3.5 fold overexpression of pfk-1 in s. cerevisiae , but \n surprisingly he observed that the rate of ethanol production was not modified . subsequent experiments for increasing the \n ethanol production rate by overexpressing either each of the presumed limiting \n steps , or in combination with other glycolytic enzymes ( table 1 ) , have been \n unsuccessful and , even in some cases , a slight decrease in flux has been \n attained . for instance , the simultaneous \n overexpression of seven enzymes of the final section of glycolysis induced only \n a 21% increase in ethanol production after 2 hours of culture ( table 1 ) . this was accompanied by a 1020% decrease in pfk-1 \n expression , which might have attenuated the flux increase . in yeasts , hk is not product inhibited \n by g6p or adp ; instead , it is strongly feedback inhibited by trehalose-6-phosphate \n ( tre6p ) . deletion \n of the tre6p synthase gene does not \n bring about an increased ethanol production , but it rather induces a defective cellular \n growth on glucose and fructose and a lowered ethanol production , as a result of \n a highly active hk that leads to hyperaccumulation of hexose phosphate \n metabolites ( particularly f1,6bp ) and fast depletion of atp , pi , and downstream \n metabolites . the explanation for this event is that , in the tre6p synthase \n mutants , the rate of glucose phosphorylation exceeds the rate of glycolytic atp \n synthesis ( named turbo effect ) . heterologous expression of a tre6p - insensitive hk does not recover \n completely the wild - type phenotype . furthermore , deletion of the tre6p synthase gene in the \n tre6p - insensitive hk strain did affect growth , suggesting other interactions \n and functions of tre6p synthase in the control of sugar metabolism , at \n least in schizosaccharomyces pombe . davies and brindle obtained a 5-fold overexpression of \n pfk-1 in s. cerevisiae , but the increase in ethanol production was not attained \n under anaerobic conditions . there was a slight \n increase in ethanol production in resting cells in aerobic conditions , under \n which the mitochondrial metabolism contributes to the atp supply . in all these works , it may be noted that \n enzyme overexpression indeed affects the concentration of several intermediaries , \n but this effect has not been further examined . it is worth noting that the \n experiments described in table 1 do not rigorously reproduce the physiological \n situation , in which overexpression of all the enzymes should be carried out in \n the proportions found in the organisms . the \n rationale behind this observation is that overexpression of only one limiting \n step leads to a flux control redistribution , a condition at which other steps \n now become rate limiting . thus , the \n concept of rate - limiting step offers no simple answer to the question of \n increasing the yeast glycolytic flux , and it rather makes this problem to \n appear as a difficult task to solve . in contrast , it seems that all relevant controlling steps \n have to be overexpressed , thus reproducing what natural \n selection has already successfully accomplished . in addition to s. cerevisiae , overexpression of glycolytic enzymes in other \n organisms such as e. coli [ 15 , 16 ] , \n lactobacteria , tomato , potato , and \n hamster ovary cells has been accomplished , although without increasing \n flux ( table 1 ) . it is somewhat surprising to note that in the glycolytic enzyme \n overexpression experiments , the strong inhibitory effect of g6p ( or tre6p in s. cerevisiae ) , and citrate on hk and pfk-1 , respectively , have been neglected . this \n regulatory mechanism does not disappear in the cells overexpressing the enzymes \n but , on the contrary , it is exacerbated . then , what would be the aim of overexpressing hk , pfk-1 or any other \n allosteric , or strongly product - inhibited enzyme if they will be more \n inhibited ? a successful experiment of increasing \n the glycolytic flux was performed in primary cultures of rat hepatocytes . the transformed \n hepatocytes showed higher activity of 18.7- and 7.1-times for hk and gk , \n respectively , at 3 mm glucose , and of 6.3- and 7.1-times at 20 mm glucose . however , at 20 mm glucose , the flux to \n lactate was not modified in hk - transformed cells , just like the experiments \n described above ( table 1 ) . in contrast , \n with gk overexpression , a 3-fold increase in flux was achieved . the mechanistic difference is the hk \n inhibition by g6p ( 10 mm g6p inhibits hk activity by 90% ) , whereas gk is not product inhibited . \n the end products , lactate and h , are \n expelled and acidify the external medium which contributes to cheese flavor and \n texture and inhibits the growth of other bacteria . similarly to yeast , the lack of carbon source \n in lactobacteria promotes a metabolic change that leads to the production of \n formic and acetic acids , ethanol , and , in a lower proportion , l - lactic acid , \n altering the product quality . thus , from a commercial point of view , it does \n not seem important to know what controls the flux to lactate ( because its rate \n of production is adequate ) , but what controls the branching flux . to understand the process , and to eventually inhibit the production of secondary acids , andersen et al . constructed ldh mutants , \n using a synthetic promoter library for tuning the gene expression . in mutants \n lacking this enzyme , most of the pyruvate was transformed into acetic and \n formic acids ( figure 1 ) . in turn , flux \n to lactate was affected in mutants expressing only 10% or less of wild - type ldh \n levels , which indicated that ldh exerts no control of the glycolytic flux in \n wild - type bacteria . only with a normal content of this enzyme ( 100% ) , therefore , the flux to formic and acetic \n acids is negatively controlled by ldh , and positively by pyk [ 17 , 25 ] . as in s. cerevisiae , overexpression of \n pfk-1 , pyk , or gapdh in lactobacteria did not increase the flux to l - lactic \n acid [ 17 , 25 ] . similarly to e. coli glycolysis , glycolysis in l. lactis was controlled by the atp \n demand when working below its maximum capacity [ 27 , 28 ] , whereas , under high - rate \n conditions , the glucose and lactate transporters exerted the main flux control . furthermore , this kind of observations indicates \n that the flux control may reside outside the pathway [ 2729 ] , and it also supports the proposal \n by hofmeyr and cornish - bowden that the end - product demand ( which is \n usually overlooked in studies of metabolism because these metabolites are \n frequently not considered as part of the pathway ) might be essential in flux \n control . glutathione ( -glu - cys - gly ; gsh ) is the most abundant nonproteinaceous \n thiol compound ( 110 mm ) \n in almost all living cells . gsh is \n involved in the oxidative stress processing , xenobiotic detoxification , and , in \n some plants and yeasts , in the inactivation of toxic heavy metals ( for a recent \n revision see ) . gsh is synthesized by two enzymes : -glutamylcysteine synthetase \n ( -ecs ) and glutathione synthetase ( gs ) ( figure 2 ) , \n which catalyze reactions with high - equilibrium constants ( keq > 1000 ) . under a low gsh demand ( unstressed \n conditions ) , the producing block of enzymes has to receive information from the \n last part of the pathway to ( i ) avoid the excessive and toxic accumulation of \n the intermediary -ec and ( ii ) reach a stable steady state . this information transfer is mediated by gsh , \n which exerts strong competitive inhibition of -ecs ( figure 2 ) . gsh and cys also exert inhibition on the \n atp - sulfurylase ( atps ) and on sulfate transporters ( figure 2 ) ( for a review , \n see ) . the feedback inhibition of -ecs has led several researchers to propose \n that this enzyme is the rate - limiting step of gsh synthesis [ 3335 ] . although there are no studies about the \n pathway 's behavior under stressed conditions , which means under a high gsh \n demand , the proposal that -ecs is the key enzyme has been automatically \n extended to any environmental condition such as heavy metal exposure . by assuming that -ecs is the rate - limiting step , many research groups \n have tried to increase , in plants and yeasts , the rate of synthesis and the \n concentration of gsh and phytochelatins ( pcs ) with the aim of fortifying their \n heavy metal resistance and storage capacity , mainly toward cd . the development of organisms able to grow in \n soils and water systems contaminated with heavy metals , which may have the \n ability of accumulating toxic metal ions , is of biotechnological interest for \n bioremediation strategies . with this goal in mind , \n researchers have then overexpressed -ecs and other pathway enzymes , including \n phytochelatin synthase ( pcs ) ( table 2 ) . some of these experiments have been \n partially successful in increasing gsh levels , although this has been rather \n marginal with no correlation between enzyme levels and gsh concentration . unfortunately , these overexpression experiments \n have not been accompanied by determinations of fluxes or other relevant metabolite \n concentrations such as pcs or cys . on \n the other hand , the overexpression of pcs has surprisingly induced oxidative stress \n and necrosis instead of increasing cd accumulation and resistance \n . this result suggests that , under high \n gsh demand ( i.e. , for pcs synthesis and for direct heavy metal \n sequestration by gsh ) , the gsh concentration does not suffice for maintaining \n the other essential gsh functions such as oxidative stress management and \n xenobiotic detoxification . another problem in the study \n of gsh biosynthesis for its eventual manipulation is that the pathway has been \n analyzed considering only the gsh - synthetic reactions without taking into \n account the gsh - consuming reactions ( figure 2 ) , . the analysis of an incomplete pathway leads to \n misleading conclusions about the control of flux . metabolic modeling has shown that only with \n the incorporation of the consuming reactions of the pathway end products , a true \n steady state can be established . in \n conclusion , without a solid theoretical framework , the overexpression of only \n one enzyme ( the rate - limiting step ) , or of many arbitrarily selected enzymes \n ( tables 1 and 2 ) , the problem of increasing the flux or metabolite \n concentrations can not be solved . there are some successful examples of the genetic engineering approach to manipulate \n metabolism : overexpression \n ( approx . 23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] . in these transformed bacteria , the end products \n are indeed overproduced and their excretion is accelerated;overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol . \n overexpression \n ( approx . 23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] . in these transformed bacteria , the end products \n are indeed overproduced and their excretion is accelerated ; overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol .", "several glycolytic enzymes are overexpressed in \n at least 70% of human cancers . except \n for glucose transporter 1 ( glut-1 ) , the other 11 glycolytic enzymes ( hk to ldh ) \n are overexpressed in brain and nervous system cancers . prostate and lymphatic nodule cancers ( hodgkin \n and non - hodgkin lymphomas ; myelomas ) overexpress 10 glycolytic enzymes ( except \n for hk ; in prostate cancer glut1 is also overexpressed ) . there is a second group of cancers that overexpresses 68 glycolytic \n genes ( skin , kidney , stomach , testicles , lung , liver , placenta , pancreas , \n uterus , ovary , eye , head and neck , and mammary gland ) . a third group includes those cancers overexpressing \n 1 or 2 glycolytic genes ( bone , bone marrow , cervix , and cartilage ) . in animals , gene expression of glycolytic enzymes is regulated ( both coordinately \n and individually ) under hypoxic conditions by hypoxia - responsive transcription \n factors such as hif-1 ( hypoxia - inducible factor 1 ) , sp family factors , ap-1 , and possibly mre \n ( metal response elements ) [ 8 , 5961 ] . . there are binding sites ( consensus sequence acgt ) for hif-1 in the promoters of genes for hk , pfk-1 , \n aldo , gapdh , pgk , eno , pyk , and ldh ( reviewed in ) . tpi and perhaps hpi and pgam are also induced \n by hypoxia , but it is not clear whether hif-1 mediates this induction , and whether this \n factor regulates other metabolic pathways associated with glucose \n catabolism . for example , although \n glycogen phosphorylase is overexpressed under hypoxia in human tissues , \n the role of hif-1 has not been demonstrated . if direct manipulation of \n pathway genes becomes difficult , then the overexpression or repression of \n transcription factors such as hif-1 , ap1 , and mres might solve the problem of changing \n flux , although overexpression of transcription factors may also be difficult \n due to the numerous upstream and downstream factors involved . the kinetoplastid parasites trypanosoma \n cruzi , trypanosoma brucei , and leishmania are the causative agents of \n chagas disease , african trypanosomiasis , and leishmaniasis , respectively . the \n available drugs to treat these diseases are highly toxic for humans . moreover , the \n parasites may become resistant , and hence the search for new drugs and drug \n targets is relevant for solving these public health problems . in these parasites , the \n metabolism is organized in a peculiar way ; they have a subcellular structure \n called glycosome in which several metabolic pathways take place : \n gluconeogenesis , reactions of the pentose phosphate pathway , purine salvage and \n pyrimidine biosynthesis , -oxidation \n of fatty acids , fatty acid elongation , biosynthesis of ether lipids , and the \n first seven steps of glycolysis . glycosomal glycolytic enzymes have unique structural , kinetic , and \n regulatory features not found in their human counterparts , and therefore have \n been the subject of extensive biochemical studies to use them as drug targets . the rationale behind this is to synthesize \n inhibitors that affect mainly the parasitic enzymes with relatively low effect \n on the human enzymes since the infective parasite stages rely mostly on \n glycolysis for atp supply . there are reports on the \n design of presumed specific inhibitors for some of the t. brucei glycolytic \n enzymes : glut ( bromoacetyl-2-glucose ) , hk , hpi , pfk , aldo , tpi , gapdh , \n pgk , pyk , and glycerol-3-phosphate dehydrogenase . although the purified enzymes display \n very low ki values for these \n inhibitors and some of them inhibit \n parasite growth or infective capabilities , their effect on inhibiting the \n glycolytic flux has not been explored . therefore , it is not yet possible to \n directly ascribe the effects seen in parasite culture with the in vitro effects \n on the isolated enzymes . to identify the \n best drug targets , determination of the flux control steps of glycolysis \n in t. brucei has been recently initiated . trypanothione ( tsh2 ) \n is a reducing agent present in trypanosomatids that is synthesized from one \n spermidine and two gsh molecules by tsh2 synthetase ( trys ) \n ( figure 3 ) . this metabolite and its reducing enzyme , \n tsh2 reductase ( tryr ) , replace the antioxidant and metabolic \n functions of the more common gsh / gsh reductase system present in mammals . in \n fact , most of the antioxidant metabolism of these parasites depend on tsh2 ( figure 3 ) [ 69 , 70 ] . thus , the enzymes of this metabolic pathway have been \n proposed as drug targets for killing the parasites . diminution in its gene transcription yields a \n loss of activity between 5690% , depending on \n the genetic technique [ 7275 ] . in \n knockdown t. brucei cells ( i.e. , when tryr activity has diminished \n to less than 10% of the wild - type level ) , the parasites show growth diminution \n and higher sensitivity to h2o2 in culture and loss of \n infectiveness in mice . however , tsh2 and thiol compound contents \n were not affected . tryr \n downregulation by > 85% in leishmania species causes inability to survive under \n oxidative stress inside macrophages [ 7274 ] . in contrast , when tryr is 14- and 10 fold \n overexpressed in leishmania and t. cruzi , respectively , there are no \n significant differences in h2o2 susceptibility between \n control and transfected cells ; both types of cells are also equally resistant \n to the oxidative stress - inducers gentian violet , and nitrofurans . intriguingly , the cellular levels of tsh2 , \n gsh , and glutathionyl - spermidine , determined in both types of experiments ( tryr \n suppression and overexpression ) were similar in control and transformed cells . knockdown \n of trys by sirna in procyclic t. brucei causes ( i ) viability impairment and arrest of proliferation when tsh2 levels decrease to 15% of the wild - type level , ( ii ) increased sensitivity to h2o2 and alkyl hydroperoxides , ( iii ) damage to the plasma membrane , and ( iv ) \n diminution of the tsh2 content and accumulation of gsh and \n glutathionyl - spermidine . a similar metabolite variation ( lower tsh2 ; \n higher gsh ) was attained with a trys knockdown induced by sirna in the \n bloodstream form of t. brucei . this \n trys knockdown also induced an increased sensitivity to different compounds \n that affect tsh2 metabolism such as arsenicals , melarsen oxide , \n trivalent antimonials , and nifurtimox . indeed , western blot analysis showed , \n in addition to the expected ( 10-fold ) decrease in trys protein , a 2 - 3-folds increase in -ecs and tryr . the changes in expression of \n other enzymes suggest unveiled compensatory or pleiotropic effects on tsh2 metabolism . other researchers have selected -glutamylcysteine synthetase ( -ecs ) , the presumed rate - limiting step of gsh \n synthesis , as an alternative drug target of tsh2 synthesis in t. brucei ( figure 3 ) . knockdown of -ecs gene in the parasite induces cell death \n and depletion of gsh and tsh2 only after 80% decrease in the enzyme \n content . the -ecs knockdown cells are rescued from death by \n adding external gsh , which elevates the cellular gsh and tsh2 levels \n . glutathione synthetase ( gs ) has not been manipulated in trypanosomatids , or in any other organism , perhaps \n because it has been considered as a nonrate - limiting step of gsh and tsh2 biosynthesis . however , dna microarray \n analysis of antimonite - resistant leishmania tarentolae shows increased \n transcription of -ecs , gs , and p - glycoprotein a rnas . \n although it was not evaluated whether increase in gene transcription correlated \n with an increase in enzyme activity , it may be possible that under high gsh \n demand ( i.e. , under oxidative stress conditions ) gs might exert control of tsh2 synthesis . on the other hand , ornithine \n decarboxylase ( odc ) overexpression in t. brucei ( the presumed limiting \n step of spermidine synthesis ) causes no change in tsh2 levels \n . therefore , odc does not seem to be \n a controlling step of tsh2 synthesis . although almost full inhibition ( > 80% ) of gene transcription or activity of any of \n these enzymes results in parasite death , the question remains of how tsh2 metabolism is affected when the enzymes are less inhibited . for example , in the therapeutic treatment of \n patients it is certain that drugs have to be administered for long periods of \n time . if the parasites are not \n completely cleared from the patient , disease recurrence and generation of drug - resistant \n parasites are possible . the results described \n above indicate that each enzyme by itself has low control on tsh2 synthesis and concentration ; therefore , highly specific and very potent \n inhibitors have to be designed in order to attain the required full activity blockade \n to affect tsh2 metabolism in these parasites .", "the metabolic \n control analysis ( mca ) was initially developed by kacser and burns in scotland [ 82 , 83 ] and by heinrich and rapoport \n in east germany \n [ 84 , 85 ] . this analysis establishes a \n theoretical framework that explains the results observed with the enzyme \n overexpression and downregulation experiments . in addition , it helps to identify and design \n experimental strategies for the manipulation of a given process in an organism \n ( heavy metal hyperaccumulation ; increased production of ethanol , co2 , \n lactate or acetate ; or inhibition of a metabolic pathway flux with therapeutic \n purposes ) . mca rationalizes the quantitative \n determination of the degree of control that a given enzyme exerts on flux and \n on the concentration of metabolites . different \n experimental approaches have been developed to detect and direct what has to be \n done and measured , in order to identify and understand why an enzyme exerts a \n significant or a negligible control on flux and metabolite concentration in a \n metabolic pathway . experiments for identifying and manipulating the conceptually wrong rate - limiting \n step . to \n understand how a metabolic pathway is controlled and could be manipulated , its \n control structure has to be evaluated . the \n control structure of a pathway is constituted by the flux control coefficient ( cvi ) , which is the degree of control that the rate ( v ) of a given enzyme i exerts \n on flux j ; the concentration control coefficient ( cvi ) , which is the degree of control that a given enzyme i exerts on the concentration of a metabolite \n ( x ) ; and the elasticity coefficients . the control coefficients are systemic \n properties of the pathway that are mechanistically determined by the elasticity \n coefficients ( x ) , which are defined as the degree of sensitivity \n of a given enzyme vi \n ( i.e. , the enzyme 's ability to change \n its rate ) when any of its ligands ( x : substrate , products or allosteric \n modulators ) is varied . viojo , in which the expression dj / dvi describes \n the variation in flux ( j ) when an infinitesimal change is done in the enzyme i concentration or activity . in practice , \n the infinitesimal changes in vi \n are undetectable , and hence measurable noninfinitesimal changes are undertaken . \n if a small change in vi \n promotes a significant \n variation in j , then this enzyme exerts an elevated flux control ( figure 4 , \n position 1 ) . in contrast , if a rather small \n or negligible change in flux is observed when vi \n is greatly varied , then the enzyme does not exert \n significant flux control ( figure 4 , position 2 ) . to obtain dimensionless and normalized values \n of cvi the scaling factor vio / jo is applied , which represents the ratio between the initial values from which \n the slope dj / dvi \n is \n calculated . if all cvi of the pathway enzymes and transporters are added up , the sum comes to \n one ( summation theorem ) . the mca clearly distinguishes \n between the control exerted by a given enzyme on flux ( flux control \n coefficient ) and on the metabolite concentration ( concentration control \n coefficient ) . thus , an enzyme can have significant control on a metabolite \n concentration but not on the pathway flux . on one hand , the use of the rate - limiting step concept \n for manipulating metabolic pathways does not make such differentiation , which \n probably has contributed to the many unsuccessful experiments reported in the \n literature ; on the other hand , it should be clearly defined whether the aim of the \n project is to increase flux and/or a metabolite concentration since mca \n establishes for each aim a different experimental design . to determine the flux control \n coefficient of a given enzyme , small variations in the enzyme content , or \n preferentially , in activity are required , without altering the rest of the \n pathway , and then the changes in flux are determined . the experimental points are plotted as shown \n in figure 4 to calculate the slope at the reference point vio / jo . this experiment , apparently easy to perform , \n has demanded great intellectual and experimental effort . several experimental strategies have been \n developed to determine cvi : \n formation of heterokarionts and heterocygots ( classical genetics),titration of flux with specific inhibitors , elasticity analysis , mathematical modeling ( in \n silico biology),in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels . \n formation of heterokarionts and heterocygots ( classical genetics ) , titration of flux with specific inhibitors , mathematical modeling ( in \n silico biology ) , in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels . the arginine biosynthesis in neurospora crassa was the first metabolic \n pathway in which flux control coefficients were experimentally determined by \n kacser 's laboratory . this fungus \n forms multinucleated mycelia that facilitate the generation of polyploid cells . \n by mixing different ratios of spores containing \n genes encoding wild ( active ) and mutant ( inactive ) enzymes of this pathway , it \n was possible to generate heterokaryont mycelia with different content , and \n activity , of four pathway enzymes . the authors built plots of enzyme activity versus \n flux ( see figure 4 ) for acetyl - ornithine aminotransferase , ornithine \n transcarbamoylase , arginine - succinate synthetase , and arginine - succinate lyase . \n all the experimental points of these \n heterokaryonts localized near to position 2 of figure 4 with cvi = 0.020.2 ( flux control by these enzymes was \n only 220% ) , which indicated that none of these enzymes exerted significant \n control on arginine synthesis . the authors did not determine the remaining flux \n control ( 75% ) , which might reside in carbamoyl - phosphate synthetase i ( this mitochondrial \n ammonium - dependent isoform can be bound to the mitochondrial inner membrane or \n form complexes with ornithine transcarbamoylase [ 87 , 88 ] ) and in mitochondrial citruline / ornithine \n transporter , both of which have been proposed as limiting steps , or might be in \n the arginine demand for protein synthesis . organisms \n with many alleles of one enzyme may form homo - and heterozygotes expressing \n different activity levels . drosophila \n melanogaster has three adh alleles encoding for isoforms with different vmax . when three natural homozygotes , a null mutant , and \n some heterozygotes were generated , different adh activities were attained but \n the ethanol consuming rate did not change ( figure 4 , position 2 ) . oxidative phosphorylation ( oxphos ) is \n the only pathway for which specific and potent inhibitors for many enzymes and \n transporters are available . oxphos is divided in two segments ( figure 5 ) : the \n oxidative system ( os ) formed by substrate transporters ( pyruvate , \n 2-oxoglutarate , glutamate , glutamate / aspartate , dicarboxylates ) , krebs cycle \n enzymes , and the respiratory chain complexes ; and the phosphorylating system \n ( ps ) constituted by the atp / adp ( ant ) and pi ( pit ) transporters , and atp \n synthase . when the flux ( atp synthesis ) \n is titrated by adding increasing concentrations of each specific inhibitor , \n plots are generated in which the enzyme activity is progressively diminished by \n increasing inhibitor concentration . hence , the cvi value depends on the type of \n inhibitor usedfor irreversible inhibition,(2)cvij = ( imaxjo)(djdi)[i]0 , \n for simple noncompetitive inhibition,(3)cvij = ( kijo)(djdi)[i]0 , \n for simple competitive inhibition,(4)cvij = ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n where jo is the pathway flux in the absence \n of inhibitor ; imax , minimal \n inhibitor concentration to reach maximal flux inhibition ; k \n i , inhibition constant ; s , \n substrate concentration ; km , \n michaelis - menten constant ; and d \n j / d \n i , \n initial slope ( [ i ] = 0 ) of inhibition titration curve . for irreversible inhibition,(2)cvij = ( imaxjo)(djdi)[i]0 , \n for simple noncompetitive inhibition,(3)cvij = ( kijo)(djdi)[i]0 , \n for simple competitive inhibition,(4)cvij = ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n to estimate flux control \n coefficients from inhibitor titration of adp - stimulated ( state 3 ) respiratory \n rates ( i.e. , mitochondrial o2 consumption coupled to atp synthesis ) , ( 2 ) for irreversible inhibitors was used \n because researchers assumed that mitochondrial inhibitors such as rotenone , \n antimycin , carboxyatractyloside , and oligomycin were however , under this assumption flux control coefficients were usually \n overestimated [ 90 , 91 ] . to solve this \n problem , gellerich et al . developed ( 5 ) for noncompetitive \n tightly - bound inhibitors and , by using nonlinear regression analysis , it was \n possible to include all experimental points from the titration curve thus \n increasing accuracy in calculating cvi:(5)j = [ n(joji)2encojoeon[(n co)jo ( nji)en ] ] + ji e2+(kd+ieo)ekdeo=0 , in which jo and ji are the \n respiration fluxes in the noninhibited ( e = eo ) and inhibited ( e = 0 ) states ; k \n d is the dissociation constant of \n the enzyme - inhibitor complex , and n is an empirical component that expresses the relationship between substrate \n concentration and the reaction catalyzed by the enzyme e. the analysis of data in table 3 shows that oxphos is not controlled by only one \n limiting step , but the flux control is rather distributed among several enzymes \n and transporters . it is worth noting \n that the value of the flux control coefficient depends on the content of enzyme \n or transporter , which varies from tissue to tissue . perhaps the atp / adp translocase in as-30d hepatoma mitochondria \n might reach the status of being the oxphos limiting step with a ca nt = 0.70 , or the pi transporter in kidney mitochondria , or the \n atp / adp translocase and the respiratory chain complex 3 in liver mitochondria , but \n it should be noted that other steps also exert significant control ( table 3 ) . although the distribution of control varies \n between tissues , the flux control mainly resides in the ps of organs with high atp \n demand such as the heart ( cpt+ant+atpsynthase = cps = 0.73 ) , kidney ( cps = 0.75 ; cos = 0.31 ) , and fast - growing tumors ( cps = 0.98 ) . in contrast , in the \n liver ( cos = 0.80 ; cps = 0.65 ) and brain ( cos = 0.35 ; cps = 0.41 ) , the control is shared by both systems . determined that the oxphos \n control was shared by the ps ( cps = 0.62 ) and the atp demand ( purified atpase ) . in turn , rossignol et al . concluded that \n the os exerted the main control ( cos = 0.68 ) , but these authors apparently used low - quality mitochondria \n ( low respiratory control values that lead to low rates of atp synthesis \n associated with high rates of respiration ) that were not incubated under near physiological \n conditions ( 10 mm pyruvate , 10 mm malate , 10 mm pi , ph 7.4 in tris buffer ) , and the authors incorrectly assumed that rotenone and antimycin \n were irreversible inhibitors . it is notorious \n that in all works shown in table 3 at least one of these mistakes is evident . there are some inhibitors for enzymes and transporters from other pathways , but they \n are not quite specific and may affect other sites . due to the fact that there are no inhibitors \n for every step in these pathways , examples \n of these inhibitors are 6-chloro-6-deoxyglucose for glucose transporters in \n bacteria , 2-deoxyglucose for hpi , iodoacetate for gapdh , 1,4-dideoxy-1,4-imino - d - arabinitol \n for glycogen phosphorylase , oxalate and oxamate for ldh , 6-amino \n nicotinamide for the phosphate pentose pathway , amino - oxyacetate for \n aminotransferases and kirureninase ( tryptophan synthesis ) , norvaline for \n ornithine transcarbamylase , mercaptopycolinate for pep carboxykinase , acetazolamide \n for carbonic anhydrase , and isobutyramide for adh ( compiled by \n fell ) . potential uses of the experimental approachmitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction . mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered . the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis.mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways . thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) . considering that the brain is a fully aerobic \n organ , whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome.titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells . the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control . this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects.the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites . these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n . mitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction . mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered . the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis . mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways . thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) . considering that the brain is a fully aerobic \n organ , whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome . titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells . the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control . this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects . the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites . these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n . xovio , which is a dimensionless number that show the rate variation v of a given enzyme or transporter i when the concentration of a ligand x ( substrate s , product p or allosteric modulator ) is varied in infinitesimal \n proportions . the elasticity coefficients are positive for those metabolites \n that increase the enzyme or transporter rate ( substrate or activator ) , and they \n are negative for the metabolites that decrease the enzyme or transporter rates \n ( product or inhibitor ) . an enzyme working , under a steady - state metabolic flux , at saturating conditions of s or p , \n is no longer sensitive to changes in these metabolites . thus , its elasticity is close to zero ( figure 6 , x = 0 ) . \n in turn , an enzyme working at s or p \n concentrations well below the michaelis constant ( kms or kmp ) \n is expected to be highly sensitive to small variations in these metabolites ( figure \n 6 , x = 1 ) . if the kinetic parameters of an enzyme are \n known ( vmf , vmr , kms , and kmp ) , then the enzyme elasticity for any given metabolite \n concentration may be calculated as shown in the following equations . for substrate,(7)svi = s / kms1 + s / kms + p / kmp + 11 /keq , and \n for product,(8)pvi = p / kmp1 + s / kms + p / kmp /keq1 /keq , in which is the mass \n action ratio , and keq is the equilibrium constant preferentially determined \n under physiological conditions . an enzyme with low elasticity can not increase ( or decrease ) its rate despite large \n variations in s ( or p ) concentration ; in consequence , such enzyme exerts a high \n flux control . in turn , an enzyme with a \n high elasticity can adjust its rate to the variation in s or p concentrations , \n and thus it does not interfere with the metabolic flux , exerting a low flux control . this inverse relationship between the \n elasticity and the flux control coefficients is expressed in a formal equation \n denominated connectivity theorem . a \n metabolic pathway can be divided in two blocks around an intermediary x : the \n producing ( synthetic , supply ) and the consuming ( demand ) enzyme blocks of x are i1 and i2 , respectively . thus , the connectivity theorem for this two - block system is(9)cv1jcv2j = xv2xv1 . the negative sign of the right part of the equation \n cancels with x , which is negative because x \n is a product of enzyme block i1 ( figure 6 ) . to obtain the flux control coefficients many strategies have been designed [ 90 , 103108 ] , but the most \n used and probably more trustworthy is that in which the initial pathway metabolite \n ( so ) concentration is varied to increase the x concentration ( any intermediary \n in the pathway ) , and measuring in parallel the variation in flux . under steady - state conditions , the flux rate \n is equal to the rate of end - product formation ( i.e. , lactate or alcohol for \n glycolysis ; oxygen consumption for oxphos ) and to the rate of any partial \n reaction . the slope , calculated at the reference \n coordinate ( xo , jo ) that is \n equivalent to ( so , vio ) , yields \n the elasticity coefficient of the consuming block of x. in another set of experiments , an inhibitor is \n added to block one or more enzymes after x. the x concentration and flux are determined and \n plotted as shown in figure 7 , from which the elasticity coefficient of the \n producing block is calculated . the flux \n control coefficients are determined by using the connectivity theorem and \n considering that the sum of the control coefficients comes to 1 , c1 + c2 = 1 ( summation theorem):(10)cv1j = xv2xv2 xv1,cv2j = xv1xv2 xv1 . \n this method for determining cvi using the elasticities of the two blocks was called double \n modulation by kacser and burns . years \n later , brand and his group [ 103 , 104 ] renamed this method as top - down approach . by applying the procedure shown in figure 7 \n and using ( 10 ) for different metabolites along the metabolic pathway , it \n is possible to identify those sites that exert a higher control ( which may be \n the sites for therapeutic use or biotechnological manipulation ) and those that \n exert a negligible control under a given physiological or pathological \n situation . elasticity analysis has been used to evaluate the oxphos control distribution \n in tumor cells . almost all studies on \n this subject have been carried out with isolated mitochondria incubated in \n sucrose - based medium at 25 or 30c or with the more physiological kcl - based medium but \n still at 30c ( table 3 ) . furthermore , \n these studies did not consider that the product , atp , never accumulates in the \n living cells , which does occur in experiments with isolated mitochondria . under such a condition , a steady state in atp production can \n never be reached as in living cells . in \n other words , the distribution of control in mitochondria ( table 3 ) has been \n determined in the absence of an atp - consuming system . a remarkable exception to this incomplete \n experimental design was the work done by wanders et al . , in which isolated liver mitochondria were incubated \n with two different atp - consuming systems ( or adp - regenerating systems ) : hk + \n glucose and creatine kinase ( ck ) + creatine . under this more physiological setting , the oxphos \n flux control distributed between ant and the atp - consuming system ; however , flux \n control by the other pathway components was not examined . therefore , to accurately evaluate oxphos control \n distribution , mitochondria should be incubated in the presence of an \n atp - consuming system or in their natural environment ( i.e. , inside the cell ) . the rate of oxphos in intact \n cells is determined from the rate of oligomycin - sensitive respiration : in the \n steady state , the enzyme rates are the same and constant ; in branched pathways \n the sum of the branched fluxes equals the flux that supplies the branches . the global elasticity of the atp - consuming processes \n ( e.g. , synthesis of protein , \n nucleic acid , and other biomolecules , as well as ion atpases to maintain the \n ionic gradients , mechanical activity such as muscular contraction or flagellum \n and cilium movement , and secretion of hormones , digestive enzymes and \n neurotransmitters ) is estimated by inhibiting flux with low concentrations of \n oligomycin or a respiratory chain inhibitor . to determine the elasticity of the \n atp - producing block , flux , and [ atp ] are varied with streptomycin , an inhibitor \n of protein synthesis ( figure 7 ) . the \n elasticity coefficients are calculated from the initial coordinate slopes \n ( without inhibitors ) of each titration . with \n this procedure , it has been determined that the atp - consuming block exerts a \n significant flux control of 34% . remarkably , \n this flux control value obtained in cells is quite similar to the flux control \n coefficients of the atp - consuming system ( hk or ck ) reported by wanders \n et al . with isolated \n mitochondria . elasticity analysis by enzyme \n blocks allows the inclusion of the end - product demand as another pathway block . \n the conclusions obtained from this \n analysis have formulated the supply - demand theory , which proposes that when \n flux is controlled by one block ( demand ) , the concentration of the end - product \n is determined by the other block ( supply ) . the ratio of elasticities determines the distribution of flux control \n between supply and demand blocks . for \n instance , if x > x ( i.e. , demand becomes saturated by the end - product x , \n and hence its elasticity is near zero ) , then the demand block exerts the main \n flux control . for concentration control , \n at larger x x , smaller absolute values of both csupply and cdemand are attained ; hence , under demand saturation , \n the supply elasticity fully governs the magnitude of the variation in the \n end - product concentration . on the other \n hand , when demand increases , it loses flux control and induces a diminution in \n the end - product concentration . in turn , supply gains flux control and loses \n concentration control . in the presence of feed - back inhibition , the system can \n maintain the end - product concentration orders of magnitude away from \n equilibrium ( at a concentration around the k0.5 of the allosteric enzyme ) . as mentioned before , the \n demand is not usually included in the pathway because it is erroneously thought \n that it is not part of it . but then , is \n it valid to analyze the control of a metabolite synthesis if its demand is not \n considered ? when the demand block is not \n included , it is assumed that the metabolic pathway produces a metabolite at the \n same rate regardless whether the metabolite demand is high or low . this reasoning is incorrect because a \n metabolic pathway indeed responds to changes in the metabolite demand and , more \n importantly , a pathway without end - products consumption reactions is unable to \n reach a steady state . the intermediary x linking the two blocks is one \n of the end - products of the producing block ( e.g. , \n pyruvate or lactate or ethanol , and atp for glycolysis ) . the variation in rate of the two blocks in \n response to a variation in x can be theoretical or experimentally determined ( figure 8(a ) ) . it is worth noting that , for this \n supply - demand approach , it is not necessary to know the kinetics of each \n pathway enzyme because the rate response of each block reflects the global kinetics \n of all participating enzymes . when the x \n concentration is increased , the rate of the supply block decreases ( i ) because x \n is its product and ( ii ) because usually an enzyme within this block receives \n information from the final part of the pathway , decreasing its rate through \n feedback inhibition . in turn to better \n visualize the effect of large rate changes , the kinetics of both blocks are \n plotted in a logarithmic scale . figure 8(b ) shows the kinetics described in figure 8(a ) converted to natural \n logarithm . the intersection point \n between kinetic curves , at which the supply and demand rates are identical , \n represents the pathway steady - state flux ( in the y axis ) and end - product \n concentration ( in the x axis ) . since the \n elasticity is also defined as x = dlnvi / dlnx , the slope at the intersection point represents the elasticity of each \n block towards the intermediary x. here , \n the use of the scalar factor is not necessary because it is included in the \n logarithmic equation . with the elasticity \n coefficients calculated from plots like those shown in figure 8 , and the \n connectivity theorem , the example in figure 8(b ) shows that \n the demand exerts a high flux control ( and has low elasticity ) and the supply \n block exerts low control ( and has high elasticity ) . the \n fact that the demand may exert a high flux control in metabolite pathways has \n at least three important implications : ( a ) the supply block responds to variations \n in the demand ( high elasticity ) ; ( b ) the demand block has information transfer \n mechanisms towards the supply block that avoid the unrestricted intermediary accumulation under a low demand , \n particularly when the supply block has reactions with large keq ( > 100 ; g > 3 kcal mol at 37c ) ; and ( c ) \n if the main flux control resides in the demand block , then the supply block may \n only exert control on the intermediary concentration but not on the flux [ 30 , 32 ] . this last conclusion explains why it \n is incorrect to consider that an enzyme that controls flux must also control \n the intermediary concentration . regulatory mechanisms of \n enzyme activity are modulation of protein concentration by synthesis and \n degradation , as well as covalent modification and variation in the substrate or \n product concentrations ( which are components of the pathway ) . in addition , another regulatory mechanism is the \n modulation by molecules that are not part of the pathway , that is , through allosteric interaction with cooperative ( sigmoidal \n kinetics ) or noncooperative enzymes ( hyperbolic kinetics ) ( e.g. , ca activates some krebs cycle dehydrogenases ; citrate inhibits pfk-1 ; malonyl - coa inhibits \n the mitochondrial transporter of acyl - carnitine / carnitine ; or the initial \n substrate of a pathway that has not entered the system ) . for these last cases , kacser and burns \n proposed the use of the response coefficient r which is defined by the \n following expression:(11)rm j = cvijmvi , where m is the external modulator of the i enzyme . the response coefficient is dj / dmmo / jo . \n if the elasticity of the sensitive \n enzyme toward the external effector is also determined , then it is possible to \n calculate cvi by using ( 11 ) . unfortunately , due to the experimental complexity for determining the \n elasticity coefficient , this coefficient is often calculated in a theoretical \n way by using the respective rate equation ( michaelis - menten or hill equations ) \n and the kinetic parameters km and vmax determined by someone else under optimal \n assay conditions , which are commonly far away from the physiological ones . therefore , for this theoretical determination \n of elasticity only the value of the external modulator concentration is required . it is convenient to emphasize that the determination \n of the flux control coefficients becomes more reliable when they are calculated \n from several experimental points ( figure 7 ) , instead of only one , as occurs \n with the theoretical elasticity analysis . determined the flux control distribution of gluconeogenesis \n from lactate in hepatocytes by using both theoretical and experimental elasticity \n analysis and the response coefficient . these authors concluded that gluconeogenesis \n stimulated by glucagon was controlled by the pyruvate carboxylase ( cpc = 0.83 ) ; in the absence of this hormone , the \n control was shared by pc , pyk , eno - pgk segment , and tpi - fructose-1,6-biphosphatase \n segment . elasticity \n analysis has been applied to elucidate the flux control of atp - producing \n pathways in fast - growing tumor cells . for oxphos , this approach showed that respiratory chain complex i and \n the atp - consuming pathways were the enzymes with higher control ( cvi = 0.7 ) . for glycolysis , the main flux control ( cvi = 0.71 ) resided in glut + hk reactions because \n hk is strongly inhibited by its product g6p despite extensive enzyme overexpression \n . examples of elasticity analysis on \n other pathways are photosynthesis , ketogenesis , serine and \n threonine synthesis in e. coli , glycolysis in yeast , glucose \n transport in yeast , dna supercoiling , glycogen synthesis in muscle , \n and galactose synthesis in yeast . in conclusion , the elasticity \n analysis is the most frequently used method for determining flux control \n coefficients because it does not need a group of specific inhibitors for all \n the enzymes and transporters of the pathway , neither does it require knowledge \n of the inhibitory mechanisms or kinetic constants . it is only necessary to produce a variation in \n the intermediary concentration x by using an inhibitor of either block or by \n directly varying the x concentration . in agreement with fell , it seems impossible for a researcher to analyze one by \n one the rate equation of each enzyme in a metabolic pathway to predict and \n explain the system behavior as a whole . to deal with this problem , in the last three \n decades some scientists have constructed mathematical models for some metabolic \n pathways using several software programs . thus , the specific variation of a single enzyme \n activity without altering the rest of the pathway ( figure 4 ) , which has been an \n experimentally difficult task for applying mca , becomes easier to achieve with \n reliable computing models . there are two basic types of modeling : ( a ) structural modeling and ( b ) kinetic \n modeling . the former is related to the pathway chemical reaction structure and \n does not involve kinetic information . the information obtained with structural modeling is the \n description of the following : the exact determination of which reactions and \n metabolites interact among them;the conservation reactions . there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) . the identification of \n conserved metabolites might not be obvious;enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes;elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) . \n the exact determination of which reactions and \n metabolites interact among them ; the conservation reactions . there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) . the identification of \n conserved metabolites might not be obvious ; enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes ; elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) . in addition to an appropriate computing \n program , this approach requires the knowledge of the stoichiometries , rate \n equations , and keq values of each reaction in the pathway ( or the vmax in the forward and reverse \n reactions ) , as well as the intermediary concentrations reached under a given \n steady state . some currently used \n softwares are copasi ( http://www.copasi.org/tiki-index.php ) \n based on gepasi ( http://www.gepasi.org/ ; ) ; metamodel ; winscamp \n and jarnac ( both available at http://www.sys-bio.org/ ) ; and pysces \n ( http://pysces.sourcesforge.net/ ; ) . for other programs and links , go to http://sbml.org/index.psp . to reach a steady - state flux , it is necessary \n to fix the initial metabolite concentration to a constant value and the \n irreversible and constant removal of the end products . except for the final reactions in which their \n products have to be removed from the system , all pathway reactions have to be \n considered as reversible , notwithstanding whether they have large keq ( if there \n is an irreversible reaction under physiological conditions , then a reversible rate \n equation that includes the keq suffices to maintain the reaction as practically \n irreversible ) . care should be taken to include the enzyme 's sensitivity toward \n its products because this property is related with the enzyme elasticity and hence \n with its flux control ; omission of this parameter may very likely lead to \n erroneous conclusions . it \n should be pointed out that the purpose of kinetic modeling is not merely to \n replicate experimental data but also to explain them . thus , pathway modeling is a powerful tool \n that allows for ( i ) the detection of those properties of the pathway that are \n not so obvious to visualize when the individual kinetic characteristics of the \n participating enzymes are examined ; and ( ii ) the understanding of the biochemical \n mechanisms involved in flux and intermediary concentration control . modeling requires the consideration of all \n reported experimental data and interactions that have been described for the \n components of a specific pathway , thus allowing for the integration of disperse \n data , discarding irrelevant facts . although all models are oversimplifications of complex cellular \n processes , they are useful for the deduction of essential relationships , for \n the design of experimental strategies that evaluate the control of a metabolic \n pathway , and for the detection of incompatibilities in the kinetic parameters \n of the participating enzymes and transporters , which may prompt the \n experimental revision of the most critical uncertainties . with \n the model initially constructed , the simulation results do not usually concur \n with the experimental results ; in consequence , the model normally requires \n refinement , a point at which the researcher 's thinking and knowledge of biology \n plays a fundamental role in modifying the structure and parameters of the model . \n the discrepancies observed between modeling \n and experimentation unequivocally pinpoint what elements or factors have to be re - evaluated \n or incorporated so that the model approximates more closely reality ( i.e. , experimental data ) . the comparison of the experimentally obtained \n intermediary concentrations and fluxes with those obtained by simulation is an \n appropriate validating index of the model ; this index indicates whether the \n model approximation to the physiological situation is acceptable or whether \n re - evaluation of the kinetic properties of some enzymes and transporters and/or \n incorporation of other reactions or factors is required . a reason to why the results obtained by modeling may substantially differ from the \n experimental results is that the kinetic parameters of the pathway enzyme and \n transporters and the keq values used \n were determined by different research groups , under different experimental \n conditions and in different cell types . moreover , \n enzyme kinetic assays are carried out at low , diluted enzyme \n concentrations ( thus discarding or ignoring relevant protein - protein \n interactions ) , and at optimal ( but not physiological ) ph and room temperature \n ( which may be far away from the physiological values ) . in addition , no experimental information is \n usually available regarding the reactions reversibility and the product \n inhibition of the enzymes and transporters ( particularly for physiological \n irreversible reactions , i.e. , reactions with large keq ) . with worrisome \n frequency , the researcher has to adjust the experimentally determined vm and km values to achieve a model behavior that acceptably resembles \n that observed in the biological system . apparently , \n this type of limitations as well as the sometimes overwhelming amount of \n kinetic data necessary for the construction of a kinetic model has restricted \n the number of reliable models that can be used for the prediction of the \n pathway control structure . once the kinetic model stability , robustness , structural and dynamic properties have \n been evaluated , and experimentally validated , the model may become a virtual \n laboratory in which any parameter or component can be modified or replaced and \n any aspect of the pathway behavior can be explored within a wide diversity of \n circumstances or limits . at this \n stage , glycolysis \n in s. bayanus , s. cerevisiae [ 113 , 123 , 124 ] , and trypanosoma brucei [ 125 , 126 ] is the \n metabolic pathway that has been more extensively modeled . both cell types \n have a very active glycolysis and are fully dependent on this metabolic pathway \n for atp supply , under anaerobiosis and aerobiosis , respectively . one advantage \n of modeling glycolysis in these cell types is that most of the kinetic \n parameters used have been experimentally determined by the same groups under \n the same experimental conditions . however , \n the kinetics of the reverse reactions has not been determined and thus these \n authors used kmp and keq \n values reported by others and obtained in other cell types under rather different \n experimental conditions , or they were adjusted to improve model fitting . nevertheless , the simulation \n results yielded relevant information on the control of the glycolytic flux . in both cases , the enzymes traditionally considered \n the rate - limiting steps , hk , atp - pfk-1 , and pyk did not contribute to the flux \n control , whereas the main control resided in glut ( 54% in the parasite and 85100% in yeast ) . under some conditions , hk may exert some \n control ( 15% ) in s. cerevisiae and some nonallosteric enzymes such as \n aldo , gapdh , and pgk may also exert some flux control in t. brucei . mca \n through kinetic modeling has been applied to several pathways : glycolysis in \n erythrocytes in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29%);carbohydrate \n metabolism during differentiation in dictyostelium discoideum with \n cellulose synthase ( 86% ) as the main controlling step;sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50%);threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger \n arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) . model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch . \n glycolysis in \n erythrocytes in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29% ) ; carbohydrate \n metabolism during differentiation in dictyostelium discoideum with \n cellulose synthase ( 86% ) as the main controlling step ; sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50% ) ; threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger \n arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) . model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch . we modeled \n the gsh and pcs biosynthesis ( figure 2 ) to determine and understand the control \n structure of the pathway and thus be able to identify potential sites for genetic \n engineering manipulation that might lead to the generation of improved species \n in heavy metal resistance and accumulation . two models were constructed , one \n for higher plants and the other for yeast , both exposed to high concentrations \n of cd . due to the \n similarity in the results , only the plant results are analyzed below . an interesting conclusion from the gsh - pcs synthesis modeling is that control of flux \n ( and gsh concentration ) is shared between the gsh supply and demand under both unstressed \n and cd exposure \n conditions ( table 4 ) . this observation strongly differs from the \n idea that -ecs is the rate - limiting step [ 3335 ] . for many researchers , the concept of -ecs being the key controlling step has seemed to be \n correct because ( a ) -ecs receives information from the final part \n of the pathway , as it is potently inhibited by gsh , the pathway end - product ; \n and ( b ) -ecs is localized in the first part of the \n pathway ( figure 2 ) . in addition however , in most of the \n studies on the control of gsh synthesis , the gsh demand has not been \n considered . the gsh synthesis modeling shows that under a physiological feedback \n inhibition of -ecs by gsh a small increase in demand increases \n flux because the gsh concentration decreases and the -ecs inhibition attenuates . in contrast , if the demand remains constant , then \n an increase in -ecs activity or content ( by overexpression ) does \n not increase flux because the gsh inhibition is still there and operates on \n both new and old enzymes . the same pattern is also observed when hk is \n overexpressed to increase \n glycolytic flux since it is still inhibited by g6p ( see section 3 ) . on the other hand , -ecs indeed exerts significant concentration control \n on gsh , which means that a -ecs increase results in higher gsh \n concentration ( table 4 ) . this last \n observation demonstrates that an enzyme controlling a metabolite concentration does \n not necessarily control the flux . cd exposure promotes a high gsh demand because significant oxidative stress surges , \n thus causing oxidation of gsh through gsh peroxidases , and because gsh and pcs \n are used for sequestering the toxic metal ion ; hence , a higher gsh consuming \n rate sets up . under this condition , \n modeling predicted that control was almost equally shared between the supply \n and demand blocks , but particularly between -ecs and pcs ( see figure 2 ) . modeling was also able to explain why pcs overexpression \n can have toxic effects on the cell . an increase in the gsh demand ( pcs \n overexpression ) under high - demand conditions ( cd stress ) leads to \n gsh depletion that severely compromises other processes such as the oxidative \n stress control and xenobiotic detoxification . the \n conclusions drawn by this model led us to propose that , to significantly increase \n the cd resistance and accumulation , -ecs and pcs should be simultaneously overexpressed \n ( table 4 ; figure 9 ) . this particular \n manipulation promotes an increase in the rate of gsh and pcs synthesis \n ( determined by the high - to - low transition of their flux control coefficients ) \n and in the gsh and pcs concentrations \n ( determined by their high concentration control coefficients ) . the model predicts that a 2-fold increase in \n the simultaneous overexpression of -ecs and pcs brings about a 1.92.4-fold \n increase in flux to gsh ( jgs ) \n and pcs ( jpcs ) and in pcs \n concentration ( figure 9 ) ; a 5-fold overexpression further increases by 4.58.1 \n times the fluxes and pcs concentration . this proposed enzyme overexpression \n should not exceed the gs and the complex pc - cd ( or gs - cd - gs ) vacuolar \n transporters ' maximal activities , in order to keep the cell away from a severe \n oxidative stress caused by gsh depletion or -ec accumulation . indeed , the concentration of gsh was maintained high and constant although \n -ec accumulated with the simultaneous overexpression \n ( figure 9 ) . furthermore , this enzyme \n manipulation should avoid the increase of the pc - cd and gs - cd - gs complexes in \n cytosol to toxic levels . in other words , \n excessive enzyme overexpression should be avoided , unless this is accompanied \n by compensating overexpression of consuming enzymes ( gs for -ecs overexpression and pcs vacuolar transporters for \n that of pcs ) . in yeasts and plants , cd is ultimately inactivated by the additional interaction with s and \n the subsequent formation of stable high molecular weight complexes with pcs , cd , \n s , and gsh [ 138 , 139 ] . in \n parallel to the -ecs and pcs overexpression , moderate repression \n of gsh - s - transferases , which compete for the available gsh ( figure 2 ) , may also \n promote an increase in gsh concentration and pcs formation flux . in contrast , gene overexpression induces large \n changes in activity ; hence , further theoretical background has been developed for \n predicting the effect on flux and metabolite concentrations induced by large \n enzyme changes . such a theoretical background was initially developed by small \n and kacser , who depicted ( 12 ) based on the flux control coefficients to \n predict the effect promoted by large changes in enzyme activity:(12)fejmrj = 11 i = jm(cvi0jo ( ri 1)/ri ) , in which f is the amplification factor ( the flux increase ) , and r represents how many times the enzyme is overexpressed . to predict the flux changes , promoted by identical \n overexpression of two enzymes ( same r value ) with different cvi , \n the equation is \n ( 13)fejmrj = 11 ( cij + cjj ) ( ( r 1)/r ) . \n \n figure 10 shows the effect on flux when one or more enzymes with different cvi are changed by the same r factor . if the sum of cvi \n of one or more enzymes is less than 0.25 , the \n impact on flux is discrete when the expression increases 5 folds ( which is the \n most common variation in the overexpression experiments analyzed in section \n 2 ) . but for a 3-fold overexpression of \n a group of enzymes , for which their sum of cvi is more than 0.5 , then a significant flux \n change is achieved . if the sum of cvi is 1 , the flux varies in a linear proportion \n with the degree of overexpression . it \n has to be remarked , however , that the predicted change in flux ( figure 10 ) will \n be valid until certain degree , the limits of which being determined by the \n other pathway enzymes that should stay as noncontrolling steps . \n figure 10 also shows the effect on flux of decreasing an enzyme activity ( third \n quadrant ) . this segment plot is useful when inhibition of pathway flux is being \n pursued for therapeutic purposes or for understanding the molecular basis of the \n genetic dominance and recessivity . like \n in the enzyme overexpression experiment , only a significant effect on flux is \n achieved when the enzymes with high cvi values are inhibited . for an enzyme or group of enzymes with cvi \n of 0.25 , greater than 80% inhibition has to be \n attained to decrease 50% the pathway flux . in this context , it seems feasible \n to explain why knockdown of enzymes involved in tsh2 synthesis has \n to be almost total to detect an effect on tsh2 content or to alter \n functional or pathogenic properties of the parasites ( section 4.3 ) . the knockdown or knockout experiments in \n trypanosomatids suggest that -ecs , trys , and tryr most probably have low flux control \n and concentration - control coefficients since their contents or activities have \n to be reduced > 80% of the normal levels to reach changes in intermediary \n levels or in oxidative stress handling . contrary \n to the several unsuccessful overexpression experiments carried out to increase \n the flux or metabolites of a metabolic pathway , modeling may allow for a more \n focused and appropriate design of experimental strategies of genetic \n engineering to increase flux or a given metabolite , and for selecting drug \n targets to decrease flux or metabolite concentration . for these predictions , modeling considers \n that overexpression of a controlling enzyme or transporter may promote flux or \n metabolite control redistributions . \n thus , a low - control step may become a \n controlling point when overexpressing another step and , in consequence , the \n prediction shown in figure 10 based on ( 11 ) and ( 12 ) may be inaccurate . by considering the whole pathway components , \n modeling is also a powerful tool for predicting the effects on flux and \n metabolite concentration of varying an enzyme activity ( by overexpression or \n drug inhibition ) . model predictions to inhibit a pathway fluxkinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes . interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities . therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work . an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells . moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) .we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite . the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite . the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast , the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux . the amebal model allowed evaluating the \n effect on flux of inhibiting the pathway enzymes . the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) . in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) . therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite . they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism . kinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes . interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities . therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work . an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells . moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) . we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite . the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite . the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast , the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux . the amebal model allowed evaluating the \n effect on flux of inhibiting the pathway enzymes . the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) . in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) . therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite . they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism . another experimental approach \n for determining the enzyme control coefficients is the in vitro reconstitution \n of segments of metabolic pathways . it is \n recalled that for determining the flux control coefficient exerted by a given \n step on a metabolic pathway the enzyme activity has to be varied , without \n altering the other components in the system , and the flux variations are to be \n measured ( figure 4 ) . such an experiment can be readily made if a pathway is \n reconstituted with purified enzymes . some advantages of this approach are that the \n pathway structure is known , in which the components concentration may be \n manipulated and analyzed separately , and the enzyme effectors can be assayed . as \n the system composition is strictly controlled , the results may be highly \n reproducible . the main disadvantage is \n that the enzyme concentrations in the assays are diluted and thus the enzyme \n interactions are not favored . if this interaction \n is important for activity , the in vitro reconstitution may limit the \n extrapolation to the metabolic pathway inside the cell . there are not many studies \n describing this type of experiments , most probably due to the fact that for \n applying mca the pathway must be working under steady - state conditions . in a reconstituted system , only a quasi steady \n state may be reached because there is net substrate , and cofactors consumption , \n as well as product accumulation , since it is difficult to attain a constant \n substrate supply and release of products . one of the first experimental \n reports on control coefficient determination in a reconstituted system was \n carried out for the upper glycolytic segment with the commercially available \n rabbit muscle hk , hpi , pfk-1 , aldo , and tpi . each enzyme was separately titrated and the \n flux variation to glycerol-3-phosphate ( by coupling the reconstituted system to \n an excess of -gpdh ) was measured in the presence of ck to maintain \n the atp concentration constant . the results showed that pfk-1 and hk exerted the \n main flux control ( 65% and 20% , resp . ) , whereas the remaining 15% resided in \n the other enzymes . these authors observed that the addition of f1,6bp , a pfk-1 \n activator slightly diminished the flux control exerted by pfk-1 and increases \n that of hk . the lower glycolytic segment \n has also been reconstituted with commercial enzymes for determining the flux \n control coefficients . the results \n showed that flux was mainly controlled by pyk ( 60100% ) , although under some \n conditions control was shared with pgam ; eno did not contribute to the flux \n control . another important limitation \n of the reconstitution experiments is that the commercial availability of the \n purified enzymes from the same organism is restricted or inexistent . however , by using the information from the \n genome sequence projects and the recombinant dna technology , it is now possible \n to access all the enzyme genes from a metabolic pathway in the same organism , thus \n facilitating their cloning , overexpression , and purification . with this strategy , we cloned , overexpressed , \n and purified the 10 glycolytic enzymes of entamoeba histolytica for \n studying the flux control distribution in this organism by using kinetic \n modeling and pathway reconstitution . the reconstitution \n experiments of the lower amebal glycolytic segment , under near physiological \n conditions of ph , temperature , and enzyme activity ( figure 12 ) showed that pgam \n and , to a lesser extent , ppdk exert the main flux control ( these amebal enzymes \n are genetically and kinetically different from their human counterparts ) with \n eno exhibiting negligible control . in turn , reconstitution of the upper amebal \n glycolytic segment has revealed that hk and , to a much lesser extent hpi , \n ppi - pfk , and ald , exerted the main flux control , with tpi having negligible \n control . these results strongly \n correlate with the enzyme catalytic efficiencies previously reported , in \n which hk is highly sensitive to amp inhibition , ald , and pgam have the lowest \n catalytic efficiencies among the glycolytic enzymes , leading to high flux control \n coefficients and thus becoming suitable candidates for therapeutic \n intervention . the reconstitution results \n also agree with the pathway modeling predictions previously analyzed ( section \n 5.4 ) , in which hk and pgam are two of the main controlling steps . the in vitro reconstitution \n experiments are also useful for studying the effect on control redistribution of \n an enzyme modulation that is particularly difficult to manage in vivo ; the main controlling steps \n identified with the reconstitution experiments should be further analyzed with \n other experimental strategies such as elasticity analysis in the in vivo \n systems . this experimental approach for determining the control coefficients could be part of \n the genetic approach analyzed in section 5.1 , but it was separated due to its \n recent methodological development and because it actually belongs to the \n molecular genetics rather than to the mendelian genetics . this approach is based on the in vivo modulation of the enzyme levels using the rna \n antisense technology . there are at least \n three strategies to \n inhibit gene expression : ( a ) the use of single stranded antisense \n oligonucleotides , which form a double stranded rna that might be degraded by rnase \n h ; ( b ) target rna degradation with catalytically active oligonucleotides , known \n as ribozymes that bind to their specific rna ; and ( c ) rna degradation using sirnas \n ( 2123 nucleotides ) . the rna antisense technology was applied for control coefficient determination of the \n ribulose - bisphosphate - carboxylase ( rubisco ) that fixes co2 in the \n plant calvin cycle . this enzyme considered \n the rate - limiting step of the calvin cycle and of the whole photosynthetic \n process , despite its high concentration ( 4 mm ) in the chloroplasts stroma that compensates \n its low catalytic efficiency . attempts to make rubisco a nonlimiting step , either by modifying its catalytic efficiency \n or by overexpressing it , have been unsuccessful . the plants were transformed with dna antisense against the \n mrna of the enzyme 's small subunit , thus promoting its degradation . for calvin cycle enzymes , the pleiotropic \n effects were minimal . the results showed \n that rubisco may indeed be the photosynthesis limiting step with a crubisco = 0.690.83 when plants are exposed to high \n illumination ( 1050 mol quanta ms ) , high \n humidity ( 85% ) , and low co2 concentrations ( 25 pa ) . however , this flux control decreases to \n 0.050.12 under moderate illumination or high co2 levels . unfortunately , the authors did not determine \n the control coefficients of the other pathway enzymes or the branches fluxes which \n may be significant . as described in section 5.4 , the results of the t. brucei glycolysis \n modeling indicated that glut was the main flux control step ( cglut 50% ) , [ 125 , 126 ] . this model predicted a large overcapacity for \n hk , pfk-1 , aldo , gapdh , pgam , eno , and pyk over the glycolytic flux leading to \n low flux control coefficients [ 125 , 126 ] . to validate the modeling results , the concentrations of hk , pfk-1 , pgam , \n eno , and pyk these knockdown expression experiments showed \n overcapacity of hk and pyk over the flux , although at lower levels than predicted \n by the model . a good correlation for \n pgam and eno was obtained between model predictions and experimental results . however , a large difference ( 9 folds ) was \n obtained for pfk-1 . this discrepancy is \n perhaps related to pleiotropic effects of pfk-1 downregulation , as these mutants \n also displayed diminution in the activities of other enzymes ( hk , eno , and pyk ) . \n the combination of these two approaches , \n in silico modeling and in vivo experimentation , is complementary : on one \n hand , modeling identifies the enzymes ( out of 19 that contain the model ) that \n display the highest flux control coefficients , whereas in vivo experimentation validates the \n accuracy of the model to establish predictions about the pathway 's behavior . the knockdown experiments described above usually yield only two experimental points \n of the plot shown in figure 4 : the \n wild - type and the knockdown strain protein levels or enzyme activities . thus , with \n such an approach high levels of inhibition ( > 80% ) are mostly analyzed , \n whereas intermediate levels of downregulation ( if obtained ) are generally \n overlooked . therefore , knockdown \n experiments are not very useful to obtain the complete set of experimental data \n ( above and below the wild - type levels of enzyme activity with the corresponding \n flux ) for determining reliable control coefficients . a strategy to determine flux \n control coefficients from several protein levels has been developed by using \n adenovirus - mediated glucose-6-phosphatase ( g6pase ) overexpression under the \n control of the cytomegalovirus promoter in rat hepatocytes . a 2-fold g6pase overexpression did not alter cg6pase or cgk ( gk , glucokinase ) . however , if g6pase is overexpressed by 4 folds , then cgk \n diminished from 2.8 to 1.8 and there was a 35% lowering in glycogen synthesis . however , this approach allows titration of flux only above the basal \n enzyme activities found in the cell , but not below . these experimental inconveniences have been circumvented by using inducible gene \n expression systems based in the lac , \n lambda , nisin , gal , tetracycline , and other inducible promoters , in bacteria \n and yeast [ 151 , 152 ] . however , a problem \n frequently encountered with inducible promoters is that a steady - state of \n protein expression is difficult to attain [ 151 , 152 ] . recently , \n jensen and hammer described the design of synthetic promoter libraries ( spl ) , in \n particular for l. lactis metabolic \n optimization . these promoters \n maintain constant the array of the known consensus sequences for l. lactis gene transcription ( 10 and \n 35 boxes ) , while the nucleotide sequence between these boxes ( a spacer \n sequence of 17 1 bp ) is randomized , thus producing a set of promoters with \n different transcriptional strength . these promoter libraries allow the transcription and protein expression \n several folds above and below the wild - type levels of enzyme activity , \n thus enhancing the usefulness of this approach for mca studies . the \n control distribution of glycolysis in e. \n coli and l. lactis , as discussed \n in section 3.2 [ 17 , 24 , 27 , 151 ] , has been determined by using the spl \n technology . spl for yeast , mammalian and \n plant cells are also under development [ 151 , 152 ] . certainly , the advances in genetic \n engineering in combination with mca allow better experimental designs for \n metabolic optimization of micro - organisms of biotechnological interest . concluding remarks \n the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step . mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control . a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments , it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway . mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) . for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them . if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients . these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks . it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis . however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n \n the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step . mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control . a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments , it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway . mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) . for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them . if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients . these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks . it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis . however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step . mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control . a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments , it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway . mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) . for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them . if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes . if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients . these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks . it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis . however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest ." ]

[ "network - driven spindle - like oscillations are a functional hallmark of the developing cerebral cortex . during late prenatal and early postnatal stages of development , spontaneous spindle - like oscillations have been identified as physiological activity patterns in various neocortical areas of different mammalian species [ 14 ] . in humans , so - called delta brushes can be observed in eeg recordings from preterm babies already at gestational week 28 , that is ~12 weeks before normal birth of a full - term neonate ( for review see ) . in ac - filtered eeg recordings , delta brushes are brief rhythmic delta waves ( 0.31.5 hz ) of 50300 v amplitude with a superimposed burst of fast rhythm ( > 8 hz , the brush ) . it has been suggested that delta brushes in human preterm infants correlate with so - called spindle bursts recorded in rodents during the early postnatal period . from a developmental point of view , these early spontaneous activity patterns in developing rodent and human cerebral cortex are probably of similar or even identical origin . rats and mice are altricial - born in a far less mature condition than humans . in rodents the degree of neocortical development at the day of birth ( postnatal day [ p ] 0 ) can be compared to the stage of human cortex between gestational weeks 28 and 32 and the cerebral cortex of a p12p14 rat is comparable to that of the full - term newborn human baby [ 6 , 8 ] . in preterm infants born between gestational weeks 28 and 32 , milh et al . and colonnese et al . recorded eeg signals containing spontaneous and stimulus - evoked delta brushes with oscillatory activity in the frequency range of 8 to 25 hz , suggesting that spontaneous delta brushes may represent a physiological neocortical activity pattern of the human fetus in utero . in the cerebral cortex of rodents , spindle bursts ( beside short gamma oscillations ) constitute the majority of spontaneous activity during the first postnatal week ( figure 1 ) . these spindle bursts resemble in their appearance spindles recorded in the adult brain during sleep . these sleep spindles are one of the hallmarks of human stage 2 sleep for comprehensive overviews the reader is referred to recent reviews by lthi and by mccormick et al . . however , in humans sleep spindles appear 4 to 9 weeks after birth , which is much later than the disappearance of delta brushes around the end of the first postnatal week , thus excluding the hypothesis that delta brushes or spindle bursts gradually develop into sleep spindles . in addition , the frequency profile of spindle bursts and delta brushes displays a rather broad frequency distribution up to 25 hz , whereas sleep spindles present oscillations in a narrower band of ~1015 hz . the present review focuses on spindle burst activity in the cerebral cortex of the developing rat during the first postnatal week and summarizes our current understanding ( i ) on the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cerebral networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development .", "two distinct activity patterns can be observed in the neonatal rat cerebral cortex in vivo : gamma oscillations and spindles bursts ( figures 1 and 2 ) [ 1419 ] . gamma oscillations have a duration of 100 to 300 ms , a frequency of 30 to 40 hz and appear spontaneously every 10 to 30 s ( figures 1 and 2(b ) ) . the properties and functional role of gamma oscillations as well as the mechanisms underlying their generation spindle bursts are characterized by their spindle - like oscillatory appearance , have a duration of 0.5 to 3 s and a frequency in the range of 8 to 30 hz , and occur spontaneously every ~10 s ( figures 1 and 2(a ) ) . as recognized in full - band direct - current ( dc ) coupled recordings , spindle bursts are nested in slow ( delta ) waves , which in infant rats , and preterm human babies [ 22 , 23 ] have been termed spontaneous activity transients ( sats ) . spindle bursts have been described in primary somatosensory cortex ( s1 ) including barrel cortex [ 14 , 1619 ] , in primary visual cortex ( v1 ) [ 10 , 15 ] , in primary motor cortex ( m1 ) , and in prefrontal cortex of anesthetized and awake rats during the first postnatal week . in s1 , spontaneous and stimulus - evoked spindle bursts can be observed as early as p0 [ 17 , 18 ] . in rats , the incidence of spindle bursts declines during the second postnatal week and sporadic spindle bursts are obscured by the ongoing neocortical activity . likewise in humans , delta brushes are replaced by more continuous eeg activity at birth of a full - term infant . this gradual developmental shift from a highly synchronized state of spontaneous activity to a desynchronized state seems to be a fundamental network property of the cerebral cortex during early neonatal stages . spindle bursts synchronize the activity of a local neuronal network of 200 to 400 m in diameter , which resembles the dimension of a whisker - related neocortical column in the immature barrel cortex ( figure 3 ) [ 18 , 25 ] . using a combination of voltage - sensitive dye imaging and high - density multielectrode recordings in p0-p1 rat barrel cortex in vivo , yang et al . could demonstrate that this early spontaneous activity constitutes the later emergence of the whisker - related barrel field map . these data indicate that spontaneous activity patterns at this early age form functional precolumns , supporting the concept of the existence of ontogenetic columns in the radial unit hypothesis . further support for this hypothesis comes from in vivo two - photon calcium imaging in the barrel cortex of both anesthetized and nonanesthetized newborn mice , demonstrating highly synchronous spontaneous burst activity , reminiscent of spindle bursts , in local networks of 100 to 200 m in diameter . these data strongly indicate that early spindle bursts , and probably also gamma oscillations ( for review ) , synchronize early neocortical networks into functional columns at a developmental stage when the upper layers 2/3 have not even been formed ( i.e. , in rats at p0 ) . at this developmental stage , thalamocortical afferents have not reached layer 4 and instead transiently innervate the subplate ( for review [ 28 , 29 ] ) . thalamic afferents form transient glutamatergic synapses with surprisingly mature properties including ampa and nmda receptors [ 3032 ] . in vitro studies in acute brain slice preparations and in intact whole cortical hemisphere preparations have demonstrated that oscillatory network activity in the frequency range of spindle bursts depend on an intact subplate [ 34 , 35 ] . selective removal of the subplate in s1 in vivo causes a significant decline in the occurrence of spontaneous spindle bursts and disturbances in the development of the cortical architecture in the barrel cortex . these data further support the hypothesis that spindle bursts in developing cerebral cortex fulfill an important role in the maturation of the neocortical architecture . in the next section we will discuss the network and molecular mechanisms underlying the generation of spindle bursts in neonatal cerebral cortex .", "the rodent cerebral cortex develops rapidly during late prenatal and early postnatal stages and at least four different activity patterns may occur sequentially between birth and the end of the first postnatal week ( for review ) . furthermore , the cortex shows a mediolateral and anterior - posterior gradient in development and within the same neocortical area neurons in upper layers 2/3 are ~2 days younger compared to lower cortical layers . many inconsistencies in the literature on the properties of spontaneous activity patterns in newborn rodents and their underlying mechanisms can be explained by the fact that these important developmental differences are often ignored . in addition , 2 days in early rodent cortical development make a large difference , so that the neocortex of a p0 rat ( without layer 2/3 ) can not be compared to that of a p2 rat ( with almost complete lamination ) . already in newborn ( p0-p1 ) rat barrel cortex in vivo , mechanical stimulation of a single whisker elicits in field potential recordings and to some extent detectable also in voltage - sensitive dye imaging ( vsdi ) a sequence of an early gamma oscillation followed by a spindle burst ( figure 3(b ) ) . at this age , the thalamocortical activity reaches the developing cortical network largely via the subplate [ 17 , 31 , 32 ] and is amplified by an intrinsic gap - junction coupled network within the subplate and cortical plate [ 28 , 34 ] . spontaneous and evoked delta brushes can be observed in premature human neonates of 2832 weeks postconceptional age , a developmental stage when the human cerebral cortex resembles that of a newborn rat . impressive examples of large delta brushes are provided in the supplementary eeg videos of milh et al . , demonstrating that a single touch elicits a large oscillatory response in the somatosensory evoked potential ( sep ) recorded above the contralateral parietal cortex . in mature human cortex , seps with smaller amplitudes and shorter durations can be only obtained after averaging of at least 100 epochs . thus , in both species , rats and humans , at a comparable stage of cortical development mechanical stimulation of the sensory periphery elicits in s1 spindle bursts and delta brushes , respectively . simultaneous multielectrode recordings in the barrel cortex and in the ventral posteromedial nucleus ( vpm ) of the somatosensory thalamus of p0-p1 rats in vivo have demonstrated that the majority of spontaneous cortical spindle bursts and also gamma oscillations are not generated within s1 , but rather in subcortical structures or outside of s1 ( figures 4(a ) and 4(b ) ) . at this age a local , functionally defined lesion in the vpm blocks the whisker stimulation - induced cortical responses and also profoundly reduces the spontaneously occurring cortical burst activity , further demonstrating that the majority of the spontaneous spindle bursts in p0-p1 rat barrel cortex are generated in subcortical structures of the whisker - to - barrel cortex pathway ( for further information see ) . silencing the sensory periphery by injection of lidocaine into the whisker pad causes a significant reduction in the occurrence of spontaneous spindle bursts and gamma oscillations by ~50% ( figure 4(c ) ) , indicating that during this developmental period at least half of the spontaneous burst activity in s1 is related to activity in the sensory periphery . a similar peripheral generation of spontaneous burst activity may occur in human preterms and fetuses between gestational weeks 28 and 32 [ 9 , 22 ] . similar to the spindle bursts in s1 , spontaneous spindle bursts in v1 are also largely generated in the sensory periphery . ( for review ) , provide the primary drive for spindle bursts in newborn rat v1 , as demonstrated by simultaneous recordings from the retina and v1 . intraocular injection of forskolin , which augmented retinal waves , increased the occurrence of v1 spindle bursts , and removal of the retina reduced the spindle bursts frequency . as in the somatosensory system , spindle bursts in v1 can be also evoked by stimulation of the sensory periphery . however , since rod- and cone - mediated visual signaling is not functional in rats during the first postnatal week , spindle bursts can not be evoked by light flashes before p8 . at that age , the neocortical response in v1 consists of an early visual evoked response followed by an evoked spindle burst . similar responses could be observed in v1 of preterm infants once photoreceptor mediated light responses occur in the retina ( for review ) . whereas a large amount of experimental data has shown that retinal waves provide the main trigger for the cortical v1 spindles bursts , it is not completely understood which pacemaker drives the spontaneous activity in s1 and how spontaneous activity is generated in the somatosensory periphery . in p3p6 rats spontaneous whisker movements occur during active sleep and are correlated with activation of whisker - related cortical columns in the barrel cortex . in newborn rats the proprioceptive feedback from self - generated myoclonic movements trigger spindle bursts in s1 ( for review ) . spontaneous limb movements of the human fetus during the third trimester of gestation , or those of the preterm infant associated with delta brushes in s1 , are similar to these twitching movements of the neonatal rat and can be also triggered by sensory feedback . kreider and blumberg have demonstrated in 1-week - old rats that the mesopontine region plays a central role in the generation of myoclonic twitching . have shown in newborn rats that spatially confined spindle bursts in s1 are triggered in a somatotopic manner by sensory feedback signals from spontaneous muscle twitches . these spontaneous movements are generated by neuronal networks in the spinal cord , but spindle bursts persisted at a reduced frequency after sensory deafferentation ( spinal cord transection ) in s1 , indicating that spindle burst activity can be also generated in neocortical or thalamocortical circuits during this early period of development . this assumption is also supported by the observation that silencing of the sensory periphery causes only a ~50% reduction in the occurrence of spontaneous spindle bursts ( figure 4(c ) ) . however , since it can not be excluded that the remaining portion of spindle bursts actually conveys activity from adjacent or distant sensory areas ( transmitted via inter- and intrahemispheric connections , see next chapter ) , the outcome of this experiment may underestimate the contribution of the sensory periphery . simultaneous monitoring of forepaw movements , vsdi , and extracellular multielectrode recordings in s1 and m1 of p3p5 rats under light urethane anesthesia have demonstrated that tactile forepaw stimulation triggers spindle bursts in s1 , followed by gamma and spindle bursts in m1 ( figure 5 ) . focal electrical stimulation of corticospinal tract neurons in layer 5 of m1 mimicking physiologically relevant 40 hz gamma or 10 hz spindle burst activity reliably elicited forepaw movements , indicating that m1 cortical spindle bursts are capable of triggering muscle twitches at this age . however , only 23.7% of the spontaneous bursts in m1 triggered forepaw movements and were followed by spindle bursts in s1 ( figures 5(b)(a ) and 5(c ) ) , indicating that only a fraction of m1 activity transients triggers motor responses directly . in 40.7% of the cases , spontaneous movements preceded the burst activity in m1 and s1 ( figures 5(b)(b ) and 5(c ) ) , suggesting that this activity may arise from subcortical regions in the brainstem or spinal cord . the remaining 35.6% of the m1 bursts were unrelated to any movements ( figure 5(c ) ) . the finding that 23.7% of the movements were triggered by m1 bursts as observed by an et al . is in contrast to previous observations , which demonstrated that dissection of neocortical inputs fails to suppress muscle twitches in rat pups . in summary , these data indicate that neocortical spindle bursts in newborn rodents ( and delta brushes in human fetus during the third trimester or in preterms ) are generated by central pattern generator ( cpg ) circuits in spinal cord , brainstem , and motor cortex ( for cpg circuits in mature brain see [ 4244 ] ) . neuropharmacological studies provided insights into the molecular mechanisms underlying the generation and modulation of neocortical spindle bursts . in vivo and in vitro data suggest that gabaergic synapses are not crucial for the generation of spindle bursts or spindle burst - like activity , respectively , but are essential for their spatial confinement to a cortical ( pre- ) column . in contrast , spindle bursts depend on intact glutamatergic synapses including alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) and n - methyl - d - aspartate ( nmda ) receptors [ 34 , 45 ] . carbachol - induced spindle - like oscillations in p0p3 mouse neocortex in vitro and spindle bursts in p0p2 rat in vivo are blocked or significantly reduced by different gap junction blockers , indicating that electrical synapses are critically involved in the generation of spindle bursts at this neonatal period . however , minlebaev et al . reported for p1p3 rats a significant increase in the occurrence of spontaneous spindle bursts in s1 following application of the gap - junction blocker mefloquine . single - cell recordings revealed additional insights into the mechanisms underlying the generation of spindle bursts . spindle bursts in v1 and s1 are accompanied by a barrage of glutamatergic and gabaergic postsynaptic currents ( pscs ) that are phase - locked to the spindle oscillations ( figure 6 ) . in prefrontal cortex , glutamatergic and gabaergic synaptic inputs to excitatory pyramidal neurons are phase locked to the theta - band component of the spindle , while pscs of inhibitory interneurons are phase locked to the higher beta and gamma frequencies , suggesting that excitatory and inhibitory neurons differentially modulate the distinct components of the spindle bursts . further , the application of cnqx eliminates the glutamatergic pscs and completely blocks the occurrence of spindle bursts , indicating a major causal role of ampa - receptor mediated glutamatergic inputs in the generation of spindle bursts . additional insights have been obtained by experiments in which subplate cells were selectively ablated in s1 shortly after birth . in these animals these in vivo results support previous in vitro studies , in which spindle bursts elicited by cholinergic stimulation were suppressed after removal of the subplate ( see below ) [ 34 , 35 ] . taken together , these data suggest that thalamocortical inputs relayed and amplified by the subplate [ 28 , 29]play an important role in the generation of spindle bursts ( e.g. , [ 17 , 45 ] ) . spindle bursts in the cerebral cortex of newborn rats can be elicited [ 34 , 35 ] and modulated by cholinergic mechanisms . in vitro , spindle burst - like oscillations can be reliably induced by activation of muscarinic acetylcholine receptors , predominantly of the m1 and m5 type . in vivo , spindle bursts in v1 are decreased by ~50% following the application of the muscarinic receptor antagonist atropine . furthermore , blockade of acetylcholine esterase with physostigmine or direct stimulation of the cholinergic basal forebrain nuclei augmented the occurrence v1 spindle bursts , indicating that the cholinergic system facilitates spindle burst activity in developing cerebral cortex .", "as discussed above , spatially confined spindle bursts in newborn rat cortex synchronize a local neuronal network resembling a neocortical ( pre- ) column ( figure 3 ) . beside these intra - areal synchronization , spindle bursts are also synchronized between different cortical regions within one hemisphere ( intrahemispheric ) . as described above , a tight functional correlation in spontaneous and stimulus - evoked spindle burst activity exists between s1 and m1 cortex ( figure 5 ) . spindle bursts with similar properties as those in v1 and s1 have been also recorded in vivo in the prefrontal cortex of urethane - anesthetized rats older than p2 . the same authors demonstrate that the hippocampus drives this early activity in the prefrontal cortex . beside this intrahemispheric synchronization between different cortical regions , spindle bursts also interact between both hemispheres ( interhemispheric ) . in vivo simultaneous recordings of spontaneous activity in homotopic cortical areas in both hemispheres at the same stereotaxic coordinates and depth have demonstrated that the amount of interhemispheric synchronization in newborn rats is initially rather low and increases during the first postnatal week . this interhemispheric communication of spindle burst activity depends on an intact corpus callosum . in this regard , in unanesthetized newborn rats callosotomy doubled the occurrence of spontaneous spindle bursts , suggesting that the corpus callosum modulates functionally inhibitory interactions between homotopic regions in both hemispheres during the occurrence of spindle burst activity . experiments in p2p15 rats demonstrated that this callosotomy - induced disinhibition is a transient feature of early development that disappears abruptly after p6 . it is not surprising that intra- and interhemispheric interactions of spontaneous activity in the spindle burst frequency range can be also observed in developing human cerebral cortex at early stages . using eeg and functional magnetic resonance imaging ( fmri ) omidvarnia et al . electric resting - state network that shows functional intra- and interhemispheric interactions in the 815 hz frequency range . in summary , several in vitro and in vivo studies have demonstrated that spindle bursts represent elementary states of intra- and interhemispheric synchronization in the very immature cerebral cortex .", "an increasing amount of experimental and clinical data strongly indicate that spindle bursts play an important role in the physiological development of the cerebral cortex . experimental evidence indicates that spindle bursts may be particularly suited to interfere with early neurodevelopmental processes , and thus disturbances in spindle burst activity may cause long - term structural and functional disorders . at early stages of development spontaneous and sensory - evoked activity patterns influence a variety of developmental processes , such as neurogenesis , apoptosis , neuronal migration , cellular differentiation , network formation , and myelination ( for review , see ) . it is not completely understood how electrical activity controls these different developmental processes and whether distinct activity patterns , such as spindle bursts , play a specific role . however , for the control of apoptotic cell death of immature neurons in vitro and in vivo the essential role of spontaneous network bursts to provide antiapoptotic signals has been demonstrated [ 52 , 56 , 57 ] . for this activity - dependent control of neuronal survival the phosphatidylinositol 3-kinase pathway plays an important role , while the mapk / extracellular signal - regulated kinase or the calcium / calmodulin - dependent protein kinase pathway is not directly involved . since one spontaneous spindle burst is associated at the single neuron level with 510 action potentials and the frequency of spontaneous spindle bursts is ~5 per minute [ 16 , 17 ] , a single neuron discharges with 2550 action potentials per minute . under in vitro conditions this discharge frequency supports neuronal survival of developing neocortical neurons , suggesting that spontaneous spindle bursts in vivo provide an important physiological signal for the control of neuronal survival versus apoptosis in the neonatal cerebral cortex . notably , spindle burst and gamma activity provides an ideal physiological stimulus for the activity - dependent release of bdnf , an important antiapoptotic signal . balkowiec and katz demonstrated for neuronal cultures that 3060 min of electrical burst stimulation ( 50 pulses at 2050 hz at intervals of 20 s ) increased extracellular bdnf levels by 20-fold , whereas stimulation patterns at lower frequency ( albeit producing the same number of extracellular electric shocks ) were ineffective ( for review , see ) . these data indicate that spontaneous spindle bursts represent a physiological trigger for the release of bdnf , which plays an important role in several aspects of development ( for review , see ) . using in vitro and in vivo models it has been recently shown that an experimentally induced inflammation by application of the endotoxin lipopolysaccharide induces rapid ( < 2 h ) alterations in the pattern of spontaneous spindle bursts and gamma oscillations in neonatal rodent cerebral cortex , which subsequently leads to increased apoptotic cell death . these inflammatory effects are specifically initiated by the microglia - derived proinflammatory cytokine tumor necrosis factor alpha and to a lesser extent by the chemokine macrophage inflammatory protein 2 . thus , inflammation causes a fast dysfunction in the pattern of spontaneous burst activity , which subsequently leads to increased apoptotic cell death , most likely by disturbances in the release of survival factors such as brain - derived neurotrophic factor ( bdnf ) acting on neurotrophin tropomyosin - related kinase b / c receptor . furthermore , removal of the subplate massively reduced spindle burst activity and led to a persistent loss of the typical barrel - like whisker representation within layer 4 , indicating that spindle bursts play a role in the development of the neocortical architecture . in summary , these experimental data suggest that any disturbances in the spontaneous activity of the developing cerebral cortex ( including spontaneous spindle bursts ) induce acute dysfunctions , which may cause long - term disorders . however , it remains to be elucidated whether spindle burst in particular can be causally related to neurodevelopmental disturbances . it has been recently shown in extremely preterm infants that the properties of neocortical bursts recorded with eeg and their scaling relationships correlate significantly with later cognitive development . these clinical data suggest that analyses of burst shapes obtained in eeg recordings from preterm and full - term newborn babies may have diagnostic use in neonatal intensive care units and predict the clinical outcome .", "while it is generally accepted that early electrical activity shapes the maturation of neocortical circuits , it remains an open question whether the specific properties of spindle burst are required or fulfill a distinct role in development . in this regard , it is possible that the spatiotemporal patterns of spindle bursts translate into a local molecular signal which fulfills an important developmental role . in particular as gaba does not seem to be necessary for the generation or maintenance of spindle bursts , and since gaba is essential for neuronal differentiation [ 64 , 65 ] , it is tempting to speculate that spindle bursts control the spatially confined release of gaba in developing local networks . another unresolved issue is that both spindle bursts and gamma oscillations can be observed during the same period of early development . they can be observed in early postnatal rodent brain from the day of birth (; also see ) and occur spontaneously as well as after sensory stimulation . moreover , both can be observed mainly during the critical periods of the primary sensory areas , although spindle bursts probably persist for slightly longer periods . in addition , spindle bursts and early gamma oscillations in newborn cortex are proposed to rely on thalamocortical inputs , in contrast to gamma oscillations in adult cortex which depend on perisomatic gabaergic inhibition . beside their differences in duration , occurrence , and frequency ( see above ) , spindle bursts and gamma oscillations in newborn cortex reveal a number of additional distinctions . in the immature barrel cortex the vast majority ( > 90% ) of spontaneously occurring spindle burst spans several barrel - related columns , whereas the majority ( ~70% ) of spontaneous gamma oscillations are restricted to a single or two barrel related columns . in line with these observations , gamma oscillations evoked by tactile stimulation are also closely related to a single functional column , while evoked spindle bursts span over more than one column . thus , one reason for the coexistence of spindle bursts and gamma oscillations during early postnatal development might be a distinct role in neocortical maturation . gamma bursts may reflect local information processing mostly within a single functional column , thus supporting the maturation of a column related network . in contrast , spindle bursts probably reflect larger local network events and may thus serve to promote the connectivity between neighboring columns . although spindle bursts as well as gamma oscillations are spatially confined to a small network in one neocortical area , it is unclear to what extent and how this activity connects to other cortical and subcortical regions ( e.g. , see [ 18 , 19 ] ) . further , it remains to be studied whether the immature brain shows a spindle burst related resting state and how this network state is altered by sensory activation or by pathophysiological events . finally , it would be most interesting and important to correlate specific patterns of spontaneous activity ( e.g. , delta brush ) recorded by means of full - band direct - current eeg in preterm und full - term human neonates with the acute functional state and with the further development of the child , as impressively done by vanhatalo and colleagues [ 62 , 67 ] .", "spatially confined spindle bursts and delta brushes represent the most prominent physiological activity patterns in the developing cerebral cortex of newborn rodents and preterm human infants , respectively . spontaneous and stimulus - evoked spindle bursts can be observed in various neocortical areas of different mammalian species and play important roles in the early development of cortical networks . however , it remains to be studied in more detail how exactly spindle bursts influence the maturation of the cortex and how a potential long - term dysfunction due to disturbances in spindle burst activity can be prevented by early intervention . since this important type of brain activity is already present in the human fetus in utero ( either spontaneously occurring or related to sensory inputs from the uterine environment ) , a better understanding of the physiological relevance of spindle burst oscillations is of major clinical relevance ." ]

[ "atrazine , 6-chloro - n2-ethyl - n4-isopropyl-1 , 3 , 5 atrazine , 4-diamine , is a selective herbicide that has been extensively used in corn production to control many broad - leaf and some grassy weeds . atrazine has long - term reproductive and endocrine - disrupting effects and a probable human carcinogen . international agency for research on cancer ( iarc ) has concluded atrazine as a group 2b carcinogen . the maximum contaminant level ( mcl ) for atrazine in drinking water established by the usepa is 3.0 gl . atrazine is moderately persistent in the environment and despite its low solubility , water resources contamination , it has become an international issue . the major dissipation route for atrazine is biodegradation , runoff , and leaching [ 4 , 5 ] . the rate of biodegradation of atrazine is reduced due to the adsorption , and desorption , and its bioavailability is the rate - limiting step in biodegradation . bioaugmentation is the addition of acclimated indigenous bacteria that can degrade the contaminant at accelerated rates . the bacteria are isolated from the heavily polluted soil in the laboratory through the enrichment process . the enrichment culture technique developed a mixed consortium of bacteria that are able to degrade the herbicide . a research was done by dehghani and colleague confirmed that atrazine biodegradation was enhanced in kavar corn field soil compared to the other soils that had not been exposed to the herbicide . therefore , several applications of atrazine on soil resulted in an enhancement of atrazine degradation . the addition of nutrient causes an increase of microbial populations , thereby , increasing the number of indigenous microorganisms capable of degrading the pollutant . n - containing compounds such as atrazine have been shown to serve as sources of nitrogen . in theory , addition of high c / n should induce nitrogen limitation and increase selective pressure for utilization of recalcitrant n sources like s - triazines , which contain n that can be used by certain bacteria and microbial consortia . cometabolic biotransformation can be enhanced by an increase in microbial activity which is stimulated by addition of organic matter . in the past few decades , intensive use of agricultural fertilization and herbicides has contributed to increasing concentration of n and herbicides . mixed microbial consortia [ 8 , 11 ] and strains have been isolated from soils and with a capability to complete the mineralization of the ring . the agrobacterium strain j14 , rhodococcus erythropolis , pseudaminobacter , and nocardioides can use atrazine as sole carbon and nitrogen source [ 1214 ] . laboratory experiments indicated that addition of carbon to soil inhibited atrazine biodegradation , but inorganic phosphate stimulates atrazine biodegradation . atrazine degradation rate was increased by addition of carbon sources to pure and mixed bacterial culture . ostrofsky and his coworkers ( 2001 ) found that cyanuric acid amendment increased atrazine mineralization by stimulation of general microbial population and activity . in liquid culture and in the presence of simple carbon sources , ( 1996 ) showed that the effect of n addition varies with the form and amount of added n . however , another study showed that the utilization of atrazine by pseudomonas adp in the presence of exogenous nitrogen ( nh4 , no3 , urea and glycine ) was not affected in a great amount by the presence of exogenous nitrogen . degradation of atrazine ( about 87% ) by p. adp and a. radiobacter was unaffected by the presence of n source , whereas no degradation occurred with bacterium m91 - 3 in media containing urea or nh4-n . another study reported that mineralization of atrazine by indigenous soil bacteria was inhibited by the addition of inorganic nitrogen . a study showed that addition of other organic amendments containing nitrogen suppressed atrazine . however , there are some limitations to the use of acclimated microbial cultures to degrade organic compound in a real field . physiochemical conditions of soils , and competition with native microorganisms , may destroy or reduce the inoculums and limit its degradative capacity . brandon and his coworkers ( 1997 ) showed that atrazine biodegradation was higher in liquid cultures than soil . he found that bacterial consortium in soil culture degraded 78% and 21% of atrazine at initial concentrations of 0.046 and 0.23 moles in 100 days . however , in liquid cultures , 90% and 56% of atrazine degraded in 80 days , respectively . in liquid culture , since fars is an agricultural province and enjoys the top rank in wheat and corn production in the country in recent years , atrazine has been widely used as a selective herbicide to control broad - leaf and grassy weeds in agricultural corn fields . the high incidence of atrazine to contaminate water resources and the increasing concern about the toxicological properties of atrazine has made researches directed toward bioremediation of atrazine in polluted sites . therefore , the main objectives of this research are evaluating the effects of carbon and nitrogen sources on atrazine biodegradation using mixed bacterial consortium isolated from corn field soil located in south of shiraz ( kavar ) and determining the efficiency of atrazine biodegradation process in soil culture . kavar corn field soil with a long history of atrazine application in fars province of iran has been explored for their potential of atrazine biodegradation .", "soil samples for liquid cultures were taken from south of shiraz ( kavar ) corn field with a long history of atrazine application ( more than 10 years ) in fars province . soil samples for soil cultures were also taken from a field in bajgah which has been under alfalfa cultivation for 3 years and has not received atrazine in the past 10 years . disturbed soil samples were collected from 0 to 20 cm of soil depth with a hand - driven soil auger and stored at 4c until they were used . the soil samples were air - dried and passed through 2 mm sieve to be prepared for further microbiological examinations . other soil characteristics such as soil solution ph and organic matter content were determined . the soil texture in kavar corn field was loam and the amount of sand , silt , and clay distribution were in order of 47.44% , 31.5% and 17.06% . the native soil characteristics at kavar site was fine loamy , mixed , thermic typic haploxerepts . at bajgah field , the soil texture was clay - loam and the amount of sand , silt and clay distribution were in order of 28.7% , 33.3% and 32.0% . the native soil characteristics at bajgah site was fine loamy , mixed , and thermic , typic calcixerepts . the soil samples were transported to the laboratory in zipped plastic bags and were kept frozen at 20c until they were ready for chemical analysis . the soil samples were thawed and air - dried at dark in room temperature and screened through a 2.0 mm sieve for maintaining homogeneity of soil in order to reduce the variability of adsorption data . 30 ml of dichloromethane was added to 10 g of the soil sample and shaken in a reciprocal shaker for 20 minutes . after filtration , the organic phase was transferred to a separating funnel and then atrazine was back extracted with 20 ml hcl ( 0.01 n ) . afterwards , the liquid phase was collected and transferred to a 15 ml glass vial and stored in a refrigerator prior to electrochemistry analysis . square wave voltammetry with the hanging mercury drop electrode ( auto lab type analyzer equipped with metrohm va stand 663 and gpes 4.9 software ) was used in this study to determine atrazine residual concentration in soil samples . atrazine recovery percent from soil with this method of extraction was 98% . in order to isolate mixed bacterial consortiums capable of growth on atrazine as a carbon source , the selective enrichment culture and basal salt medium were prepared as described in rousseaus , . ten grams of wet soil was inoculated into 90 ml of atrazine medium and supplemented with sodium citrate and delvocid ( 25 mg l ) after autoclaving . delvocid was used to prevent the growth of fungi and ph was also adjusted to 7.5 . cultures were incubated aerobically on a reciprocal shaker ( 150 rpm ) at room temperature in dark to preclude photolysis reactions . all enrichment cultures were subcultured on the same medium at one - week interval . from a one - week - old culture , 10 ml after culture was subculture for 30 and 300 days under conditions of nitrogen limitation , the remained atrazine after inoculation of the media for 10 days was quantified by electrochemistry . the bacterial consortium was harvested by centrifugation ( 6000 g at 40c for 20 minutes ) washed twice with 0.1 ml phosphate buffer ( ph = 7.3 ) . different carbon compounds such as glucose ( g ) , sodium citrate ( sc ) , sucrose ( su ) , starch ( st ) with three replications each at a concentration of 2 g l , and also the combination of these mentioned carbon sources such as g + sc , st + su , su + sc , st + sc , su + g , and st + g each at a total concentration of 2 after inoculation of the media supported with carbon sources , they were incubated at room temperature and placed in dark for 10 days . control and blank without bacteria inoculation and no carbon sources ( ncs ) were also used for this study . after 10 days , the remained atrazine was measured . to measure the influence of nitrogen sources on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture , nitrogen sources as routine fertilizers were added to atrazine mineral salt broth containing sucrose and sodium citrate . ammonium nitrate and urea as fertilizers were applied to corn field at a concentration of 600825 and 200400 kg ha , the nitrogen percent for these fertilizers were 34% and 46% , respectively . urea was added to atrazine minimal salt media at a concentration of 138690 mg after inoculation of the media supported with nitrogen sources , they were incubated at room temperature in dark for 10 days . a laboratory experiment was arranged to measure the influence of ph on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture . a different ph from 5.5 to 8.5 with three replications was used in atrazine mineral salt broth containing sucrose and sodium citrate and no nitrogen sources . atrazine degradation rate by the bacterial consortium was measured in 100 ml capped erlenmeyer flask containing soil samples . ten grams of soil sample was brought to the desired soil moisture ( 7% and 25% ) by the addition of sterile deionized water . the experimental design consisted of 36 flasks with 12 treatment and three replications for each treatment . the initial atrazine concentrations of 1.3 and 6.7 mg g soil are corresponded to 0.5 and 2.5 kg ha , respectively . however , atrazine concentration of 20 mg g soil is related to 7.5 kg ha(4.43 kg a. i. ha ) which is considered a relatively high atrazine concentration and might have occurred due to an accidental spillage . after one day of incubation to allow herbicide sorption to the soil , inoculation with 300 l phosphate buffer containing the selected bacterial consortium was added to soil to yield 7.5 10 bacterial cell g soils as determined by plating on soil extract agar . the soil samples were mixed until they were homogeneously wet and then incubated at room temperature in dark until the end of the experiment . soil moisture was maintained constant through the incubation by weighing and correcting for any weight loss by adding sterile deionized water . to maintaining high population of atrazine degraders , the inoculation with the consortium was made every 5 days over a 30-day period for a total of 6 inoculations . soil samples at the initial atrazine concentration of 6.7 mg g soil were extracted at the incubation time of 2 , 10 , 20 , and 30-day and analyzed to determine the remained atrazine concentration at each time interval .", "by using a mixed bacterial consortium with a high capability of atrazine degradation isolated from kavar corn field soil , the effect of different carbon sources on atrazine biodegradation was studied ( figure 1 ) . according to this figure , the percentage of atrazine biodegradation rate for different carbon sources was in the range of 9.47% to 87.72% . a blank sample did not adequately support the growth of consortium bacteria ( due to low turbidity ) , and the rate of atrazine degradation is lass than 3% . linear regression showed that there was a significant difference between different carbon sources and atrazine biodegradation rate ( p < 0.001 ) . the effect of different nitrogen sources on atrazine biodegradation was shown in tables 1 and 2 . according to table 1 , the percent of atrazine biodegradation rate decreased from 87.72% to 29.58% as the ammonium nitrate concentration increased from 0.0 to 900 kg ha soil . the rate of atrazine biodegradation for ammonium nitrate decreased quickly when the concentration of ammonium nitrate increased from zero to 600 kg ha soil , after that the reduction rate of atrazine degradation was getting slower . the same trend has been observed for atrazine biodegradation in the presence of urea . according to table 2 , the percentage of atrazine biodegradation rate decreased from 87.72% to 26.76% as the urea concentration increased from 0.0 to 500 kg ha soil . for urea , atrazine biodegradation rate under nitrogen amendment showed an initial sharp decreasing slope and then reaching constant with relative slower rate . variations of ph on atrazine degradation rate were examined when sucrose and sodium citrate were used as a carbon source , while there was no nitrogen source available ( figure 2 ) . according to regression analysis , it can be concluded that there was a significant difference between ph and atrazine biodegradation ( p < 0.05 ) . \n table 3 showed the effect of initial atrazine concentration and soil moisture on the biodegradation of the inoculated soil . according to data on this table , after 30 days of incubations atrazine degradation rate for noninoculated soil samples was low and atrazine reductions were only 7% to 19% . however , for inoculated soil samples , degradation rate was higher and its reductions were 19.572% . the inoculated soil samples at 25% soil moisture at initial concentration of 1.3 , 6.7 , and 20 mg g soil , atrazine reductions were 69.5% , 60.5% and 30.5% within 30 days of incubation , respectively . figure 3 showed that atrazine biodegradation rate during the different time intervals in soil culture . \n figure 4 depicted the plot of the semilogarithm of initialized atrazine concentration ( c / c0 ) versus time . during the 30 days of incubation period , atrazine concentration decreased from an initial concentration of 6.7 mg gsoil to a final concentration of 2.67 mg gsoil . only a 5% decrease in atrazine concentration however , figure 3 showed that at 20 days 40% of the atrazine had degraded . after that a very slow increase in atrazine degradation was observed and remained constant until the end of the incubation period . assuming a pseudo - first - order reaction for the disappearance of atrazine , a plot of the natural logarithm of initialized atrazine concentration ( c / c0 ) versus time resulted in a rate constant equal to 0.0328 d ( figure 4 ) . the t1/2 for atrazine calculated from the plot was 21.13 d. the initial slow degradation rate of atrazine was followed by a much faster degradation rate that lasted about 5 day and then the degradation rate became slower and finally remained constant until the end of incubation period .", "carbon sources of sodium citrate and sucrose had the highest atrazine biodegradation rate . according to data , mandelbaum and his coworkers ( 1993a ) used sodium citrate and sucrose as the carbon source . according to data in this research , the percentage of atrazine biodegradation rate with no carbon sources was only 5.5% . past studies showed that carbon in atrazine ring can not be used by many bacteria as energy sources ; however , carbon in alkylated group provided carbon for bacterial growth . the low amount of atrazine can not support bacterial growth , and therefore , a supplemental carbon was needed . according to data on tables 1 and 2 , atrazine utilization is repressed under nitrogen - sufficient growth condition and activated under nitrogen limitation . many investigations also showed a negative effect of nitrogen amendment on atrazine biodegradation by indigenous populations in soils [ 34 , 35 ] . however , other study reported that organic nitrogen supplied in dairy manure increased atrazine mineralization . data regarding the effect of ph shows that as ph increased from 5.0 to 7.0 , the rate of atrazine biodegradation increased . however , as ph increased from 7.0 to 8.5 this caused a reduction rate in atrazine biodegradation . atrazine biodegradation rates were significantly enhanced in the inoculated soils as compared to uninoculated control soils . after 30 days , the percent of atrazine reduction was only 12% for uninoculated soils at initial atrazine concentration of 6.7 mg g soil and the soil moisture of 25% . however , soil that was inoculated every 5 days with the bacterial consortium had reduced atrazine up to 60.5% at the same initial atrazine concentration and the soil moisture by day 30 ( table 3 ) . as initial atrazine concentration increased from 1.30 to 20 mg g soil , the percentage of atrazine reduction decreased from 69.5% to 30.5% ( inoculated soils and soil moisture 25% ) . the decrease in atrazine reduction at 20 g g soil was possibly due to the result of complex interaction between microbial activity and nutrient availability . therefore , unbalanced nutrient supply and not atrazine toxicity was probably responsible for the decrease . figure 3 showed that atrazine degradation rate increased as time passed . according to ( table 3 ) , enhanced atrazine biodegradation rates occurred with an increase in soil moisture from 7% to 25% . the percent of atrazine reduction increased from 32.5% to 69.5% as the soil moisture increased from 7% to 25% ( at initial atrazine concentration of 1.30 mg g soil ) . many researchers found that higher atrazine biodegradation rate occurred with an increase in soil moisture . soil moisture influences microbial processes through direct effect ( e.g. , water availability ) or indirect effects , for example , solute diffusion , chemical availability , and aeration . therefore , the positive effect of increasing soil moisture was probably due to increased microbial mobility , solute diffusion , and chemical availability which all had an indirect effect on atrazine degradation . in this study , more than 60% of atrazine was degraded in 30 days of incubation periods ( figure 3 ) . atrazine degradation exhibited a half - life of approximately 21.13 d. in conclusion , our results confirmed that atrazine biodegradation was higher in liquid cultures than soil . the mixed bacterial consortium in soil culture degraded 69.5% of atrazine at initial concentrations of 1.3 mg g soil in 30 days . however , in liquid cultures , 87.72% of all atrazine degraded in 10 days . results of this study suggested that atrazine bioremediation in soil utilizing atrazine - degrading bacterial consortium could be accomplished across a wide range of atrazine concentration if the soil moistures swere enough and also nutrient availability was balanced , too . the bacterial consortium with the ability to degrade atrazine provided a good opportunity for increasing the degradation rate of this herbicide through bioaugmentation . it is very important to note that these bacteria in the controlled laboratory conditions ( with the addition of carbon and nitrogen sources and without the interaction of environmental factors ) had been able to use atrazine . however , there are some limitations to the use of acclimated microbial cultures to degrade the herbicide in a real field . therefore , it is highly recommended that the feasibility of atrazine degradation to be studied by the bioaugmentation of mixed bacterial isolates to the real contaminated soil . the mixed bacterial consortium successful in laboratory studies may fail in the real field because of their sensitivity to high concentrations of other compounds ." ]

[ "neurons , oligodendrocytes ( ols ) , and astrocytes are unique cell types in the central nervous system ( cns ) of vertebrates.1 neurons process and transmit information electrochemically.2 ols generate myelin sheaths around most axons of the vertebrate cns , enabling a faster conduction of the nerve impulses . astrocytes provide support and nutrients for the nerve tissue.3 the different cell types in the nervous system are regulated by the precise spatiotemporal expression of specific genes.4,5 transcriptional regulation plays an important role in the determination of neural cell differentiation.6,7 \n olig genes belong to the basic helix loop helix transcription factor family , which encode ol lineage transcription factors 1 , 2 , and 3 ( olig1 , olig2 , and olig3 ) . with the exception of olig3 , olig genes are specifically expressed in the cns , and play a critical role in cns development by controlling differentiation and maturation of ols , motor neurons ( mns ) , and astrocytes.8,9 olig2 null mice die at birth from a lack of mns.10 both gain- and loss - of - function studies were performed in an olig1 null mouse with normal myelin during development , but which were unable to remyelinate on experimental challenge.11 a second olig1 null mouse with less compensatory effect by olig2 had a more severe phenotype and died around postnatal day 14 from a complete lack of myelin . this mutant had mature ols , but failed to wrap myelin or even deposit lipid around axons.12 knocking out olig1 and olig2 individually or together affected differentiation and maturation of ols , suggesting functional overlap in the cns.10,13,14 until now , the role of olig2 during development of spinal cord attracted more attention . however , studies investigating the expression and function of olig1 in development and disease are limited . although it is widely known that olig1 promotes the differentiation and maturation of ols , it is unclear how these occur during development . exploring temporal and spatial expression and distribution of olig1 will contribute to our understanding of the role of olig1 in specialization of neural cells during development . therefore , in this study , we determined the expression pattern of olig1 in neural cells during rat spinal cord development .", "sprague dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) . all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e ) was designated as day 0.5 ( when vaginal plugs in female rat were observed ) . eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) . each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) , the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot . spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats . spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use . for immunohistochemistry staining , the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes . they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt . the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) . the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c . the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei . at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments , western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant . the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) . the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) . protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) . the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst , the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions . films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) . the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin . the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software . the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups .", "sprague dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) . all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e ) was designated as day 0.5 ( when vaginal plugs in female rat were observed ) . eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) . each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) , the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot . spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats . spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use .", "for immunohistochemistry staining , the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes . they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt . the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) . the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c . the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei . at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments ,", "western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant . the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) . the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) . protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) . the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst , the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions . films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) . the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin .", "the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software . the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups .", "western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood . our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points . however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) . to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively . our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points . furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) . mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) . the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood . we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development .", "western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood . our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points . however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) .", "to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively . our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points . furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) . mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) . the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood . we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development .", "the cns originates in the neuroepithelial stem cells ( nscs ) , which ultimately generate the three major cell types : neurons , ols , and astrocytes.1 in this process , transcriptional regulation plays an important role.6,7 in mice , genetic studies revealed that olig transcription factors play key roles in the genesis of mns and ols . gain- and loss - of - function analysis confirmed the fundamental role of olig1 in the survival and maturation of opcs.12,1517 regulation of olig1 gene expression in spinal cord of demyelinating disease promotes myelin repair after spinal cord injury . however , the distribution and expression of olig1 in neural cells during development is unclear . in this study , we systematically investigated the expression pattern of olig1 in neural cells during rat spinal cord development . first , using the western blot , we demonstrated the differential expression of olig1 in rat spinal cord development . olig1 expression increased and peaked at e18.5 , which may contribute to the differentiation of a large number of neural cells into opcs and mn precursors at this stage . recently , it has been reported that olig1 protein expression was significantly decreased during mouse ol progenitor differentiation.18 however , the expression of olig1 increased again from p2w , which was consistent with a recent report that olig1 was expressed in human ols during process outgrowth and maintained at a high level prior to myelination.19 next , we identified the expression of olig1 in neural cells at several time points during spinal cord development . we confirmed that olig1 was expressed not only in opcs but also in neurons from e14.5 to adulthood . cns development starts from nscs , which generate the three major cell types ( neurons , ols , and astrocytes ) of the cns.20,21 however , the intermediate steps in the differentiation of nscs in vivo have not been clarified . in the embryonic ventral spinal cord , mns and ols are derived from a common progenitor pool , suggesting that these lineages share intrinsic and extrinsic regulatory mechanisms.10,13,21,22 ol progenitors were more closely aligned with neuronal subtype progenitors than with astrocytes , at least in the developing cns.23 a study demonstrated that olig2 was more likely an early - stage factor for ol precursor cells , while olig1 played a critical role in late - stage ol maturation and myelin formation.19 however , jakovcevski and zecevic24 found olig1 protein expressed in primitive neuroepithelia that can give rise to interneurons in the human fetal brain . a recent study suggests that olig1 regulates neuron - glial fate choice in the embryonic telencephalon.25 in conclusion , olig family members do not act independently but interact with other transcription factors to tightly control cns development.17,26 our findings raise the possibility that olig1 has some function in the formation of mns during cns development . mbp is a major myelin protein , which is expressed in mature ols.27,28 mbp is the major protein component of myelin at 2 weeks after birth in the rat brain , expressed a week later than in spinal cord.28 in this study , we observed that mbp expression commenced from postnatal period , and olig1 was not coexpressed with mbp during rat spinal cord development . opcs undergo two types of differentiation , resulting in ols in the absence of serum or type ii astrocytes in the presence of serum in vitro.2931 fate - mapping studies have shown that opcs generate a subset of astrocytes during perinatal development.32 most neurogenic bhlh transcription factors , for example , mammalian achaete - scute homologue 1 , mammalian atonal homologue , and neurogenins 1 and 2 , are only expressed transiently in progenitor cells as needed.23 in this study , we observed the transient expression of olig1 in gfap - positive cells at e14.5 , suggesting that ols and astrocytes may derive from the same progenitor cell . both olig1 and olig2 occur in the nucleus of ols and their progenitors occur on p1 in mouse brain.33 olig2 continues to be nuclear at all developmental stages , whereas olig1 is almost completely cytoplasmic in the adult mouse brain . changes in nuclear or cytoplasmic olig1 location correspond with altered morphology of opcs.11 recently , othman et al19 reported that o1-positive multipolar opcs with nuclear expression of olig1 exhibited fewer processes , while opcs with cytoplasmic olig1 manifested more extensive membrane expansion . nuclear olig1 facilitated mbp expression , but with greatly diminished membrane expansion.19 however , these experiments were performed in mouse and human . our results demonstrated that olig1 was expressed in o4-positive opcs and -tubulin - positive neurons , but was localized in the cytoplasm during the period from e14.5 to adulthood . we speculate that the cause for this inconsistency may be attributed to differences between animal species or other unknown factors .", "in conclusion , we systematically explored the expression pattern of olig1 in neural cells during rat spinal cord development . our results showed that olig1 was coexpressed with o4-positive opcs and -tubulin - positive neurons at all time points during development . olig1 was localized in the cytoplasm of o4- or -tubulin - positive cells during the transition from e14.5 to adult . our results contribute to understanding the mechanism of developmental regulation of neural cells by olig1 ." ]

[ "neck pain is a common musculoskeletal disorder in modern society that can produce severe \n pain . 67% of the population suffers from it at least once in a lifetime and the prevalence \n is about 23%1 . the pain exacerbates and \n fades periodically , and many patients do not fully recover from the symptoms2 . neck pain occurs in the upper thoracic \n spine area including the shoulder , and it is mechanical pain caused by bad postures and \n habits in most cases3 . according to \n sharon , the upper thoracic spine is involved in the physiologic motion of the neck4 . the decreased movement of the upper \n cervical spine can cause excessive movement of the lower cervical spine , increase fatigue in \n the sternocleidomastoid , anterior scalenus , and upper trapezius , cause changes of neck \n postures and breathing patterns , and a decrease in the range of motion5 . patients with chronic neck pain experience functional \n impairments including weakening of deep bending neck muscles due to the activation of neck \n surface muscles6 , increased deformity of \n the forward head posture7 , proprioception \n impairment8 , and poor balance9 . in addition , decreased movement of the \n cervical spine restricts the range of motion of the spine and decreases breathing \n function10 , 11 . slightly bent positions like the forward head posture may cause \n mechanical neck pain , cause a greater load by affecting the mobilization order of the \n muscles operating when the arm is raised , and restrict the range of motion12 . these changes reduce the ability to \n maintain balance and increase the risk of falls and injury of the musculoskeletal \n system9 . physiotherapists are using methods such as electrotherapy , therapeutic exercise , and manual \n therapy to intervene in the neck pain13 . \n in a cochrane review study of neck pain , stretching exercises for the neck and upper limbs , \n strengthening exercises , static and dynamic stabilization exercises maitland mobilization \n grade 3 and 4 treatment for the cervical spine and upper spine significantly decreases neck \n disability index ( ndi ) , the pain index15 , \n and increases the range of motion16 . in \n addition , an the intervention combining therapeutic exercise and manual therapy was \n significantly more effective compared to manual therapy alone17 . recent evidence - based studies report that treatment combining \n therapeutic exercise and manual therapy is more effective18 , but research of combined interventions is sparse . the purpose of \n this study was to apply joint mobilization and therapeutic exercise to the cervical spine \n and upper spine , investigate the effects on functional impairments caused by the neck pain , \n and examine differences between groups by comparing the intervention group with the group to \n which only therapeutic exercises were applied .", "this study was implemented with non - specific neck pain patients with no medical findings \n who had visited the cheongju st . all participants received verbal and written information about the study and signed a \n consent form . the patients who had undergone or would have surgery in the spine , and those who \n had neurological damage , a cervical spine fracture , osteoporosis , arthritis , a malignant \n neoplasm , a vascular disease , or a psychiatric problem were excluded . the subjects were \n randomly assigned to one of two groups of nine people each . group i was the therapeutic \n exercise group , and group ii was the group to which joint mobilization was applied in \n combination with therapeutic exercise ( table \n 1table 1.general characteristics of the subjects ( n=18)group igroup iigender ( female)99age ( years)58.0 1.659.0 2.4height ( cm)158.0 3.9157.0 4.7weight ( kg)58.0 4.659.0 6.6values are expressed as mean sd . * significant difference between pre - post test ( p<0.05 ) . group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) ) . the pretest included the visual analog scale ( vas ) , neck disability index \n ( ndi ) , active cervical range of motion ( acrom ) , static balance ability , muscle tone of the \n upper trapezius , and respiratory function . after the pretest , group i performed therapeutic \n exercise and group ii performed both therapeutic exercise and joint mobilization for 60 \n minutes a day , three times a week for two weeks under the guidance of a physical therapist . \n the participants did not receive any other interventions associated with the neck pain while \n this research was being conducted . the post - test was carried out with the same protocol as \n the pretest after two weeks . values are expressed as mean sd . * significant difference between pre - post test ( p<0.05 ) . group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) therapeutic exercises involved enhancing mobility , stability and muscular strength of the \n neck , improving proprioception , and performing reeducation of movement , and the intensity of \n exercises was adjusted according to the physical abilities of the individuals . for the joint \n mobilization , after \n the painful sites were located by examination , the active movement test was conducted to \n find where joint mobilization would be applied , and the amount and quality of motion were \n examined . afterwards , passive physiological intervertebral movement ( ppivm ) testing and \n passive intervertebral accessory movement ( paivm ) test were conducted to find which joints \n had restricted or excessive movement , and determine where joint mobilization would be \n applied . the vas was employed to measure the intensity of pain19 , the ndi was used to measure neck pain disability20 , and crom instrument to assess the \n cervical range of motion21 . in order to \n check static balance , myoton pro ( myoton as , estonia ) measured \n the muscle tone of the upper trapezius22 , \n and a spirometer ( pony fx spirometer ; cosmed , rome , italy ) measured the respiratory \n function23 . , an ibm company , \n chicago , il , usa ) . for the normality test , the shapiro wilk test was conducted , confirming \n that the data are normally distributed . assuming homogeneity between groups , the independent \n samples t - test was performed , and the paired sample t - test was conducted for the \n within - group comparison of the measurements before and after the interventions . in addition , \n the independent samples t - test was conducted to compare the differences between groups . null \n hypotheses of no difference were rejected if p - values were less than 0.05 .", "vas and ndi led to significant changes in both groups , and group ii improved significantly \n more than group i ( tables 2table 2.visual analog scale of the subjectsgroup igroup iipre4.8 0.44.9 0.3post2.7 0.5 * 1.4 05**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) , 3table 3.neck disability index of the subjectsgroup igroup iipre15.1 2.317.2 3.1post8.9 1.5 * 8.6 1.9**significant difference between pre - post test ( p<0.05 ) . the acrom increased significantly in both groups , and group ii \n improved significantly more on the right lateral flexion and rightward rotation than group i \n ( table 4table 4.active cervical range of motion of the subjects ( )group igroup iiflexionpre49.2 8.748.9 4.7post54.2 4.5 56.1 6.8*extensionpre54.9 4.356.4 4.9post 64.3 3.2 * 67.6 3.9*rt . rotationpre60.8 3.259.7 5.1post 67.6 3.6 * 67.7 4.2**significant difference between pre - post test ( p<0.05 ) . both groups improved significantly in static balance , with no difference \n between groups ( tables 5table 5.center of gravity sway velocities of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre0.04 0.00.05 0.0 post0.03 0.00.04 0.0firm - eyes closedpre0.15 0.00.15 0.0post0.14 0.00.14 0.10.37 0.1post 0.30 0.1 * 0.32 0.1**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) , 6table 6.center of gravity total sway distances of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre294.6 64.1304.6 97.9 post270.4 66.4281.9 109.2foam - eyes closedpre618.2 82.7687.3 182.8post 529.1 88.2 * 548.4 172.5**significant difference between pre - post test ( p<0.05)significant difference between groups ( p<0.05 ) ) . muscle tone in the left and right upper trapezius muscle improved \n significantly in both groups , and there was no difference between groups ( table 7table 7.muscle tone of the subjectsgroup igroup iirt . upper trapeziuspre16.0 1.5 16.0 1.2post13.7 1.3 * 14.2 1.4**significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) . significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "chronic neck pain patients suffer diverse functional impairments , including a deterioration \n of balance ability , respiratory function , range of joint motion , and proprioception11 . research related to neck pain suggests \n that there are more efficient interventions based on evidence through systematic reviews , \n and the interventions combining manual therapy and therapeutic exercise are described as \n more effective in recent literature18 . in \n this study , therapeutic exercises were conducted that are reported to be effective for \n patients with mechanical neck pain , namely , stretching exercise for the neck and upper \n limbs , static and dynamic stability exercise , strengthening exercise , and exercise \n reeducation14 . in addition , as manual \n therapy , mobilization to reduce pain and improve the movements of the soft tissue exhibiting \n contracture and restricted motions of joints ( maitland grade iii and iv)13 , 24 \n were applied to group ii . the effect of the two kinds of interventions , and group \n differences based on those interventions were examined . in this research , a significant \n reduction in the vas and ndi was observed in both groups , and the comparison between the \n groups showed that the intervention was more effective in group ii . this result is different \n from studies that found no difference between groups based on a short - term intervention17 . these differences may result from our \n smaller number of subjects and differences of individual characteristics . for the acrom , \n both groups improved significantly overall , and group ii improved more on right lateral \n flexion and rightward rotation . these results are consistent with studies that reported that \n mobilization applied to both the neck and spine at the same time improved cranial vertical \n angle and cranial rotation angle25 and \n research that showed that endurance exercise of the neck , stabilization exercise , and \n strength training improved the joint range of motion26 . in this study , static balance was measured under a variety of conditions , including hard or \n soft bearing surfaces and open or closed eyes . a significant decrease in the sway velocity \n of the center of gravity and in the sway distance occurred only under eyes closed , and there \n was no difference between groups . although it was reported that patients with neck pain and \n forward head posture may exhibit reduced balance ability9 , it was possible to measure the static balance ability most \n accurately when the soft bearing surface was used and eyes were closed to block the visual \n feedback . muscle tone of the upper trapezius decreased significantly in both groups , and \n there was no difference between the groups . this may indicate that the tension and fatigue \n of the upper trapezius were reduced due to pain reduction , enhancement of mobility of the \n neck joints , and change in the mobilization sequence of movements caused by afferent \n information activation of proprioception5 . \n deterioration of mobility of the spine and thoracic cage was reported in patients of chronic \n neck pain , and respiratory function was also affected27 . however , in this research , no significant change in the \n respiratory function was observed . this may be because the patients were not given direct \n training for functional improvement of breathing muscles , although the range of motion of \n the cervical and upper thoracic spine was improved through the study interventions . the number of subjects was small , and since the \n period of intervention was not long , it was difficult to produce a mechanical change of \n muscles . therefore , future research is required to investigate the effect of joint \n mobilization and therapeutic exercise on the functional impairments caused by chronic neck \n pain using diverse subjects and intervention periods , and research on the persistence of the \n effect also needs to be conducted . in conclusion , joint mobilization and therapeutic exercise for functional impairments \n caused by chronic neck pain significantly improved several types of functional impairment . \n in addition , in the group to which both joint mobilization and therapeutic exercise were \n applied , significantly more improvement in the pain index , neck disability levels , and acrom \n was seen than in the group that received only therapeutic exercise ." ]

[ "adss obtained de - identified data for the entire population of alberta from alberta health and wellness administrative databases ( discharge abstract database , alberta physician claims data , ambulatory care classification system and vital statistics ) . data on the adult ( over 20 years ) population has been reported elsewhere ( 5 ) . the present analysis focused on albertan youth < 20 years of age . flagged in the databases and were defined as any alberta inhabitant registered under the federal indian act and entitled to treaty status with the canadian government . the status aboriginal identifier captures first nations and inuit peoples both on- and off - reserve with treaty status , but not mtis individuals or aboriginal individuals without treaty status who are included in the general population comparison group . alberta law requires that all residents and dependants register with the alberta health care insurance plan , thus the alberta central stakeholder registry functioned as a denominator . diabetes cases were identified by applying the national diabetes surveillance system ( ndss ) algorithm ( 13 ) , which has recently been validated for the determination of pediatric diabetes rates ( 14 ) . the ndss methodology of identifying has been validated in non - aboriginal populations that include persons of varying geographical locations ( 15,16 ) , and also within an aboriginal community in alberta ( 17 ) . the algorithm requires an individual to have either 2 physician visits or 1 hospitalization for diabetes ( icd-9 codes starting with 250 and icd-10 codes e10e14 ) within 2 years to be labelled as case of diabetes ( 13 ) . in contrast to the ndss case definition , pregnant women that may have had gestational diabetes were not excluded in the current analysis due to the elevated risk of subsequent diabetes ( 18 ) . also , since the numbers of diabetes cases are small among this population , only crude , unadjusted rates are provided . for both populations , the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year . the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years ) was considered an incident year , and considered a prevalent case in subsequent years . prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year . incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) . odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville , md ) for crude values . the aapc provides a relative summary measure of the trend over a pre - specified fixed interval . tests of parallelism were then performed to determine if trends over time differed by group or sex .", "the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year . the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years ) prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year . incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) . odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville , the aapc provides a relative summary measure of the trend over a pre - specified fixed interval . tests of parallelism were then performed to determine if trends over time differed by group or sex .", "based on alberta health and wellness administrative databases , 853,733 youth ( 51.2% male ) were living in the province as of 2007 , of which 50,930 were status aboriginal ( 51.2% male ) . among these youth a total of 2,589 prevalent diabetes cases were apparent in 2007 , with 140 cases occurring amongst status aboriginal youth . the 2007 crude prevalence rate of diabetes was 0.27 and 0.31% in the status aboriginal and general population youth populations respectively ( fig . 1 , table i ) . incidence was 0.59 per 1,000 for status aboriginal and 0.49 per 1,000 for general population . no discernible differences were seen in diabetes prevalence or incidence with respect to group or sex in 2007 ( fig . 1 , crude rates of diabetes among status aboriginal and general population youth in alberta , 19952007 ( adss ) . a = prevalence ; b = incidence . crude diabetes prevalence and incidence among status aboriginal and general population youth , 2007 crude rates of both diabetes prevalence and incidence increased in status aboriginal and general population youth over the observation period ( fig . 1 , table ii ) . however , prevalence grew to a greater extent among status aboriginal youth ( aapc 6.98 ) compared to those in the general population ( aapc 3.93 ; p < 0.01 ) , with status aboriginal males experiencing the largest increase in diabetes prevalence ( aapc 9.18 ) . ethnic differences in diabetes incidence trends were only observed among the male population , which saw a significantly larger rise in incidence for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ; p < 0.01 ) . male youth , regardless of group , experienced greater increases in both prevalence and incidence over time than that of respective female youth ( table ii ) . age - specific crude prevalence and incidence of diabetes by group are shown in fig . 2 . age specific crude rates of diabetes among status aboriginal and general population youth , 1995 and 2007 . ethnicity comparisons of aapc ( average annual percent change ) in diabetes prevalence and incidence among status aboriginal and general population youth , 19952007 p < 0.05 for aapc ethnicity comparison .", "paralleling adult populations , diabetes among youth is increasing worldwide ( 20 ) . while type 1 diabetes remains the majority of cases in youth , type 2 diabetes ( a disease typically regarded as an adult phenomenon ) is becoming progressively more common in children and adolescents ( 20 ) . we report that youth - onset diabetes is an increasing problem in alberta , especially among status aboriginals . though our results are in line with international data showing increasing trends ( 20,21 ) , they contradict the recent ndss reports ( 12,22 ) , which found diabetes prevalence and incidence rates remained stable at 0.3 and 0.4 ( per 1,000 ) respectfully among general population youth between 20012006 . it is possible that differences in ethnicities and/or socioeconomic parameters contribute to these differences . among indigenous populations especially , including american indian / alaskan natives , australian aborigines , the maori of new zealand , and canadian aboriginals , youth - onset diabetes is increasingly being identified ( 14,2327 ) . in canada , longitudinal trends of diabetes in aboriginal youth had previously only been explored in manitoba . dean and colleagues ( 10 ) showed the crude type 2 diabetes incidence rate rose to 0.55/1,000 ( for ages 019 ) in 2001 . similarly , crude diabetes incidence increased from 0.22 per 1,000 to 0.59 per 1,000 over a 12 year time span in the current analysis . the observed accelerated increase in diabetes among status aboriginal youth is consistent with reports of a decrease in the age of diagnosis of diabetes among aboriginal adults . in saskatchewan type 2 diabetes incidence was highest among status aboriginal adults aged 4049 , while most new diabetes cases among the general population were in those > 70 years of age ( 4 ) . also , as indicated by the first nations and inuit regional health survey , the majority ( 53% ) of those with diabetes in aboriginal communities were < 40 years of age ( 28 ) . compared to general population , diabetes grew approximately twice as much among status aboriginal youth . ethnic comparisons have remained largely unexplored in youth , despite numerous studies documenting the divergence in diabetes epidemiology in adult populations . in the beginning of adss observation 1995 , incidence and prevalence of diabetes were distinctly higher among general population . by 2007 however , status aboriginal youth have caught up to the point where no detectable differences were apparent , likely ( as mentioned above ) due to type 2 diabetes . reinforcing this notion , a recent national surveillance study amongst practicing physicians found the incidence of type 2 diabetes among aboriginal children ( 0.23/1,000 ) was strikingly 46-fold higher than caucasian children ( 0.005/1,000 ) over a 24 month period ( 29 ) . we observed an increased rise of diabetes among young status aboriginal males , confirming earlier studies . sex ratios of prevalence and incidence ( female : male ) of approximately 4:1 and 2:1 in 1995 decreased to 1:1 and 1:1 respectively in 2007 . additionally , the aapc in prevalence was highest amongst status aboriginal males ( aapc 9.18 ) compared to females ( aapc 5.70 ) . over a 15 year time period in manitoba , the early predominance of youth diabetes cases among aboriginal females ( 8-fold compared to males ) disappeared by 2001 ( 10 ) . from 1990 to 1998 , american indian and alaskan native young males also experienced relative increases in diabetes prevalence almost double that of their female counterparts ( 30 ) . similarly , the gap in diabetes prevalence between adult status aboriginal females and males is also diminishing ( 4,5 ) . reasons for the accelerated rise of diabetes among status aboriginal males have not been explored , but may be due in - part to escalating childhood overweight and obesity ( 31 ) with a possible gender - based lag in the obesity epidemic where it may have occurred earlier in time amongst females . in countries with universal healthcare , administrative data have become common and invaluable sources for the population - based surveillance of many chronic diseases . ndss methodology has been validated in adults and youth , and is a standard for diabetes monitoring at the population level ( 16,18,32,33 ) . for instance , guttmann et al . ( 33 ) recently found the ndss algorithm in youth ( 019 years of age ) in ontario was associated with 100% sensitivity and 94% specificity . using a similar algorithm ( 4 physician claims over a 2-year period ) , diabetes prevalence rose from 0.18 to 0.24 and incidence ( per 1,000 ) increased from 0.25 to 0.32 among ontarian youth between 1994 and 2003 ( 32 ) . in support , general population youth in the current study experienced nearly identical increases in prevalence and incidence between 1995 and 2003 : 0.19 to 0.25 and 0.22 to 0.32 ( per 1,000 ) respectively , with rates progressively increasing until the end of the study period in 2007 . we are unable to distinguish the type of diabetes in our administrative data . however , our clinical experience suggest that the majority of the diabetes in aboriginal youth is type 2 diabetes , whereas in the non - aboriginal population it is heavily dominated by type 1 diabetes . moreover , rates of type 1 diabetes are much lower among american indian and canadian aboriginal youth compared to the general population historically ( 34 ) . future research aimed at describing the contribution of both type 1 and type 2 diabetes to the observed trends is warranted . possible gestational diabetes cases were included and may have inflated rates in status aboriginal as gestational diabetes has been shown to be more common in adult aboriginal populations ( 35 ) . it is possible that increased awareness and screening in youth may have contributed to the observed increases , however for various projects ( http//:www.braiddm.ca ) we have screened 799 youth at risk ( mostly aboriginal ) in rural alberta since 2001 and have found only 3 ( 0.4% ) of those had undiagnosed diabetes ( unpublished ) . by contrast , the rates of undiagnosed diabetes are approximately 4% in at risk adults . thus , it appears that diabetes in youth is not often asymptomatic , and therefore is less likely to be detected by increased awareness and screening . our results can not be generalized to non - registered aboriginal or mtis youth , whom could not be identified and were included in the general population group . also , due to the small number of diabetes cases in youth , age - standardized prevalence and incidence rates were not calculated and only crude rates were reported . lastly , amendments to the indian act in 1985 are possibly increasing the status aboriginal denominator ( bill c-31 ) and likewise some descendants of status aboriginal people are losing their status through the three generations rule ( 36 ) . taken together , prudence is needed when interpreting the observed epidemiological findings . in conclusion , diabetes prevalence and incidence has increased among all alberta youth from 1995 to 2007 . status aboriginal youth , males in particular , experienced a disproportionate growth in diabetes . if unabated , increasing diabetes in youth will likely only further perpetuate the diabetes epidemic in the status aboriginal population . type 2 diabetes is typically the tip of the iceberg as it is often preceded by several co - morbidities ( obesity , hypertension , metabolic syndrome , pre - diabetes , etc ) , rendering its increased diagnosis in youth a potential public health crisis .", "this work was supported in part by a team grant to the alliance for canadian health outcomes research in diabetes ( achord ; reference # : otg-88588 ) , sponsored by the canadian institutes for health research institute of nutrition , metabolism and diabetes . this study is based in part on de - identified data provided by the alberta health and wellness ." ]

[ "one disability that can sufficient to interfere with activities of daily living is non - syndromic hearing loss ( nshl ) ( 1 , 2 ) . people with even mild nshl have problems hearing speech when there is background noise and identifying the sounds sources ( 3 , 4 ) . nshl is the most common sensory deficit in humans , with an incidence of about 1 in 1,000 newborns . the prevalence increases during childhood , reaching a rate of 2.7 per 1,000 children before the age of 5 years and 3.5 per 1,000 adolescents . two thirds of people with nshl worldwide live in developing countries ( 5 , 6 ) . the transmembrane channel - like 1 ( tmc1 ) gene is considered a member of a gene family predicted to encode transmembrane proteins ( 7 , 8) . mutations in the tmc1 gene have been associated with profound prelingual deafness ( dfnb7/b11 ) and progressive postlingual hearing loss ( dfna36 ) ; thy have been reported in different populations ( 9 ) . the dfna36 and dfnb7/b11 loci are located on chromosome 9q13 - q21 ( 7 ) . tmc1 and tmc2 are members of a gene family predicted to encode transmembrane proteins and are located on p13 of chromosome 20 . the tmc1 gene encodes a sodium sensor and may function as ion transport channel or pump . tmc1 mrna is specifically expressed in neurosensory hair cells of the inner ear , and it is required for normal function of cochlear hair cells , although the molecular and cellular functions of the tmc1 protein are unknown ( 8) .", "since no report has yet determined the frequency of tmc1 gene mutations in the iranian population , the present study was performed to screen and identify the mutations of this gene associated with nshl using polymerase chain reaction single - stranded conformation polymorphism ( pcr - sscp ) and heteroduplex analysis ( ha ) .", "this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran . the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan . finally , 100 patients ( one proband from each family ) were selected ( table 1 ) . nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) . known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research . total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 ) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 . mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) . site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) . the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna . pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method . for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel . the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours . first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products . then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) . finally , the heteroduplex fragments were visualized using standard silver nitrate staining . the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc . for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms . informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) . all data were collected in the statistical program for the social sciences software ( spss , inc . , the mean difference between groups was calculated using a t test . in this study , a p value of 0.05 was considered statistically significant .", "this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran . the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan . finally , 100 patients ( one proband from each family ) were selected ( table 1 ) . nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) . known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research .", "total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 )", "for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 . mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) . site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) . the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna . pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method .", "for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel . the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours .", "first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products . then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) .", "the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc .", "for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms . informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) .", "all data were collected in the statistical program for the social sciences software ( spss , inc . , the mean difference between groups was calculated using a t test . in this study ,", "in this study , blood samples of 100 patients with hearing loss ( mean age 16.5 2.01 years , 49.2% men and 50.8% women , 74.15% married ) were collected from 10 provinces in iran . the connexin 26 gene is responsible for a large proportion of deafness ( about 14.6% ) , and these patients had no mutations in this gene ( 10 , 13 ) . extracted dna with a 260/280 nm absorbance ratio of 1.8 - 2 was subjected to gene amplification . exons 7 and 13 of the tmc1 gene were amplified using the pcr technique with specific and mutated oligonucleotide primers . after page , fragments with a length of 187 and 250 bp were revealed for exons 7 and 13 , respectively , of the tmc1 gene . pcr products were used for sscp and ha for investigation of mutations in the sequences . neither pcr - sscp nor ha showed mutations in exons 7 and 13 of the tmc1 gene ( figures 1 and 2 ) . line m is a 100 bp dna ladder ( fermentas , germany ) , line 5 is a sample amplified by mutant primers with typical shift ( positive control ) , and lines 2 - 4 and 6 - 13 are deafness patients samples . all specimens have the same template bands without shifts in the sscp bands and after ha . line m is a 100 bp dna ladder ( fermentas , germany ) , line 1 is positive control , line 3 is a suspected sample containing different bands compared to other samples , and lines 2 and 4 - 15 are samples from deaf patients . in the present study , to increase the accuracy of the sscp reaction , ha and mutant control ( positive control created by sdm ) were performed . only a number of suspected fragments in exon 7 showed a different banding pattern , but after sequencing , the mutations were not confirmed ( figure 3 ) .", "in various human populations , different dominant ( e.g. , coch , dfna5 , and pou4f3 ) and recessive genes ( e.g. , gjb2 and slc26a4 ) have been reported as the cause of hearing loss worldwide ( 7 , 9 , 14 - 16 ) . the tmc1 gene is an autosomal recessive gene and a common cause of hearing loss in many countries , such as india , pakistan , algeria , iraq , lebanon , sudan , tunisia , and turkey ( 15 , 17 - 19 ) . the prevalence of non - syndromic hearing impairment due to tmc1 in the pakistani population is 4.4% , and one study has indicated that the tmc1 protein might have an important function in the k channels of the inner hair cells ( 16 ) . 2002 ) detected a 1.6 kb genomic deletion encompassing exon 14 of tmc1 in a recessive deafness mouse mutant , which lacks auditory responses and has hair - cell degeneration ( 7 ) . in the present study , the association of exons 7 and 13 mutations of tmc1 gene locus dfnb7/11 in deaf patients after gene amplification , neither sscp nor ha showed mutations in exons 7 and 13 of this gene related to hearing loss in these patients . only a suspected sample in exon 7 had a different banding pattern , but after sequencing , mutations in this exon were not identified . the strength of this study was the number of samples , as 890 blood samples were provided ; however , due to a lack of financial resources , we chose two exons of this gene . via the molecular analysis of the tmc1 gene in korean patients , one study showed that this gene was not the cause of nshl in the korean population ( 20 ) . in our study , as in the research in korea , a relation between the tmc1 gene and nshl in the iranian population was not observed . ( 2005 ) investigated four novel tmc1 ( dfnb7/dfnb11 ) mutations in turkish patients with congenital autosomal recessive nshl ( arnshl ) , and they indicated that tmc1 mutations account for at least 6% ( 4/65 ) of arnshl cases in gjb2-negative turkish families from the northeast and east of turkey ; however , in our study , mutation in exons 7 and 13 of tmc1 gene was not observed ( 14 ) . in another study in sudan , it was shown that tmc1 mutations contribute to deafness ; this confirmed and extended previous reports on the role of tmc1 in recessive non - syndromic deafness ( 20 , 21 ) . meanwhile , in the present study on the iranian population , no significant relation between exon 7 and 13 mutations of the tmc1 gene were observed . in pakistan , 2007 ) identified 10 new families segregating dfnb7/b11 deafness and tmc1 mutations , including three novel alleles ; moreover , they identified a c.100c > t mutation in exon 7 ( 16 ) . 2008 ) identified four new mutations in the tmc1 gene and suggested an additional deafness gene at loci dfna36 and dfnb7/11 in 51 familial of turkish patients with autosomal recessive hearing loss ; these results implied the presence of mutations outside the coding region of this gene , or alternatively , at least one additional deafness - causing gene in this region ( 9 ) . another study in turkey reported five novel mutations in the tmc1 gene related to arnshl ( 19 ) . in comparison with the other populations discussed , in a review study in iran by mahdieh et al . the frequencies and distributions of nshl included gjb2 , gjb6 ( large deletion ) , tecta , slc26a4 , and pejvk mutations . the researchers indicated that mutations in gjb2 , slc26 , tecta , and pjvk genes have an important role in deafness in iran , and a screening test should be generated for better intervention and diagnostic programs ( 22 ) . the study of hildebrand et al . in iranian families showed that two families are related to locus dfnb7/11 , and one of them had a c.776 + 1 g > a mutation in exon 7 ( 23 ) . in 2014 , lin et al . identified novel compound heterozygous mutant alleles of tmc1 responsible for arnshl in a tibetan chinese family ( 24 ) ; meanwhile , in the present work , we did not observe a relation with mutations of exons 7 and 13 of the tmc1 gene in iranian nshl . the findings of the current study indicate that mutations in exons 7 and 13 of tmc1 gene are not related to hearing loss in the iranian population . therefore , the tmc1 gene may not related to nshl , but further studies investigating related mutations in other parts of this gene in iranian population are necessary and could help in the genetic counseling of patients and design of practical strategies for the management of auditory disorder ." ]

[ "microleakage between the root canal filling and root - canal walls may adversely affects the results of root - canal treatment.1 apical leakage is considered to be common cause of endodontic failure.2 hence , different endodontic filling materials , sealers and techniques have been introduced to the dental community in an attempt to improve apical seal.1 various materials have been used in root canal treatment in an attempt to achieve success . but , a combination of gutta - percha and a sealer are used most commonly . gutta - percha is considered an impermeable core material ; therefore , leakage through an obturated root canal is expected to take place at the interfaces between the sealer and dentin or the sealer and gutta - percha , or through voids within the sealer.3 apical sealing is desirable to prevent passage of bacteria and their endodotoxin apically . in vitro evaluation of apical dye penetration is used to estimate the sealing ability which is corresponding to in vivo amount of micro leakage with particular sealer.2 many techniques were used to evaluate the leakage of sealers such as ; colored dye penetration radio labeled tracer penetration dissolution of hard tissue clearing of teeth , spectrometry of radioisotopes electrochemical and gas chromatography . however , many studies showed no significant difference between these techniques.1 the aim of this in vitro study was to quantitatively evaluate the sealing properties of three different root - canal sealers ; tubliseal , sealapex and ah26 using a spectrophotometric method .", "thirty - six extracted sound mandibular molar natural teeth specimens with complete root and free from caries or cracks were collected , stored , disinfected and handled as per the recommendations and guidelines laid down by occupational safety and health administration and centers for disease control and prevention.4 the teeth were then placed in 0.9% physiologic saline solution for ten days prior to access cavity preparation . the samples were divided into three experimental groups ; group i : sealapex , group ii : tubliseal and group iii : ah26 with 12 samples in each group . the samples were then coated with nail varnish all over the root surface except 2 mm around the apical foramen . 2 ml of freshly prepared 2% methylene blue dye was taken in each vial and the apical third of the root was suspended in the dye for 72 h. samples were washed with distilled water , nail varnish removed and then placed in 20 ml of 35% nitric acid for 72 h. standard solutions of 1% , 0.5% , 0.2% , 0.05% , 0.02% and 0.01% of methylene blue in 35% nitric acid were prepared and stored for 72 h. the standard solutions and the nitric acid solutions were filtered and centrifuged for 1 min after 72 h. the supernatant was subjected to spectrophotometric analysis using a filter of 670 nm . the amount of leakage was extrapolated from a standard linear regression curve constructed from stock standard methylene blue dye solutions . obtained data were statistical analyzed with kruskal wallis and mann whitney u - tests using spss software version 20 ( ibm ) .", "the results of the quantitative evaluation of the sealing properties of the three root - canal sealers are shown in table 1 . , tubliseal showed a significant difference ( p > 0.005 ) . in comparison to sealapex comparisons made between tubliseal and ah26 showed no significant difference ( table 2 , chart 1 ) . mean and standard deviation values of volumetric dye penetration in groups with reference to transmission .", "three - dimensional obturation of the root canal system with a fluid impervious seal is an important factor for successful endodontic therapy . the root canal filling should seal the canal both apically and coronally to prevent the passage of microorganism to apex or vice versa.5 most reliable method is the use of gutta - percha cones with sealer cement . sealers based on zinc oxide - eugenol ( tubliseal ) , calcium hydroxide ( sealapex ) , epoxy resins ( ah26 ) were included in the present study . in present quantitative dye leakage study , tubliseal demonstrated least dye leakage in comparison with other experimental groups ( table 1 ) . our results were in contrary to study by masoud and saleh where they found more microleakage in tubliseal group than other groups.6 sealapex is a calcium hydroxide type sealer . calcium hydroxide used as root canal sealer since it stimulates periapical tissues in order to maintain health or promote healing and secondly for its antimicrobial effects.7 it has been observed in some studies that , calcium hydroxide sealers showed a significant volumetric expansion during setting because of water absorption , which increases its solubility . the present in vitro investigation indicated maximum leakage value with sealapex among the experimental groups . in a contradictory to our results , cobankara et al . ( 2006 ) observed sealapex with better apical sealing than the other sealers ( ah plus and rc sealer ) at 7 , 14 , and 21 days.3 ah26 is an epoxy resin based sealer that provides easy handling characteristics , good flow , good sealing to dentin and prominent antimicrobial activity.6 kumar et al . observed more micro leakage with zinc oxide - based sealer and least with resin based sealers , this is contradictory to our study.8 in our study , there was no statistical significant difference between tubliseal and ah26 in micro leakage . observed least amount of dye penetration for ah plus and endorez group.9 even though , the current study did not indicate any statistically significant difference between ah26 and sealapex , it is time to question the overall efficacy of calcium hydroxide sealers on the grounds that the reparative and the calcification capabilities attributed to calcium hydroxide are generally desirable before completing the obturation . ah26 , resin based sealer provided a better apical seal when compared with sealapex even though the results were statistically not significant . it mixes easily , flows well , and has ample working time , good radio - opacity , comparable solubility , good adhesion and good biocompatibility.3 in the present study , there was no significant difference between group ii and iii and group i and iii . did nt find any significant difference between the tested three groups ; sealer 26 , enfoflas and resin group . but they observed higher microleakage in sealer 26 group compared with control.11 joseph and sing evaluated the apical sealing with four root canal sealers ; ah26 , sealapex , endoflas fs and ah plus and observed no significant differences between all groups except between ah plus and endoflas.5 nagas et al . observed significantly lower overall leakage with ah plus group , whereas no difference was found between master cone points.12 kopper et al . observed significant dye penetration for ah plus , endofill and sealer 26.13 cobankara et al . observed better sealing values for roekoseal after 21 days when compared to ketac - endo and ah plus , and there was no statistically significant difference.1 dultra et al . found no statistical difference between groups for apical leakage ( endofill , ah plus , endorez and epiphany).14 in addition , before accepting a new material for routine clinical use further experiments should also be performed to evaluate the other aspects of the materials physical and biological properties such as biocompatibility , solubility , disintegration , radio - opacity and dimensional stability . however , these in vitro studies do provide comparative information of the relative performance of sealers tested under the same conditions in each particular study and clinicians can use this information to possibly choose a better sealer .", "in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer . it is important to remember before declaring any root canal sealer as most acceptable that the results of the dye penetration studies indicate only the relative sealing ability of root canal fillings in vitro and they do not indicate their ability to prevent the penetration of bacteria into filled root canals in vivo ." ]

[ "targeting type 4 phosphodiesterase ( pde4 ) has been recognized as a promising approach to managing copd by relieving the symptoms , slowing the progress of the disease , increasing exercise tolerance , reducing exacerbation rate , and improving quality of life ( giembycz 2001 , 2005 ; mehats et al 2003 ; spina 2003 , 2004 ; lagente et al 2005 ; lipworth 2005 ; soto and hanania 2005 ) the pressing need to develop drugs that control symptoms and reduce mortality ( pauwels et al 2001 ; gold 2005 ) and the billion - dollar marketing potential for management of copd have pushed the r&d of pde4 inhibitors into the product development pipelines of major pharmaceutical companies in the recent years . the early clinical trial data for the second - generation pde4 inhibitors cilomilast ( ariflo , glaxosmithkline , usa ) and roflumilast ( daxas , altana , germany ) all pointed to a successful introduction of a novel non - steroid anti - inflammatory therapy to clinicians in combating severe copd ( gamble et al 2003 ; rabe et al 2005 ) nevertheless , while the progression of developing cilomilast has idled at the approvable stage for more than two years , the announcement of the termination of the agreement to develop roflumilast between altana and pfizer has raised concerns about the therapeutic efficacy of selectively inhibiting one or two isoenzymes in the pde4 family for copd management ( pharmiweb 2005 ) . in the early six - month record phase iii trial , roflumilast ( 500 mg daily ) clearly improved lung function ( ie , increased fev1 by + 97 ml ) and significantly reduced exacerbations ( acute worsening of symptoms ) compared with placebo ( rabe et al 2005 ) . however , in the follow - up one - year phase iii trials using exacerbations as one of the key endpoints , the results from the european copd ratio study that included 1513 patients with severe and very severe copd have failed to repeat the previously claimed efficacy . in addition , the new trial data confirmed that the pde4 inhibitor roflumilast s efficacy was considerably lower than the approved therapies such as fluticasone / salmeterol ( a combination therapy of glucocorticosteroid and long - acting 2-agonist ) and tiotropium bromide ( long - acting anticholinergic ) . the unexpectedly low long - term efficacy on exacerbation rate from roflumilast therapy made the r&d community re - examine the role of targeting pde4 in copd because one of the highest unmet needs in treating the disease is to reduce or eliminate exacerbations ( pharmiweb 2005 ) . in november of 2005 , altana announced the withdrawal of the european marketing authorization application ( maa ) for roflumilast and decided to wait for more clinical trial data for submission of a future maa ( altana 2005a ) . this holdup no doubt sets back the r&d of the most promising pde4 inhibitor in development for copd .", "copd is a complex disease with pathophysiological features including inflammation ( neutrophils , macrophages , cd8 + lymphocytes infiltration , and inflammatory mediator tnf- and il-8 release ) , airway obstruction ( smooth muscle contraction , elevated cholinergic tone ) , respiratory bronchiolar alveolar vasculature remodeling ( loss of elastic recoil , alveolar destruction , and fibrosis ) , pulmonary hyperinflation , gas - exchange abnormalities , and pulmonary hypertension . the progressive loss of lung function leads to reductions in patients quality of life and results in exacerbations , cor pulmonale , and death . it is believed that the chronic non - infectious inflammation underlies the pathogenesis and the steady progression of the disease ( pauwels 2001 ; gold 2005 ) . the pathological changes in the patients with copd are not fully reversible and it often takes many years for a patient at risk ( cough , sputum production ) to progress into suffering from mild airflow limitation , to moderate , severe , and very severe copd ( with chronic respiratory failure ) . in the absence of a magical therapy that can stop the disease progression and reverse the abnormalities of pulmonary function , the management , including drug therapy , for copd is long - term care . inhibition of pde4 has been established as an effective and reliable approach to increasing intracellular camp ( conti et al 2003 ) that underlines the signaling mechanisms for the treatment of copd . in recent years , numerous in vitro , in vivo , and clinical trial studies demonstrated that pde4 inhibitors ( eg , rolipram , cilomilast , and roflumilast ) relax airway smooth muscles to increase air flow ( holbrook et al 1996 ; bundschuh et al 2001 ) and improve pulmonary circulation ( schermuly 2000 ; de witt 2000 ) , inhibit bronchiolar alveolar vasculature remodeling , and fibrosis ( kumar et al 2003 ) , reduce neutrophils macrophages / cd8 + t cells infiltration and pro - inflammatory mediator release ( kumar et al 2003 ; profita et al 2003 ; wollin et al 2005 ) , improve patients exercise capacity and quality of life , and prevent the progressive loss of pulmonary function ( rabe et al 2005 ; gamble et al 2005 ) . with all these preferred outcomes , it seems that the pde4 inhibitors in development ( cilomilast and roflumilast ) would be an ideal armory for the healthcare community to combat copd . why , then , has the long - term trial with roflumilast failed to produce the expected results ? it could be due to a dose regimen ( 500 mg daily ) that was effective for patients with moderate to severe copd ( rabe et al 2005 ) but not adequate for those patients suffering from severe and very severe copd ( altana 2005b ) or the intrinsic low efficacy of the narrow - spectrum pde4 inhibitors .", "developing pde4 inhibitor as a therapy for copd is based on the fact that theophylline dilates airway smooth muscles and improves pulmonary function by inhibition of pde activity ( barnes 2003 ; spina 2004 ) the dose - limiting adverse reactions ( nausea , emesis , cardiac arrhythmias ) with the non - selective pde inhibitor theophylline and the first - generation pde4 inhibitor rolipram ( huang et al 2001 ; lagente et al 2005 ) directed the r&d of pde inhibitors to discover the second - generation of pde4 inhibitors cilomilast and roflumilast that have been successfully brought to the final stage for administration approval ( spina 2003 , 2004 ; lipworth 2005 ) based on the fact that the emetogenic reaction to pde4 inhibition is due to reticence of the pde4d isoenzyme ( lamontagne et al 2001 ; robichaud et al 2002 ) , several researchers in the field proposed to develop isoform - specific pde4 inhibitors that reduce or completely avoid disturbing pde4d activity and therefore do not trigger the emetic responses in the nervous system ( giembycz 2002 ; robichaud et al 2002 ; card et al 2004 ) . structural studies have provided evidence that the folding of catalytic domains of pde4 has a conformation involved in binding of selective inhibitors with a common scheme : ( i ) a hydrophobic sub - pocket sandwiching an inhibitor in the active site ; ( ii ) hydrogen bond(s ) to an invariant glutamine controlling the orientation inhibitor binding ( therefore the affinity or potency ) ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) ( figure 1 ) . the scaffold of individual pde4 isoenzyme and the structure of a given selective inhibitor govern isoenzyme - selective inhibition , depict the binding affinity , and determine the therapeutic window and rank order of potency in clinical use for the treatment of copd . enhancement of isoenzyme selectivity is critical for reducing side - effects of the pde4 inhibitors . the strength of the interaction ( hydrogen bond ) between the oxygen group(s ) of an inhibitor and the amide nitrogen group of glutamine ( gln ) 369 for pde4d and gln 443 for pde4b plays a pivotal role in determination of the potency and isoenzyme selectivity of an inhibitor ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) . in addition , selective pde4 inhibitors such as cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space of the pocket to yield extra binding energy ( increasing potency ) and result in greater isoenzyme selectivity ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) for example , while cilomilast s additional functional groups interact with 10 almost identical residues that form the hydrophobic sub - pocket in pde4d and pde4b , the oxygen atoms of the cyclopentyloxy and methoxy groups of cilomilast form two hydrogen bonds with the gln369 of pde4d , whereas there is only one hydrogen bond being formed between the methoxy group of cilomilast and the gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) . this difference may partially explain the fact that cilomilast is approximately 10-fold more selective for pde4d than for pde4b , despite over 90% identity between the pde4b and pde4d catalytic domains . ( giembycz 2001 ; odingo 2005 ) roflumilast shows a better fitting to the hydrophobic sub - pocket in the pde4d catalytic site than cilomilast ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xoq&template=ligands.html&l=2.1 ) , which depicts the experimental finding that roflumilast inhibits pde4d 338-fold more potently than cilomilast ( table 1 ) . as for pde4b inhibition , roflumilast s cyclopropylmethoxy and difluoromethoxy oxygen groups form two hydrogen bonds with gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xmu&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) , which may partially explain its 585-fold greater inhibition potency than cilomilast toward pde4b ( table 1 ) . the dichloropyridyl substitution of roflumilast also increases its potency compared with cilomilast in terms of pde4b inhibition ( card et al 2004 ) . the rank order of potency for inhibition of pde4 activity and lipopolysaccharide - stimulated tnfa release , for relaxing bronchoconstriction in guinea pigs , and the daily doses for treatment of copd by roflumilast , cilomilast , rolipram , and theophylline are summarized in table 2 . by improving the inhibition ratio on pde4b / pde4d , roflumilast indeed lowers the emetic profile without compromising its therapeutic efficacy in comparison to cilomilast ( huang et al 2001 ; spina 2003 ) the pde4b - specific single - molecule targeting can undoubtedly reduce or eliminate an inhibitor s unwanted effects . however , this approach may compromise the efficacy of a pde4 inhibitor because airway and vascular smooth muscles express multiple pde4d isoforms ( eg , d1 , d2 , and d5 ) and pde4d plays a crucial role in bronchoconstriction and vascular smooth muscle contraction ( liu et al 2000 ; baillie et al 2001 ; mehats et al 2003 ) . an agent without 4d inhibition may end up lacking sufficient therapeutic efficacy for controlling copd ( spina 2004 ) .", "the beneficial effects on copd via relaxing airway smooth muscles and anti - inflammation mediated by pde4 inhibition with cilomilast and roflumilast have been emphasized and reviewed extensively ( spina 2004 ; lagente et al 2005 ; lipworth 2005 ; soto 2005 ) . improving pulmonary circulation has not been considered as an important therapeutic approach for managing patients with copd . however , the facts are 1 ) during copd exacerbations , pulmonary hypertension ( ph ) is increased , 2 ) the presence of ph is recognized as the single strongest indicator of prognosis in copd patients among the numerous clinically used lung function parameters ( doi et al 2003 ; alp et al 2006 ) , and 3 ) clinically , the higher the pulmonary arterial pressure , the shorter the life expectancy of copd patients ( barbera et al 2003 ) . although inhaling nitric oxide vasodilator can worsen gas exchange because of altered hypoxic regulation of ventilation - balance ( barbera 1996 ) in patients with stable copd , and vasodilators are considered as contraindications for copd patients , in their preliminary clinical trial , alp et al ( 2006 ) demonstrated that reduction of pulmonary vasculature resistance using the pde5 inhibitor sildenafil was able to significantly improve the exercise capacity ( 6-minute walk test ) of patients suffering from severe copd . a double - blinded , placebo - controlled , crossover clinical trial of sildenafil in patients ( n=10 ) with copd is going on to evaluate the effect of pde5 inhibition on patients exercise function , pulmonary function , and quality of life ( national institutes of health ) . although there is a lack of reports about cilomilast- or roflumilast - caused pde4 inhibition on the improvement of pulmonary circulation , in isolated perfused lung preparations , intravascular or transbronchial administration of subthreshold doses of the wide - spectrum pde4 inhibitor rolipram synergistically amplified the pulmonary vasodilatory response to inhaled pgi2 and concomitantly improved ventilation more interestingly , in anesthetized cats , de witt et al ( 2000 ) found that rolipram was more potent than either siguazodan ( pde3 inhibitor ) or zaprinast ( pde5 inhibitor ) in reducing pulmonary lobar arterial pressure . when the tone in the pulmonary vascular bed was raised to a high steady level with a constant infusion of the thromboxane mimic u46619 ( 9,11-dideoxy-11 , alpha9alpha - epoxymethano prostaglandin f2 ) , intralobar injections of rolipram caused dose - related decreases in systemic arterial pressure and pulmonary arterial pressure . the preference reduction in pulmonary arterial pressure by inhibition of pde4 suggests that administration of pde4 inhibitors may benefit copd patients by increasing ventilation ( airway relaxation ) and improving alveolar perfusion ( vasodilation ) , therefore improving blood gas exchange capacity and patients pulmonary function . thus , it is highly likely that the maximal therapeutic efficacy of targeting pde4 in the treatment of severe copd depends upon three effectors of downstream intracellular camp elevation : 1 ) anti - inflammation ( lipworth 2005 ) , 2 ) airway relaxation ( spina 2004 ) , and 3 ) vasodilation . sacrificing any one of these effectors with an isoenzyme - specific narrow - spectrum pde4 inhibitor will compromise the effectiveness of the therapy . how , then , is the dose - limiting dilemma ( nausea , vomiting , diarrhea , and arteritis ) ( giembycz 2005 ; spina 2004 ; lipworth 2005 ) associated with the wide - spectrum pde4 inhibition overcome ?", "the fact that there are over 60 pde isoenzymes encoded by 21 human pde genes and at least 16 pde4 isoenzymes from 4 pde4 genes ( soderling and beavo 2000 ; conti et al 2003 ; houslay and adams 2003 ; huai et al 2004 ) may render it highly possible that searching for isoenzyme - specific pde4 inhibitors will yield low - efficacy agents . in addition , mechanisms for upregulation of pde4 activity by camp - induced pde4 gene expression and pka - catalyzed phosphorylation activation of pde4 isoforms ( conti et al 2003 ; wallace et al 2005 ) may very likely reverse an isoenzyme - specific , pde4 inhibitor - produced elevation of intracellular camp level , and therefore the associated biological beneficial effects . noticing the low therapeutic ratio and insufficient clinical efficacy of the current generation of pde4 inhibitors ( cilomilast and roflumilast ) , giembycz ( 2005 ) assumed that one potential means of improving the therapeutic ratio and safety of pde4 inhibitors may lie in the development of compounds that have broader phosphodiesterase specificity and suggested dually targeting pde4 and pde1 , pde3 , or pde7 to enhance clinical efficacy . this approach seems to revisit the previously well - described pde inhibitors such as theophylline or zardvarine ( schmidt et al 2000 ; barnes 2003 ) . it may lead to the dose - limiting drawback cycle again , because it is known that targeting camp - specific pde3 is associated with an increase in morbidity and mortality in heart failure patients ( packer et al 1991 ) . in a news feature on pharmiweb.com , there is a remark about pfizer s development of an inhaled dual - action pde4/spiriva ( long - lasting muscarinic antagonist tiotropium ) combination product for copd ( pharmiweb 2005 ) . this combined dual action modality is a favorable approach for managing patients with severe copd considering the presence of bronchoconstricting and inflammatory pathologies in the disease . in general , moderately targeting two mechanisms to reach the therapeutic goal should be more effective and safer than exploiting a single mechanism to its extended degree . we have proposed to co - administer ca channel antagonist ( cca ) to overcome pde4 inhibitor - caused adverse effects , especially emetic responses ( wang and wang 2005 ) because 1 ) stimuli that upregulate the camp pathway can increase the excitability of the neurons in the locus coeruleus ( lc ) that plays an important role in mediating neuronal emesis ( nestler et al 1999 ; takeda et al 2001 ) ; 2 ) the pde4d isoform is localized to neurons in the structures of the medulla including lc ( lamontagne et al 2001 ) , which are consistent with a role for pde4d in the emetic response ; and 3 ) the lc neurons fire action potentials spontaneously , resulting from endogenous properties of the membrane conductance to a persistent inward ca current , which can be blocked by diltiazem ( williams et al 1984 ; tokuyama and ho 1996 ; filosa and putnam 2003 ) . thus , in the presence of cca , even when a complete inhibition of pde4d leads to an elevation of camp in the lc neurons , the lc cells will not be able to fire action potentials due to a blockade of the depolarizing l - type ca currents , therefore eliminating the intrinsic dose - limiting emetic pharmacological profile of the wide - spectrum pde4 inhibition . in addition , ccas also relax airway smooth muscles and exhibit anti - inflammatory effects , which may synergistically augment a pde4 inhibitor s therapeutic effects on copd ( worley and kotlikoff 1990 ; szabo et al 1997 ; brown et al 2004 ) . the clinical use of ccas in treatment of pulmonary hypertension in patients with copd further support the combination therapy of a pde4 inhibitor and a cca ( sajkov et al 1997 ) . one concern regarding the combined therapy is the possible difference between the pharmacokinetics of the two drugs that may compromise the expected outcomes . this pitfall can be eliminated by developing an agent with two pharmacophores in one chemical structure , therefore being able to simultaneously target two therapeutic mechanisms such as l - type ca channels and pde4s ( wang and wang 2005 ) . we believe that it is worthwhile to carry out a randomized clinical trial to evaluate the safety and efficacy of dually targeting pde4 and ca channels in managing the patients with severe copd .", "the unsatisfied efficacy of using pde4 inhibitor roflumilast in treatment of severe and very severe copd has raised concerns in the r&d community about the administrative approvability for the highly expected novel therapeutic modality in combating copd . the extensive in vitro , in vivo , and clinical trial study data and the established clinical beneficial effects ( anti - inflammation , bronchorelaxation , pulmonary vasodilation ) associated with pde4 inhibition strongly validate targeting pde4 for controlling copd . development of an inhaled dual - action therapy such as pde4 inhibitor and muscarinic antagonist may be a correct approach to bringing a pde4 inhibitor to the demanding market . the other approach is using cca to overcome pde4 inhibitor - caused adverse effects , especially emetic responses and simultaneously enhance pde4 inhibitor s anti - inflammatory and bronchorelaxation , pulmonary vasodilation effects . of course , development of a dual agent that has with two pharmacophores in one chemical structure , therefore being able to target pde4 and l - type ca channels should also be able to improve the therapeutic window of pde4 inhibition and may make available a new therapeutic approach to managing copd ." ]

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

[ "hepatic angiomyolipoma ( haml ) is a rare benign mesenchymal liver tumor first described by ishak in 1976 ; it belongs to a group of perivascular epithelioid cell tumors called pecoma . until date , approximately 300 cases have been reported [ 310 ] ; however , its natural history has not been clarified . the tumor composed of blood vessels , smooth muscle , and adipose cells and due to the variety of predominance of these tissues , its patterns in imaging studies have resulted in a difficulty in diagnosis and misdiagnosis of the tumor as hepatocellular carcinoma ( hcc ) in some cases [ 6 , 11 ] . therefore , the preoperative correct diagnosis has been difficult ; however , recent advances in imaging diagnosis through a combination of ultrasonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and angiography and specific immunohistochemical analysis of this tumor using human melanoma black-45 antigen ( hmb-45 ) staining have resulted in accurate diagnosis and it is reported that the current accurate preoperative diagnosis was made in 25%52% of cases [ 8 , 9 ] . the majority of these tumors are believed to be clinically benign during a mean follow - up period of 6.8 years ; however , an increasing number of cases and aggressive changes including growth in size , recurrence after surgical resection , metastasis , and invasive growth pattern into the parenchyma and along the vessels have been reported . in this paper , we have focused on the characteristic features of this tumor shown in imaging studies and by histological analysis , summarized these cases showing aggressive patterns , and discussed management of the patients and indications for surgical treatment .", "most cases were found as incidental liver tumors upon health screening or imaging examinations for other diseases . it usually follows a benign clinical course while some patients visited hospitals with unspecific symptoms of abdominal discomfort , fullness , and other such complaints . more than half of the renal aml are considered to be associated with tuberous sclerosis which features the loss of heterozygosity at tsc1 ( 9q34 ) and tsc2 ( 16p13 ) , while it is estimated to be 515% in the liver . thus the etiology of most of these tumors in the liver is unknown ; most cases have no history of liver diseases or specific symptoms , and no changes in laboratory data are seen . moreover , serum levels of the tumor markers alpha - fetoprotein , protein induced by vitamin k absence or antagonist ii , carcinoembryonic antigen , and carbohydrate antigen 19 - 9 were normal .", "since this tumor is composed of various tissues , such as lipomatous , myomatous , and angiomatous tissue in various proportions , the imaging studies show a wide array of features depending on the predominance of each tissue . the tumor showing the predominance of lipomatous tissue is likely to be correctly diagnosed ; however , myomatous and angiomatous variant poses diagnostic problems since it is difficult to be distinguished from malignant tumors . us images may vary depending on the tissue components affected by the tumor . high echogenic lesions can be observed because of lipomatous and myomatous tissue , and angiomatous tissue may result in low echoic lesions in tumor images ( figure 1(a ) ) . if the tumor has predominance of lipomatous tissue , the differential diagnosis with hepatic hemangioma is difficult by sonography alone . color doppler sonography shows punctiform or filiform vascular distribution pattern if the tumor has predominance of angiomatous tissue . recent reports showed the diagnostic effectiveness of contrast - enhanced us ( ceus ) [ 10 , 13 ] . reported that ceus revealed the typical imaging characteristics of haml , that is , an inhomogeneous hyperenhancing pattern in the arterial phase and prolonged enhancement during the portal and kupffer phases . plain ct showed homogeneous or heterogeneous low - density lesions , and contrast - enhanced dynamic ct showed highly enhanced lesions in the arterial phase , prolonged enhancement in the portal phase , and , occasionally , defective lesions in the late venous phase depending on the component of the tumor tissue . a density of less than 20 hounsfield units in plain ct is useful to determine the involvement of lipomatous tissue . the difficulty is , however , to diagnose the tumor that is myomatous tissue and angiomatous variant . for this point , modification of size of region of interest in ct was reported to be effective for accurately diagnose renal angiomyolipoma from renal cell carcinoma . abdominal angiography showed marked tumor staining in the arterial phase , which remained in the portal phase ( figure 1(b ) ) . in some tumors , drainage of the hepatic veins can be observed in the late vascular phase . the first phase of ct during hepatic arteriography showed significant hypervascular lesions in the tumor ( figure 1(c ) ) , and the second phase showed the remains of staining and defective lesions in other areas ( figure 1(d ) ) . ct during arterial portography showed areas of defective tumors ( figure 1(d ) ) . mri is considered to be the best modality to determine the components of the tumor . hyperintensity on the t2-weighted image and hyper- or hypointensity on the t1-weighted image are observed depending on the component of tumor tissue [ 810 ] . lipomatous lesions may be determined as hyperintensity on the t1-weighted image ; they may also be determined by the chemical shift imaging technique . contrast - enhanced dynamic mri using gadolinium or the hepatocyte - specific contrast agent gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid showed early enhancement in the arterial phase followed by the prolonged enhancement in the portal phase and defective lesions in the hepatobiliary phase .", "macroscopic and magnifying glass view of the tumor showed a soft , white to yellow , and well - circumscribed tumor and range in size from 0.1 cm to greater than 36 cm . no signs of chronic inflammation or fibrosis were seen in the surrounding liver tissue . because haml comprises lipomatous , myomatous , and angiomatous tissues , microscopic examination showed a mixture of blood vessels , specialized smooth muscle cells , and adipose cells with atypical changes in classic haml ( figures 2(a ) and 2(b ) ) . this variation of mixture levels in one tumor reflected the differences in imaging studies and made accurate diagnosis difficult . it is believed that majority of the hamls behave in a benign fashion and even if some cases showed invasive growth pattern into the adjacent hepatic parenchyma , portal triad ( figure 2(c ) ) , and hepatic vein . tumor cells , especially myomatous cells , were stained positive for hmb-45 in most tumors ( figure 2(d ) ) , for cd34 in the endothelial cells of the blood vessels and for smooth muscle actin in spindle smooth muscle cells . hmb-45 is an antibody that reacts with an oncofetal premelanosome - associated glycoprotein 2 , found in neoplastic melanocytes . also cd63 , cd67 , desmin , s100 , and ema may also be positive but not specific . these cells were negative for cytokeratin 18 , cytokeratin 19 , cam 5.2 , hepatocyte paraffin-1 . therefore , the identification of lipomatous , myomatous , and angiomatous tissue by a positive reaction to hmb-45 currently provides the only evidence of haml [ 46 , 18 ] and can be useful for defining from the other liver tumors such as hcc after the tumor biopsy and surgical resection followed by these immunohistochemical stainings . although favorable prognosis can be expected for this tumor since the majority of the tumors are benign , however recently , the number of reports of haml showing malignant potential has increased [ 3 , 5 , 8 , 9 , 12 , 1624 ] which revealed significant growth , recurrence , metastasis , and poor prognosis summarized in table 1 . among those cases , deng et al . reported in their case that haml showed atypical angiomatous , epithelioid components with pleomorphic and frequent mitosis in the center of the large tumor displayed p53 immunoreactivity , and mutation at exon 7 for p53 and resulted in vascular invasion , distant metastasis , and fatal outcomes . these results indicate that the hmb-45 staining , mitotic analysis by mib-1 ( ki67 ) , and p53 reactivity following the fine - needle biopsy of the tumor might be useful for the diagnosis of the tumor and its malignant potential .", "since haml usually follows benign clinical courses , the majority of the cases can be conservatively treated . however , as not a few cases showed aggressive pattern in their courses [ 3 , 5 , 8 , 9 , 12 , 1624 ] and because of the low level of accurate diagnoses by imaging studies , and because of the possibility of dissemination of tumor cells into the peritoneal cavity if a tumor is malignant , tumor biopsy has been avoided , and many hamls have been surgically resected [ 3 , 8 , 9 ] . with the increasing number of resected samples , careful comparisons of imaging studies and pathological findings therefore , once haml has been diagnosed by imaging studies , a fine - needle biopsy should be performed to make an accurate diagnosis and to confirm the predominance of the tissue components , the existence of pleomorphic nuclei with high proliferation activity , p53 immunoreactivity , and mutation in p53 if possible . if the aggressive patterns such as vascular invasion , high proliferation of the tumor cells , p53 immunoreactivity were marked , or when the imaging findings and biopsy can not provide a definitive diagnosis , or if the patients have abdominal symptoms , surgical resection should be considered .", "haml is considered to be a benign mesenchymal tumor , and nonsurgical treatment with conservative management involving close followup is therefore suggested for patients with asymptomatic tumors smaller than 5 cm , which have been proved to be a typical haml by fine - needle aspiration biopsy . as a fact the renal angiomyolipoma often showed perirenal invasion , involvement of the renal vein , and the inferior vena cava , that are not considered as signs of malignant behavior and the angiomatous , epithelioid monophasic or pleomorphic variant might be associated with the aggressive behavior and cellular atypia , mitotic activity , and metastatic lesions are the criteria for the malignant renal angiomyolipoma . however , due to the difficulties in diagnosis by imaging studies , many hamls have been surgically resected and have been followed closely for a long period . in addition , by careful analysis of these recent cases [ 3 , 5 , 8 , 9 , 12 , 1624 ] summarized in table 1 , it was revealed that , although rare , hamls may have malignant potential , which may be distinguished by aggressive patterns characterized by ( 1 ) significant changes in size in short period ; ( 2 ) a change of tumor composition ( cases 1 , 2 , 5 , and 6 ) [ 9 , 16 , 17 , 19 ] ; ( 3 ) metastases to the other organs ( cases 1 , 9 , and 10 ) [ 17 , 22 , 23 ] ; ( 4 ) recurrence after curative surgical resection ( cases 3 , 7 , 9 , 10 , 11 , 12 , and 13 ) [ 3 , 8 , 18 , 20 , 2224 ] ; and ( 5 ) invasive growth into the vessels ( cases 2 , 4 , 8 , 9 , and 10 ) [ 12 , 17 , 2123 ] . ohmori et al . reported the first possibly malignant case , which showed that a significant increase in the tumor size resulted in liver dysfunction . fatal progression was observed in nine cases listed by multiple recurrences [ 3 , 8 , 18 , 20 , 2224 ] and metastases to the liver , peritoneum , retroperitoneal region , gastrohepatic omentum , pancreas , and lung [ 17 , 22 , 23 ] . the cases reported by chang , rouquie , and us underwent surgical resection , with a suspicious of aggressive patterns and no recurrence has occurred . we also reported that portal thrombosis , that is , high - grade portal vein invasion , found in five cases in table 1 ( cases 2 , 8 , 9 , 10 , and 11 ) [ 3 , 17 , 2123 ] may be a clinical marker of the malignant potential and transformation of haml as it resulted in a significantly aggressive disease and fatal course in 4 cases with multiple recurrences and metastases . this finding might be able to be detected by imaging studies as similar to hcc . in addition , pathological findings of atypical epithelioid component with high proliferation activity , p53 immunoreactivity , and mutation in p53 might be predictive markers for malignant transformation . based on these reports , as recommended by previous papers [ 3 , 8 , 9 ] , long - term follow - up of haml is necessary after its diagnosis by imaging studies and biopsy specimens and curative surgical treatment . the majority of hamls behave as a benign tumors and conservative follow - up may be recommended ; however , with increasing number of the reports showing potentially malignant behavior , prompt surgical resection is essential for better prognosis of this tumor .", "we have reviewed noninvasive imaging studies and the role of histological diagnosis showing distinctive characteristics of haml to increase the rate of accurate diagnoses . in addition , we summarized the cases that showed progressive patterns of the tumor and concluded that a careful followup of the tumor even after the final diagnosis is necessary . we propose that tumor resection is indicated in the following scenarios : ( 1 ) the patients show symptoms ; ( 2 ) the tumor shows an aggressive growth ; ( 3 ) the tumor shows invasive growth into the vessels evidenced by fine - needle biopsy or imaging studies ; ( 4 ) the component of the tumor shows atypical epithelioid pattern , high proliferation activity , and/or p53 immunoreactivity ; and ( 5 ) a definitive diagnosis can not be made by imaging and pathological studies from malignant tumors ." ]

[ "the sentinel node is defined as the first tumour - draining lymphatic filter , and its involvement by metastatic disease should predict the global nodal status of the specific patient . skip metastasis in the context of a normal sentinel node is described as a very rare event . in patients with clinically node - negative disease , sentinel lymph node biopsy ( slnb ) had become the gold standard staging procedure , followed by axillary lymph node dissection ( alnd ) performed selectively in patients with previously proven ( by slnb ) sentinel node metastases . considering the therapeutic consequences , involvement of the sentinel node by metastatic disease in breast cancer patients is of great interest to the breast surgeon in order to plan the surgical approach . unfortunately , the physical examination of the axillae can be inaccurate in identifying pathologic lymph nodes . the same is also true for the pre - treatment imaging ( with or without interventional procedures on the axillary node ) , characterized by a poor negative predictive value [ 48 ] . using gamma probe combined with ultrasound- ( us ) guided percutaneous core needle biopsy ( cnb ) could increase the accuracy of identifying the sentinel node and allow a proper histopathologic diagnosis in order to avoid further slnb . the purpose of this pilot study was to assess this new pre - operative diagnostic approach .", "main inclusion criteria were : women 18 years old , invasive breast carcinoma , with no clinically positive axillary node and no history of any other malignancy . multicentric cancer and previous excisional biopsy were exclusion criteria . the sentinel node occult lesion localization ( snoll ) was performed in all patients the day before surgery . a dose of 510 ( generally 7 ) mbq of technetium 99-labelled human albumin nanocolloid particles in 0.3 ml saline were administered by us - guided percutaneous injection in the area immediately surrounding the breast lesion , followed by injection of 0.2 ml saline using a 22 g needle . we used nanocolloid particles with a size range of 5100 nm ( nanocoll , ge healthcare ) . after injection , antero - posterior and lateral lymphoscintigraphic projections were obtained to identify the presence of a sentinel node and to define whether the radiocolloid has shifted to other possible sites of drainage , such as the internal mammary , intramammary , contralateral axillary or supraclavicular nodes . the hot spot was identified over the skin by the handheld gamma probe ( navigatog gps rmd ) and was examined using ultrasound logos hi vision gold ( hitachi ) using a breast - dedicated linear array transducer . the radiologist performing the us - guided biopsy established which lymph node corresponds to the hot spot under gamma probe guidance . we evaluated suspect us findings such as cortical thickening , especially if focal , markedly hypoechoic cortex , absence of the fatty hilum , large expansion , irregular shape , round shape , extracapsular tumoral extension or increased peripheral blood flow at power doppler . lymph nodes with at least 2 of these criteria were considered pathologic , and we excluded suspicious lymph nodes . patients were placed in a supine or contralateral - side - down oblique position on the table , with the ipsilateral hand placed behind the head . biopsies were performed in all patients by a single radiologist with more than 10 years experience in ultrasonography . cnb was performed with an automatic 18 g needle ( bard core biopsy instrument , tempe , usa ) after local anesthesia with 2% mepivacaine . all biopsies with a free - hand technique were performed under us guidance with direct visualization of the needle entering into the cortex of the node to confirm position of the needle tip in the appropriate location . only 1 pass the percutaneous biopsy was performed only if the lymph node corresponded to the hot spot . we considered the biopsy procedure to be successful when the obtained sample contained a large portion of solid non - fatty tissue and/or sank in formalin to be stained with haematoxylin and eosin . the skin above the first radioactive node was marked by the radiologist with an indelible ink tattoo to assist the surgeon . the puncture zone was compressed and the presence of complications such as local pain or hematoma was evaluated . the next day , in the operating room , radioactive lymph nodes were detected in all cases using the same gamma probe that was used to detect the hot spot for the us - guided pre - operative imaging . surgical slnb was performed in all cases , followed by alnd in cases of positive percutaneous biopsy . the surgeon was able to recognize the sentinel lymph node biopsy marked by the radiologist with the skin mark and correlate with the results of the standard slnb ( validate the ability of the preoperative diagnostic method to correctly identify the sentinel node ) . because the most frequent tumor site is the upper - outer quadrant of the breast , the surgeon generally used the same type of incision of the breast to identify the sentinel node using the gamma probe guide when the conservative surgery was performed . in different cases , the surgeon used a separate incision guided by the skin marker performed by the radiologist . the final histopathological diagnosis of slnb was compared with the results of the cnb of the sentinel node . the accuracy of percutaneous biopsy guided by us and gamma probe over the skin in the pre - operative staging was correlated with final pathologic reports of slnb . information on histological type and grade and biological characteristics ( eg , receptor status or peritumoral vascular invasion of the primary carcinoma ) were obtained from the pathology report of the breast specimen . for the pathological staging of the axillae , this study used the guidelines and terminology proposed by the seventh edition of the american joint committee on cancer staging manual .", "main patient characteristics are shown in table 1 and include age , type of surgery , location of primary breast cancer , nodal stage at percutaneous biopsy , pathological nodal stage , and biological features of the breast tumor . sentinel node identification was successful in all patients , although there were no suspicious nodes at us examination . the hot spot was detected over the skin by gamma probe in all 10 patients , and was examined using us - guided cnb in all patients . multiple sentinel nodes were not detected and no patient had the sentinel node draining to different possible sites . there were no major complications ( eg , clinically important bleeding , nerve injury , or infection ) related to the cnb procedure . one patient experienced a small amount of bleeding , which was stopped by simple local compression ; this was considered a minor complication . in all patients , nodal metastases were found at final diagnosis in 2 of 10 patients ( table 1 ) . the definitive histopathological report of slnb expressed the presence of 1 micrometastasis that was not observed in the frozen section at the extemporary analysis . in the second patient , micrometastases were observed in the para - sentinel node at slnb , but no macrometastasis in the sentinel node were found . in all the surgically excised sentinel lymph nodes , the pathologist documented the sign of the previous core needle biopsy , verifying that the biopsied lymph node was really the sentinel node .", "in women with early breast cancer , slnb has proved to be a safe and accurate method for evaluating axillary disease , and is associated with less morbidity than complete alnd . nevertheless , slnb is not a problem - free procedure . patients with documented lymph node involvement at the slnb usually require further alnd as a second step during the same surgical procedure , favoring scar tissue formation and edema , and increasing the rate of complications and the surgery time . hospital stays are also increased in case of slnb + alnd compared with alnd alone as suggested by goyal et al . . pre - operative knowledge of an axillary metastatic sentinel lymph node could avoid the intraoperative slnb . in fact , in cases with positive preoperative lymph node cnb , a complete alnd can be performed directly as a primary procedure . the obvious advantages of this approach are suggested by several authors that used different methods to localize the abnormal lymph node before surgery . had avoided slnb in 30% of patients with a final pathology of metastatic lymph node by performing fine needle aspiration cytology ( fnac ) of the abnormal sonographic axillary node . sever et al10 proposed the use of intradermal peritumoral microbubble us contrast agents injection and lymphatic imaging to identify and localize the sentinel node in the preoperative stage . many studies report a moderate diagnostic sensitivity of the percutaneous biopsy of a morphologically abnormal axillary lymph node and an even lower sensitivity in cases where the biopsied lymph node has an unaltered appearance and the procedure is performed randomly . but how can you differentiate the sentinel node from a regular lymph node especially when the pathological sentinel node is not always the largest one or the morphologically abnormal one ? . found metastatic involvement in 12% of the lymph nodes with normal sonographic appearance , and declared that us - guided biopsy would miss the sentinel node in 36% of the cases , resulting in false - negative axillary cnb , partly due to failure to identify the lymph node or the abnormal region of the lymph node to biopsy . furthermore , there is little information in the literature regarding the accuracy criteria for performing an axillary us . adding gamma probe over the skin could increase the sensitivity of percutaneous us - guided biopsy , providing a good approximation of detection of true sentinel lymph node . theoretically , it is well established that a negative sentinel lymph node is currently equivalent to disease - free axillae . the majority ( about 70% ) of women with clinically negative axillae will prove to be microscopically negative as well . on the other hand , it is also documented that a certain percentage of the false - negative sentinel node cnbs are for micrometastases or isolated tumor s cells . there is no current evidence showing that submicroscopic metastases predict an adverse outcome or that they require treatment . several authors [ 1820 ] demonstrated the absence of axillary recurrence during long - term follow - up in patients with sentinel node micrometastasis in which alnd was not performed . several studies were done , and one of the most recent addressing this question participated in an ibcsg trial that compared complete alnd ( used in cases with positive slnb ) to follow - up ( in patients with node - free macrometastasis disease ) . similarly , we speculate that patients with micrometastases and false - negative sentinel node proved by cnb , gamma probe and us - guided biopsy , may not need intraoperative slnb , particularly since follow - up with ultrasonography , and eventually pet , could detected early axillary disease that can be adequately treated later by therapeutic axillary dissection . nevertheless , avoiding the morbidity of slnb must be weighed against the risk of harboring axillary micrometastases that may potentially seed occult metastatic disease after a percutaneous biopsy . in the clinical context , considering a patient s expected life span and associated health problems , this situation might be defined as a minimal acceptable risk . slnb has also a false - negative rate ( 5% according to veronesi et al . ) and this is the reason why these patients are generally subject to regular follow - up with clinical and us examination of the axilla . we also suggest that cases with negative sentinel node percutaneous gamma probe and us - guided biopsy should be similarly monitored . several studies reported that the sensitivity of us in identifying axillary adenopathy has been increased by cytology sampling of the suspicious lymph node ; the approach is limited by the high rate of false - negatives results of the aspiration tecnique . in other words , the unacceptably high rate of false - negatives results makes nodal fnac an unreliable method that can not be used to avoid intraoperative slnb . as known , there are a number of possible reasons for a false - negative result at fnac : inadequate specimen sampling , a low number of metastatic lymph nodes , small - sized metastasis , and failure to visualize true sentinel lymph node during the us examination of the axilla . the aim of this study was to verify the feasibility of this innovative method to detect and to biopsy the sentinel node in order to improve confidence in identification of the metastatic lymph node in the preoperative phase . in our opinion the decision to use large cnb rather than fnac was made because the amount of tissue taken is greater than with the latter , providing a high negative predictive value . it is reasonable that increasing the volume of tissue obtained from 1 step by cnb can be more effective than multiple samples of fnac to obtain a final diagnosis . the sensitivity of the method could be increased by performing several cnbs ( 23 or 35 samples according to garcia - ortega ) from the cortex of the presumed sentinel node . do not recommend the use of cnb because of the potential complications of this procedure ( eg , bleeding and nerve damage ) ; previous reports have shown that such interventional procedures in all breast cancer patients are not cost - effective . on the other hand , complications can be reduced if the procedures are performed by an experienced , dedicated breast radiologist ( within the breast unit ) . this feature is of major importance because , in early the stage , the lymph nodes usually have normal findings at us , and they are frequently small in size . consequently , the cutting needle must be sampling only the cortical layer if performed in a technically adequate way . the second element is identification of the axillary sentinel lymph node preoperatively using the gamma probe over the skin . combining the use of the gamma probe , with which the true sentinel node can be identified with reasonable confidence , and the us - guided cnb that can avoid the sampling error dictated by the reasons mentioned above ( in case of fnac ) , the diagnostic sensitivity should , theoretically , increase . as a minimally invasive staging procedure , us - guided lymph node biopsy under percutaneous gamma probe surveillance seems attractive because it provides information during the preoperative period that could allow avoidance of intraoperative slnb if the sentinel node is negative , and hence , avoid alnd in case of micrometastasis or isolated tumor s cells . the most relevant predictive factor for sentinel node metastases is the primary carcinoma peritumoral vascular invasion . it could be suggested that whenever this pathology ( or other findings ) finding is found in association with a negative percutaneous sentinel node biopsy performed under us gamma probe guidance , intraoperative slnb should be mandatory . have reported the pre - operative use of gamma probe over the skin to detect axillary sentinel node under us guidance . the latter had evaluated the sentinel node s location using the gamma probe and then marked it with a hook wire , while motomura used the gamma probe combined with fnac to obtain the sample . to our knowledge , no other published study evaluated gamma probe - assisted sonographic localization combined with cnb of the presumed sentinel node as a one -step procedure in the pre - treatment phase of breast cancer patients . first , this is a pilot study that analyzed the feasibility of a new pre - treatment method proposed for detecting the sentinel node . second , the patient population is small because this is a pilot study , hence the results do not have a statistical impact . however , the concepts presented could improve the quality of lymph node sampling , particularly in early breast cancer patients . this could be particularly useful in cases that need slnb before neoadjuvant chemotherapy . the pre - treatment sentinel node evaluation technique described in this pilot study had shown a perfect concordance with the histological findings , even if the number of subjects enrolled in this study was relatively low . the accuracy and the clinical implications of the method", "in conclusion , gamma probe - assisted sonographic localization associated with cnb of the sentinel node in early breast cancer patients could be a feasible and accurate new method . further studies should investigate the definitive role of this method in pre - treatment breast cancer staging . particularly , it is necessary to investigate the clinical impact of this method in avoiding slnb , especially in cases when percutaneous sentinel node cnb is negative , without primary carcinoma peritumoral vascular invasion , in clinically node - negative breast disease patients ." ]

[ "since the first fontan procedure was introduced by fontan in 1971 , the surgical technique has evolved and the survival rate has shown dramatic improvement . recently , fontan surgery has been performed as an operation of the final stage for congenital heart disease with single ventricle physiology1 ) . in fontan circulation therefore , the pulmonary vascular resistance ( pvr ) must be lower than the antegrade transpulmonary flow resistance2 ) . thus , even a mild increase in the pvr may interfere with pulmonary circulation which in turn may result in decreased preload of ventricle , diminished cardiac output , and declined exercise tolerance3,4 ) . loss of pulsatility of pulmonary flow , reduced growth of the pulmonary vasculature , micro and macro thromboembolism in pulmonary circulation and reduced ventricular function are underlying mechanisms that induce the high pvr in fontan circulation5,6,7 ) . several studies8,9,10,11,12 ) reported the pulmonary vasodilating therapy had an effective influence on fontan patients so we want to determine whether inhaled iloprost as a pulmonary vasodilator might have a role in the clinical symptoms , hemodynamics and exercise capacity of fontan patients . ethical approval for this study and permission for acquiring , analyzing and reporting of patient 's data were obtained in accordance with the guidelines of the institutional review board of gachon university gil medical center ( no .", "an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) . a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable . recently , she felt more difficult to climb the stairs due to shortness of breath . the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks . pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed . after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased . six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii . , the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr . the results are shown in table 1 . a 22-year - old male with double outlet right ventricle after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly . , the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr .", "an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) . a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable . recently , she felt more difficult to climb the stairs due to shortness of breath . the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks . pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed . after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased . six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii . , the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr .", "a 22-year - old male with double outlet right ventricle status post fontan surgery at age of five years complained of mild doe only . after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly . , the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr .", "despite the increased survival rate of patients who has congenital heart disease with single ventricular physiology due to advanced surgical technique , the fontan surgery is still a palliative procedure . development of the fontan failure state might occur gradually over time ; ventricular dysfunction , aggravated cyanosis , hepatic dysfunction , coagulopathy , protein loosing enteropathy , plastic bronchitis , arrhythmia etc.1 ) . progressive decreasing cardiac output , and increasing central venous pressure can be also observed in fontan physiology with age1 ) . most patients who underwent cardiac transplant long after fontan surgery had elevated pvr before and immediately after the operation13 ) . indeed , pvr is an important factor in the progressive decline in efficiency of the fontan physiology . the loss of pulsatile blood flow of pulmonary circulation of fontan patients is positively correlated with high pvr . the lowering of pvr may increase pulmonary blood flow and preload reserve in fontan physiology , and for this reason the selective pulmonary vasodilating therapy is interesting as a means to improve the fontan circulation8 ) . in addition , nonpulsatile pulmonary blood flow may reduce capillary recruitment and endothelial function . as a result of endothelial dysfunction , impairment in production of nitric oxide , prostacyclin and prolonged overexpression of endothelin-1 level can be observed5,14 ) . most studies were based on high level of endothelin-1 in fontan patients9,10 ) , the endothelin receptor antagonist induced improvement of clinical , hemodynamic parameters and exercise capacity . experiences with sildenafil therapy on fontan patients have also been reported11,12 ) . in a recent study , rhodes et al.15 ) reported on the effect of inhaled iloprost on exercise function of fontan patients . in this article the authors administered a single treatment with iloprost nebulizer just before exercise and concluded that iloprost improves the peak oxygen pulse and peak vo2 of patients with fontan physiology . in our two case studies , we found that 12 weeks of iloprost therapy showed an association with improvement of clinical , hemodynamic parameters and exercise capacity in patients with fontan physiology . first case was considered fontan failure because of aggravated doe , functional class and pitting edema . unlike previous study using iloprost once , we used iloprost for three month period and positive results were achieved without major side effects . in addition , we found that iloprost was effective for fontan patients with or without symptoms . although more advanced curable treatments of fontan physiology have not been developed , pulmonary vasodilator therapy on fontan circulation is one of good suggestion for symptomatic treatment of fontan failure and improvement of the cardiac output ." ]

[ "angiomyolipoma is a benign tumor that is histologically composed of groups of mature adipose tissue intermixed with convoluted thick - walled blood vessels , interlacing bundles , and irregularly arranged sheets of smooth muscle , with the kidney as the most frequent site of involvement . extrarenal angiomyolipoma can occur in organs such as the liver , lung , uterus , and skin . only two instances of this tumor arising in the tongue have previously been reported [ 1 , 2 ] .", "a 61-year - old woman was referred to our hospital with a mass in the left proglossis that was painless , but had enlarged slowly for 5 years . the mass measured 20 mm 20 mm and was soft and dome shaped with a dark violet - colored surface ( figure 1 ) . the mass was well - demarcated and easily dissected with an ultrasonic surgical aspirator ( figure 2 ) . histopathological examination showed an encapsulated lesion composed of a proliferation of an intricate mixture of mature adipose tissue , blood vessels , and smooth muscle ( figure 3(a ) ) . the patient 's postoperative course was uneventful , and there were no signs of recurrence at followup at 18 months ( figure 4 ) .", "koizumi et al . reported a case of angiomyolipoma in the centre of the tongue . described a case of a angiomyolipomatous hamartoma arising in the left lateral border of the tongue . in the present case , angiomyolipoma arose in the proglossis and it had a diagnosis of hemangioma because of its hardness and color . because the three principal components in angiomyolipoma , regardless of its location , vary greatly in proportion and distribution , its heterogeneity may cause diagnostic confusion . the differential diagnosis includes lipomatous or myolipomatous tumours , angiomyoma , angiolipoma , hemangioma , fibroma , and fibrolipomatous hyperplasia . angiomyolipoma could be considered a hamartoma , but there is no consensus that these lesions are a single entity . ide et al . reported that the present lesion may not be a classic oral angiomyolipoma because it was poorly circumscribed and not encapsulated . they suggested the term angiomyolipomatous hamartoma to designate this lesion . in agreement with ide et al . , we diagnosed the present case as an angiomyolipoma because the mass had enlarged gradually with active proliferation and was well - encapsulated . in particular , tongue cancer and hemangioma are tumors with many blood vessels in the proposed area of surgical excision . for preservation of the nervous system and avoidance of bleeding , an ultrasonic surgical aspirator is effective in the resection of these tongue tumors [ 35 ] . in our case ," ]

[ "recently , extensive reciprocal interactions between the immune and skeletal systems have been demonstrated , resulting in the establishment of a new interdisciplinary research field , named osteoimmunology , which is focused on the understanding of the crosstalk between the immune and bone systems [ 1 - 3 ] . in particular , osteoimmunology investigates how the immune system impacts on bone turnover in physiological and pathological conditions through the immunoskeletal interface . researchers recently hav - egained better understanding of the dialogue between immune and bone cells and a new reading register of bone remodeling is emerging , in which the various phases of bone formation and resorption , that coexist in a dynamic equilibrium , are under strict immunological control . bone and immune system are functionally integrated through complex homeostatic networks and , in all respects , osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other inflammatory conditions , as well as of other immune mediated diseases . in this context immune and bone systems appear to be integrated and sharing signaling pathways and regulatory mechanisms whose understanding provides a framework for obtaining new insights for the discovery of novel treatments for diseases related to both systems . thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well - known and common diseases , such as osteoporosis . its fields of interest are constantly expanding , thus enriching with an increasing number of translational implications , even in clinical practice and in various branches of medicine . here , the most important concepts in osteoimmunology are addressed in the context of physiopatological states bridging these two organ systems , including osteoporosis , ageing , menopause , inflammatory arthritis , cancer , dysmetabolism and neurological disorders . purpose of this review is to outline a new panorama of osteoimmunology that is not limited to immune mediated bone turnover but also consider , in the light of the latest findings in this field , interesting connections with other systems and regulatory functions over bone remodeling .", "bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals . antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc . ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes . moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone . ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function . the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes . there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] . the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes . they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) . the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients . they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages . ob , ocy and oc continuously communicate with each other to optimize the quality of the bone . for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors . the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption . the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal . osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption . rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways . simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation . many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 . ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis . it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis . wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors . the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage .", "bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals . antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc . ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes . moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone . ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function . the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes . there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] . the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes . they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) . the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients . they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages . ob , ocy and oc continuously communicate with each other to optimize the quality of the bone . for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors .", "the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption . the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal . osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption . rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways . simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation . many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 . ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis . it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis . wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors . the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage .", "both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . inflammation and bone turnover share the same mediators , such as cytokines and transcription factors . various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures . t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease . th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- . for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl . moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation . rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells . the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions . other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal . the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling . tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation . their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system . also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function . therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis . tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface . cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen . in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover , cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption . eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization . osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator . the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis . rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively . cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory . cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation . pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages . the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin . cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss . tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production . moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg . bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 . inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis . the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis . all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network . each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines . for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] . a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated . the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production . ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) . treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity . endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 . these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc . resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob . osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered . a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system . cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis . oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption . osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors . they secrete a variety of proinflammatory and immunosuppressive factors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production . skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif . they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells . msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions . msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules .", "both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . inflammation and bone turnover share the same mediators , such as cytokines and transcription factors . various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures . t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease . th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- . for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl . moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation . rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells . the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions . other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal . the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling . tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation . their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system . also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function . therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis . tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface . cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen . in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover , cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption . eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization . osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator . the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis . rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively . cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory . cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp", "boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation . pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages . the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin . cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss . tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production . moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg . bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 .", "inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis . the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis . all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network . each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines . for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] . a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated . the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production . ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) . treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity . endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 . these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc . resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob . osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered . a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system . cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis . oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption . osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production . skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif . they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells . msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions . msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules .", "osteoporosis is a systemic disease of the skeleton , whose main features are loss of bone mass , bone mineral density ( bmd ) decrease and disruption of bone microarchitecture , so the skeleton becomes fragile , exposing patients to increased risk of fractures . aside from senile and postmenopausal osteoporosis , the first recognized types of primary osteoporosis , many other causes of secondary osteoporosis have been subsequently recognized , for example vitamin d and calcium deficits , lack of sun exposure , immobility , drugs such as cortisone , malabsorption syndromes , endocrine and dismetabolic diseases such as diabetes , disthyroidisms , hypercortisolism , and so on . only later , clinical and experimental findings evidenced a close connection between osteoporosis and immune mediated inflammatory conditions , for example , rheumatoid arthritis ( ra ) and acquired immune deficiency syndrome ( aids ) , and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk , such as old age and estrogen deficiency [ 57 - 59 ] . more recently , other unpredictable pathological conditions , such as obesity , are coming out as potential osteoporotic risk factors . even in these cases , the main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation . from this point of view osteoporosis could be therefore regarded as an immune mediated disease in which immune activation , through the induction of cytokine production and inflammation , leads to a remodeling of oc and ob activity and dysregulation of bone turnover with consequent increased bone resorption and osteoporosis . paradigmatic examples of the link between inflammation and osteoporosis are inflammatory arthritis , mainly ra ( fig . 1 ) . ra is an autoimmune disease that is characterized by inflammation of the synovial joint , leading to severe structural damage and bone destruction . an increased bone resorption is the main pathogenetic mechanism in both disease progression leading to juxta - articular bone erosions and irreversible joint damage and systemic osteoporosis . bisphosphonates , drugs used for some time in the therapy of osteoporosis , are potent inhibitors of oc activity both in the primitive and secondary osteoporosis , such as that associated with autoimmune diseases . a decreased bmd in the spine and hip and higher prevalence of osteoporosis have been described in ra patients . in early untreated ra , bmd is related to longer symptom duration , the presence of rheumatoid factor ( rf ) and cyclic citrulinated peptide antibodies ( anti - ccp ) , disease activity score , and the presence and progression of joint damage [ 1 , 19 ] . monoclonal antibodies ( moab ) against various proinflammatory cytokines and their receptors , such as tnf- , are useful in preventing and/or reversing bone erosions as well as systemic osteoporosis . autoimmune reactions induce rankl expression and subsequent osteoclastogenesis . that the rank/ rankl / opg pathway is central to the osteoporosis pathogenesis is confirmed by the elevated antiresorptive capacity of denosumab , an anti - rankl moab , utilized in osteoporosis therapy . activated immune cells at sites of inflammation produce a wide spectrum of proinflammatory and osteoclastogenic cytokines , resulting in bone erosions , osteitis , and peri - inflammatory and systemic bone loss . local peri - inflammatory bone loss and osteitis occur early and precede and predict erosive bone destruction in ra . moreover , peri - inflammatory bone formation is impaired , resulting in non - healing of erosions , and this allows a local vicious circle of inflammation between synovitis , osteitis , and local bone loss . rankl is highly expressed in the ra synovium , and inflammation - mediated bone damage is largely attributable to its abnormally high expression . in addition to activated t lymphocytes and macrophages , a pivotal role in inducing bone erosions is also played by rankl - expressing b cells , as highlighted by the observation of the therapeutic effect of anti - cd20 antibody in ra . cd4+t - cells , especially th17 cells , play a prominent role , particularly in the initiation of systemic immune response in ra . the interaction between immune and mesenchymal cells in joints , including synovial fibroblasts , which are characterized by hyperproliferative and hyperactive properties in response to an inflammatory environment , is of paramount importance in rheumatoid inflammation . in fig . thus , in the affected joints , hyperplasia of the synovial membrane is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells , including cd4 + t cells and innate immune cells . autoimmune diseases , including arthritis , often result from an imbalance between treg cells and th17 cells . the th17 cells derived from foxp3 + t cells in ra comprise a novel th17 cell subset with a distinct pattern of gene expression and arthritogenic properties . the fate of plastic foxp3 + t cells may be a key determinant of the treg / th17 balance that is critically involved in the self - tolerance and autoimmunity . mesenchymal cells are a determinant of the development of ra that links the systemic immune response and the local disorder in the joints . mesenchymal cells contribute to the th17 mediated chronic inflammation by promoting the migration of th17 cells to the inflamed joint and concomitant increase in il-17 production . thus , the interaction of immune and mesenchymal cells plays a key role in both the chronic inflammation and bone destruction in ra . in particular , pathogenetic autoreactive immune cells migrate into joints and activate the mesenchymal cells resident in joint , such as synovial fibroblasts . moreover , since soluble inhibitors of the wnt pathway , such as dkk-1 , produced by synovial fibroblasts , are upregulated by tnf- , antibodies against dkk1 could be able to both promote bone formation and prevent bone erosions in ra .", "osteoporosis has long been regarded as simply the consequence of the menopausal estrogen decline . os- the progression of articular erosions is clearly driven by proinflammatory and osteoclastogenic cytokines produced by immune cells in the inflamed joint . both activated lymphocytes and macrophages stimulate osteoclast differentiation by producing proinflammatory mediators . activated lymphocytes of the synovial pannus overexpress rankl and tnf- that stimulate bone marrow cd11b+ ocp to proliferate and enter the bloodstream where they themselves produce inflammatory factors amplifying inflammation . activated macrophages in the inflamed joints produce various chemokines which drive the localization of periarticular bone ocp . the stimulation of osteoclastogenesis induced by the high concentrations of rankl and tnf- results in bone resorption . these cells do not produce antiosteoclastogenic cytokines , such as ifn- or il4 , while expressing high levels of rankl and secrete il-17 , that in turn stimulates ob , synoviocytes and fibroblasts to express rankl , and induces macrophages to produce proinflammatory cytokines , such as tnf- and il-6 in the synovium amplifying local inflammation . il-17 also induces the synthesis of enzymes capable of degrading the bone matrix such as matrix metalloproteinases ( mmp ) . these effects are balanced by treg that inhibit bone destruction by suppressing oc formation through both cell - cell contact and the secretion of inhibitory cytokines such as tgf- , il-4 and il-10 . teoimmunology , suggesting that immune cells take part in the bone changes typical of menopause , has led to a shift in the concept of osteoporosis , that is currently considered an inflammatory condition . post - menopausal osteoporosis is a clear example of the mutual influences between immune system , bone and endocrine system . like many other hormones , in addition to specific target organs ( breast and reproductive system ) , estrogens have their receptors also on immune cells and bone , as well as on bone marrow precursors . ( 3 ) shows the effects of estrogen deficiency on cells and molecules involved in bone metabolism . menopausal estrogen decline leads to proliferation and activation of t cells by increasing mhcii expression on monocytes and apc function and by inhibiting t cell apoptosis . these effects leads to the expansion of activated t lymphocytes resulting in hyperproduction of inflammatory cytokines and chronic oc stimulation which is responsible for bone loss and increased fracture risk . maintenance of inflammatory reactions leading to bone resorption and skeletal fragility is also present during senescence and inflammaging ( fig . 4 ) , that is the condition of chronic inflammation characterizing aging , as the result of the immune system s ability to cope with stressors . inflammaging is now considered the background of a broad range of age - related diseases with an inflammatory pathogenesis . many of the biological mechanisms implicated in the aging process , such as cell senescence , proinflammatory immune profile , apoptosis and metabolism imbalance , are also implicated in bone remodeling . also in the absence of overt inflammatory diseases , the heightened catabolic signals induced by inflammation enhance apoptosis of ob and muscle cells , causing both osteoporosis and sarcopenia . during aging , the lifelong exposure to oxidative stress and chronic antigenic load leads to the loss of the regulatory process which counteracts t cell activation induced bone resorption . in aged people , lipid oxidation mediated by ros oxidized polyunsaturated fatty acids induce the association of ppar with -catenin and promote its degradation . oxidized lipids also potentiate oxidative stress , enhance ob apoptosis and inhibit bmp-2 induced ob differentiation . antioxidant agents seem to have some action on bone remodeling : resveratrol decreases nf - k activation induced by rankl and oc differentiation and also promotes osteogenesis in msc via the sirt1/foxo3 axis stimulation . during senescence , besides the impaired treg function , the number of effector memory cells is increased . these are senescent cells with proinflammatory properties , secreting several inflammatory cytokines able to influence bone remodeling . interestingly , this immunological pattern ( accumulation of activated cells and memory / effector lymphocytes secreting proinflammatory cytokines ) characterizing immunosenescence , is also peculiar of other immunological conditions associated with osteoporosis , such as ra , aids , chronic viral infections , etc . .", "the already complex cross - talk between bone and immune system is further expanding as they emerge new mediators involved . interestingly , the scenario of osteoimmunolgy is increasingly including interconnections with other organs and systems , therefore influencing several homeostatic functions in addition to bone turnover and immunity , such as hemopoiesis , metabolic and neuro - endocrine functions . the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis . hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation . hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells . interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis . of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action . the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function . a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 . these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development . cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis . ob , together with msc , are crucial components of the niche of growth of hsc . ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling . the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells . osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation . also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support . even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression . being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells . moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling , a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation . the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement , neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand , the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement . a promising new field of interest in this regard is the osteo - immune - oncology . there is in fact a close relationship between immune regulation of bone turnover and tumor growth . often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors . then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer . osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth . tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors . adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc . shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob . in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures . leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain . leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells . adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes . adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway . leptin and adiponectin act on the bone through different signaling pathways with contrasting effects ( table 2 ) . it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction . white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin . brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding . visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone . obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile . therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals . both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) . osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism . fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling . ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway . interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome . the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) . age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage . age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . the binding of age to organic bone matrix may also increase the fragility of bones . autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . however , excessive autophagy is harmful to cells and leads to damage or massive death of cells . autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion . it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system . adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment . an association between major depression and osteoporosis has been recognized [ 96 - 99 ] . the prevalence of low bmd is higher in people with depression than the general population . interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] . neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) . leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner . it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand , 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) . there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) . the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal . the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption . the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism ( fig . a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life . small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders . amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis . the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context , bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity . inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production . serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions . it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors .", "the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis . hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation . hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells . interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis . of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action . the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function . a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 . these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development . cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis . ob , together with msc , are crucial components of the niche of growth of hsc . ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling . the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells . osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation . also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support . even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression . being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells . moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling , a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation . the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement , neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand , the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement . a promising new field of interest in this regard is the osteo - immune - oncology . there is in fact a close relationship between immune regulation of bone turnover and tumor growth . often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . 5 ) . in skeletal metastases , not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors . then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer . osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth . tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors .", "as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors . adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc . shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob . in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger containing transcription factor , shift the equilibrium towards the osteoblastogenesis . mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures . leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain . leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells . adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes . adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway . leptin and adiponectin act on the bone through different signaling pathways with contrasting effects ( table 2 ) . it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction . white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin . brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding . visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone . obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile . therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals . both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) . osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism . fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling . ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway . interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome . the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) . age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage . age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . the binding of age to organic bone matrix may also increase the fragility of bones . autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . however , excessive autophagy is harmful to cells and leads to damage or massive death of cells . autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion .", "it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system . adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment . an association between major depression and osteoporosis has been recognized [ 96 - 99 ] . the prevalence of low bmd is higher in people with depression than the general population . interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] . neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) . leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner . it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , is also likely involved in interactions between the central nervous and immune systems . in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand , 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) . there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) . the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal . the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption . the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism ( fig . a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life . small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders . amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis . the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context , bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity . inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production . serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions . it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors .", "immune system and skeletal system interact with each other both in physiological and pathological conditions . considerable progress has been made in clarifying the crosstalk between bone and immune system that occurs in a complex and dynamic bone microenvironment . a central role in this crosstalk osteoimmunology therefore represents a new approach to studying osteoporosis that in the past was not considered an inflammatory condition . moreover , the discoveries of the existence of the immunoskeletal interface has also highlighted interesting repercussions for a wide range of pathological conditions beyond osteoporosis , including infections , autoimmune diseases , and neoplasia , in which same pathogenetic pathways are shared . osteoporosis could be therefore considered as a systemic model of integrated signaling pathways and cytokines working in a cooperative fashion . further important research horizons are opened with the extension of the discoveries of osteoimmunology and the disclosure of the immunoskeletal interface , whose practical implications may provide novel therapies for diseases other than osteoporosis , by targeting specific transcription factors , cytokines and their receptors . the correct understanding and decoding of the complex language through which immune system and bone communicate , although still in its dawn , is the essential requirement for the identification of such potentially useful therapeutic targets for both osteoporosis and other correlated inflammatory conditions , which share same mediators and signaling pathways .", "" ]

[ "lymphangioleiomyomatosis ( lam ) is a rare idiopathic disease that exclusively occurs in women of childbearing age , and this disease is characterized by the proliferation of abnormal smooth muscle cells in the lungs and along the thoracic and abdominal lymphatics ( 1 - 3 ) . the disease primarily affects the lungs in the majority of cases , but extrapulmonary lam occasionally occurs with or without subsequent involvement of the lungs . lam involvement of the uterus is extremely rare and there have been only a few such reported cases ( 2 - 7 ) , and the imaging findings of uterine lam have only been briefly mentioned in two reports ( 2 , 5 ) . in this article , we report the cross - sectional imaging findings of extensive lam involving the uterus and pelvic cavity in a 29-year - old woman with tuberous sclerosis complex ( tsc ) and we propose that certain imaging features and the clinical history may suggest the diagnosis . to the best of our knowledge , this is the first report of the imaging findings of lam involvement of female pelvic organs .", "when she was ten years old , she was diagnosed with tsc based on her history of seizure and mental retardation . five years previously , she underwent a pelvic magnetic resonance imaging ( mri ) study for the evaluation of menometrorrhagia and dysmenorrhea . the mr images showed several small ( less than 3 cm ) intramural and subserosal hemorrhagic lesions in the uterus and hematometra ( fig . adenomyotic cysts and uterine leiomyomas with red degeneration were considered in the differential diagnosis based on the imaging at that time . endometrial drainage was performed for the hematometra , but no further treatment such as hormonal therapy was done . for the current visit , her serum ca-125 level was 204.01 u / ml , the ca 19 - 9 level was 50.11 u / ml and the hemoglobin level was 8.7 g / dl . 1c ) showed multiple , large , lobulated , thick - walled cystic masses involving the uterus and the entire pelvic cavity , and these masses extended to the lower abdomen . the attenuation of the cystic masses was higher than that of simple fluid , which suggested hemorrhagic contents . both kidneys were enlarged by multiple , various sized masses with attenuations matching fat and soft tissue , which are findings compatible with angiomyolipomas . pelvic mr imaging showed huge , irregularly - shaped , cystic masses involving the uterus and parauterine pelvic cavity , and these masses were predominantly hyperintense on the fat - saturated t1-weighted image ( fig . 1d ) and they were heterogeneously hypointense and hyperintense on the t2-weighted image ( fig . these cystic masses were thought to originate from the uterine myometrium on the sagittal t2-weighted image ( fig . multiple loculated fluid collections with fluid - fluid levels were seen in the cul - de - sac . on the contrast - enhanced t1-weighted image markedly enlarged lam involving the uterus with extension to the pelvic cavity was suggested as the most probable diagnosis based on the follow - up imaging and clinical findings . the chest ct showed numerous , well - defined , thin - walled cysts that were diffusely distributed throughout the lungs ( fig . 1 g ) . because the cysts were regularly round in shape and there was no associated nodular lesion , pulmonary lam was the suggested diagnosis , but she had no pulmonary symptoms . brain mr imaging showed multiple cortical and subcortical tubers as well as several subependymal nodules in the bilateral lateral ventricles , and this represented tuberous sclerosis ( fig . the intraoperative findings revealed that the uterus was enlarged and distorted by multiple subserosal and intramural hemorrhagic cystic masses . the cystic masses also involved both adnexae , the pelvic side wall and the omentum . there were severe adhesive changes between the cystic lesions and the sigmoid colon and bladder . microscopic examination revealed that the tumor was composed of atypical smooth muscle cells ( lam cells ) arranged in short fascicles around dilated lymphatics and a ramifying network of endothelium - lined spaces ( fig . immunohistochemical staining showed that the tumor cells were diffusely positive for smooth muscle actin and they were strongly multifocally positive for human melanin black-45 ( hmb-45 ) ( fig . the patient 's hospital course was uneventful and she was discharged on the tenth postoperative day .", "lam results from the proliferation of abnormal - appearing smooth muscle cells in the lymph vessels , which causes dilatation and obstruction in the lymph vessels and this results in cystic collections of chylous material ( 1 ) . these smooth muscle cells are classified in the family of perivascular epithelioid cells ( pec ) , which is a cell type that is constantly present in a group of tumors , including lam , sugar tumors of the lung and pancreas , renal angiomyolipomas and clear cell myomelanocytic tumor of the falciform ligament ( 8 , 9 ) . these so - called \" pecoma \" all express hmb-45 ( 8) . lam occurs in about 30% of the women with tuberous sclerosis complex ( tsc ) , which is an autosomal dominant multisystem neurocutaneous disorder of highly variable penetrance , and it is characterized by hamartomas , seizure and mental retardation ( 1 ) . due to the striking similarities in the pathological processes between the lam and tsc , lam has been considered a forme fruste of tsc , and lam is classified as a tsc - associated or sporadic form ( 5 , 10 ) . lymphangioleiomyomatosis predominantly affects the lung parenchyma and this is characterized by pulmonary cysts seen on ct . the extrapulmonary manifestation of lam is uncommon and this is mainly located in the retroperitoneum , pelvic cavity and the posterior mediastinum along the lymphatic channels ( 1 - 7 ) . in a large study of 80 patients with pulmonary lam ( 11 ) , the ct imaging findings of retroperitoneal lam were described as a low - attenuating ( 3 - 25 hounsfield unit [ hu ] ) , multilobulated mass , and the ultrasound findings were reported as a cystic mass with a thick echogenic rind . uterine involvement of lam is extremely rare , and to the best of our knowledge , only eight cases of pathologically proven uterine lam have currently been reported ( 2 - 7 ) . six cases of uterine lam occurred in patients with the stigmata of tsc , and two patients did not have stigmata of tsc . most uterine lams were microscopic and they are incidentally found in patients undergoing evaluation for extrauterine disease ( 2 ) . menorrhagia and/or pelvic pain have been reported in half of the cases , the same as in our case ( 2 - 7 ) . the imaging findings of uterine lam were briefly mentioned in two gynaecological reports ( 2 , 5 ) . ( 2 ) reported that uterine lam simulated high - stage endometrial stromal sarcoma , and the ct finding of uterine lam was described as a large uterine mass , which was thought to be a fibroid . however , the surgery revealed multiple subserosal and intramural hemorrhagic nodules ranging in size from 2.5 to 4.0 cm and bloody ascites due to tumor perforation . ( 5 ) recently reported on a 5.5-cm hypervascular tumor between the uterus and the right ovary that showed low signal intensity on the t1-weighted mr image and intermediate signal intensity on the t2-weighted mr image . this mri finding is quite different from that of retroperitoneal lam and it is rather similar to that of uterine leiomyoma . the pathological examination in that case revealed multiple intramural leiomyomas and several fragments of irregular soft masses composed of hmb 45 positive lam cells . the most remarkable aspects of our case are the initial involvement of lam in the uterus that gradually grew into the pelvic cavity and the intratumoral bleeding . intratumoral bleeding may be caused by overdistention and rupture of the cysts . although the ct findings showed highly attenuating cystic masses involving the uterus and pelvic cavity , the mr imaging confirmed the hemorrhagic cystic nature of the masses by their signal intensity and the mr imaging exactly localized the masses . these imaging findings are consistent with the known imaging findings of retroperitoneal lam . however , if a patient is without a clinical history of tsc , then uterine intramural and subserosal leiomyomas with hemorrhagic and cystic degeneration , adenomyotic cysts and/or endometrial cysts , and malignant uterine and/or ovarian tumors with hemorrhage should be considered in the differential diagnosis . in summary , we report here on the extraordinary radiological findings of lam involving the uterus and pelvic cavity in a young woman with tsc . although uterine lam is rare , radiologists should consider the possibility of this disease when they see multiple cystic uterine or parauterine masses with or without internal hemorrhage in a patient with a history of tsc or pulmonary lam ." ]

[ "ts is a key enzyme in the de novo synthesis of dtmp , an essential precursor of dna , which catalyses the methylation of dump to dtmp . the critical role of ts in the nucleotide metabolism has made it an important target of a variety of chemotherapeutic agents including 5-fu , 5-fu prodrugs as capecitabine , and novel folate - based ts inhibitor such as raltitrexed and pemetrexed , used in the treatment of colorectal and other solid tumours . resistance to fluoropyrimidines and other ts inhibitors may occur through a variety of mechanisms including elevated intracellular ts levels resulting from increases in ts transcription and translation . in crc patients the ts intratumoural expression may predict for the sensitivity to 5-fu and other ts inhibitor - based chemotherapies [ 5 , 6 ] and may also be an important prognostic marker . high ts expression in early stage crc patients seems to predict for poorer overall survival in both chemotherapy - treated and untreated patients following surgery . also , metastatic crc patients with high ts levels are unlikely to respond to infusional treatment with 5-fu , whereas patients with low ts levels have higher than average response rate [ 8 , 9 ] . in a previous study , however , we demonstrated that higher ts levels are favourable prognostic factors for disease free and overall survival . to date the real value of the ts level as prognostic factor is doubtful and some authors are in disagreement about it . the ts gene , containing 7 coding exons , is located in chromosome 18q , for which a high percentage of monosomic loss has been reported to be cell - cycle dependent , although some recent evidence points are more oriented towards proliferation - dependence . it can be predicted that mutations in the ts gene might result in a modification of its structure and then in its ability to interact with the fluoropyrimidines . this prediction has been born out in a variety of studies [ 1215 ] . s. h. berger and f. g. berger reported that the human colon tumour cell line hct116 produces a variant structural form of ts , in addition to the common form found in all colon cell lines tested . among the ts overproduction derivatives of hct116 , cells overexposing the novel forms are more resistant to fdurd , compared with cells overexpressing the normal form so , an association between drug response and altered ts structure could suggest that the novel ts form , which is encoded by a variant structural ts gene , confers relative resistance to fdurd ; this was supported by preliminary kinetic data indicating that this novel form has a reduced affinity for fdump . this variant form presents a replacement of an evolutionary conserved tyrosine by a histidine at residue 33 of the ts polypeptide [ 17 , 18 ] ; this mutation represents the only difference between the two ts forms and must account for the structural and functional differences between them . in an our previous paper we performed the analysis of the ts structure in patients bearing crc to try to demonstrate the presence of that specific mutation , but we did not find it in any patient . here , we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer and previously untreated and try to find relationship between any hypothetical ts variant form with the 5-fu treatment . we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade but we did not find any change in the ts - dna structure .", "ts structure was assessed in a series of 68 patients who underwent surgery for primary colorectal carcinoma confirmed histologically and previously untreated . the following exclusion criteria were applied : history of other neoplasias apart of crc and death occurring within 30 days following surgery and due to postoperative complications . the tumours were staged and graded according dukes ' system and their clinicopathological features were summarised in tables 1 , 2 , and 3 . the samples at surgery were divided in two parts : one was frozen at 80 until analysis , and the other portion was embedded in paraffin to confirm histologically the absence of contamination by normal and necrotic tissue and lymphocytes . genomic dna was extracted from tumour and mucosa samples with a commercial kit purchased from qiagen ( kjvenlo , the netherlands ) and stored at 20. the ts gene of tumour and mucosa samples was amplified in seven different pcr reactions using dna primers of 1620 bases in length placed in the adjacent intronic regions of exons 1 , 2 , 3 , 4 , 5 , 6 , and 7 and listed in table 4 . amplification was performed using a perkin elmer model 2400 thermal cycler ( boston usa ) . reaction mixture included 5 l of 10% dimethylsulfoxide , 5 l of 10 buffer , 1.5 l of 1.5 mm mgcl2 , 1 l of 10 mm dntps , 200500 ng of genomic dna , 200 pmol of both upstream and downstream primers , and 1 units of taq dna polymerase , in a final volume of 50 l . amplification was run for 35 cycles with each cycle consisting in a denaturation step at 95c for 1 minutes , a primer annealing step at 51c for 1 minute , and an extention step at 72c for 2 minutes . pcr was terminated by incubation at 72c for 10 minutes . the length of the amplification products ranged from 180 to 310 bases . dna samples , generated with independently repeated pcr products , were sequenced with the sequitherm excel ii dna sequencing kit ( epicenter , madison , wi ) on a li - cor 4000 ( mwg - biotech , ebersberg , germany ) sequencer .", "the enzyme structure obtained by sequencing the ts genomic dna of each sample was performed in 68 patients with operable crc , untreated with previous chemotherapy . primary tumours and corresponding colonic mucosa were obtained from each patient and the ts genomic structure was performed on each of them , after histological control . 7 patients had g1 histological grade , 36 had g2 , and 25 had g3 histological grade ; 10 patients had dukes ' a tumours , 27 patients had dukes ' b , and 31 patients had dukes c cancers . the median age of patients was 67 years ; 30 patients were male and 38 female . in all the tumour samples evaluated we did not find any ts - dna variant structure in tumour : all a , b , and c dukes ' patients showed stable ts - dna , and also in the germline genome no ts - dna variants have been observed .", "the structure of the macromolecular target of a cytotoxic drug is a critical determinant of cellular sensitivity to that drug . recently , ts has become the subject of several studies aimed to elucidate a possible clinical role of ts detection either as determinant of drug resistance or as prognostic marker of the disease and predictive factor of the treatment . many studies are currently examining the real value of ts expression levels as a prognostic factor , but in the mean time significant uncertainty prevails ; whatever its prognostic influence , to date no studies have been able to establish a cut - off all of these in vitro studies have promising implications in the study of the role of ts in clinical practice . moreover , the unclear meaning of levels not only as a prognostic indicator but also as marker predictive of resistance could be due to mechanisms different from over expression , as mutation . in fact some authors focused their attention on the ts structure with a double aim : development of a new classes of ts - inhibitors with a different mechanism of action and generation of ts mutants to develop gene - therapy strategies . berger and coworkers showed that a single naturally occurring change , a tyr to his replacement , in the primary structure of the ts molecule confers relative resistance to the ts - directed antimetabolite fdurd in hct 116 cells . the tyr to his mutation is the only difference between the altered form of ts and the normal form and , therefore , must be responsible for the diminished effectiveness of the enzyme as a drug target . the data in those studies , together with previously published experiments , strongly favour the notion that the tyr to his ' mutation is responsible for the relative fdurd resistance of cell line hct 116 [ 15 , 16 ] . however , as noted earlier , it is quite possible that factors in addition to this mutation contribute to the phenotype of hct116 and other colon cell lines . the frequency of the tyr33 to his33 mutation in the normal and in the pathological human populations is unknown . the altered ts may exist as a polymorphism in humans ; alternatively , it may have spontaneously arisen during tumorigenesis or during establishment of the cell lines in culture . distinguishing among these possibilities will be of great utility in assessing whether variant forms of ts identified in cultured cell lines are segregating in the human population and have an impact upon clinical response to 5-fluorouracil therapy in cancer patients . on the basis of this knowledge we hoped to see if in human crc samples it could be possible to find the same tyr33 to his33 substitution in the ts structure and eventually to correlate this finding with some clinicopathological parameters such as age and sex , tumour size and location , histological grade , dukes ' stage , 5-fu and raltitrexed treatment , and disease free and overall survivals , to see if that point mutation could be considered a reliable marker of drug resistance and of prognosis . in those samples we did not find that point mutation at the codon 33 . in this study we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer ( crc ) and previously untreated and try to find a relationship between any hypothetical ts variant form with the 5-fu treatment and prognosis . we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade , but we did not find any mutation in the ts - dna structure . the conclusions that could be drown are that in dukes ' a , b , and c crcs there are no changes in the ts - dna gene structure and that the evaluation of the ts expression is the main crc prognostic and drug response marker . what remains is to evaluate the ts gene structure of the d metastatic dukes ' crcs : in these tumours it might be possible to find ts - dna structural changes related with their higher genomic instability and this fact could give an explanation of the 5-fu drug resistance and worse prognosis ." ]

[ "colorectal cancer is one of the most common cancers , representing the second ( males ; 9% ) and third ( females ; 8% ) most prevalent cancer in the usa ( 1 ) , and the third overall leading cause of cancer - related death in the western world ( 2 ) . the past several decades have seen a dramatic increase in the incidence of colorectal cancer also in japan ( 3 ) ; in 2008 , age - standardized incidence rates in males and females in japan were 41.7 and 22.8 cases per 100 000 , with corresponding death rates of 15.2 and 8.9 per 100 000 , respectively ( 4 ) . the introduction of monoclonal antibody - based therapy targeting epidermal growth factor receptor ( egfr ) has provided new alternative treatment options for various types of malignancy , including colorectal cancer ( 58 ) . panitumumab is a high - affinity , fully human igg2 monoclonal antibody specific to the extracellular domain of egfr ( 5,9 ) . the clinical efficacy and tolerability of panitumumab have been well established in large , randomized , controlled clinical trials as monotherapy ( 1013 ) or in combination with cytotoxic agents ( 1417 ) , in the first- , second- and third - line , or later , settings for patients with unresectable , advanced or recurrent colorectal cancer with wild - type kirsten rat sarcoma-2 virus oncogene ( kras ) . panitumumab was first approved in the usa ( september 2006 ) and european union ( eu ) ( december 2007 ) as monotherapy and for use in combination therapy in the eu ( november 2011 ) . in japan , panitumumab was approved ( april 2010 ) for the treatment of wild - type kras gene unresectable , advanced and recurrent colorectal cancer as monotherapy , and for use in combination therapy in the first- , second- and third - line , or later , settings based on global clinical trials ( 1012,1417 ) . the approval of panitumumab in japan for use in combination therapy represents the first such approval in the world . however , because the number of japanese individuals enrolled in the global clinical trials was relatively limited ( 13 ) , the japanese ministry of health , labour and welfare required the market authorization holders to conduct a post - marketing surveillance study , called all cases surveillance ( 18 ) , as a condition of its approval to evaluate the safety and effectiveness of all japanese patients treated with panitumumab . the main purpose of this study was to collect detailed information , particularly with regard to safety issues , on all panitumumab - treated patients ; additionally , by evaluating a large number of patients , the study aimed to identify rare adverse drug reactions ( adrs ) , which are often undetectable in smaller clinical trials .", "this post - marketing surveillance study was planned to include all patients treated with panitumumab from the start date ( 15 june 2010 ) of its launch in japan . to promote the appropriate use and evaluate safety information , a vectibix appropriate use committee , a vectibix safety evaluation committee and a vectibix interstitial lung disease ( ild ) review subcommittee all reported ild - like events were assessed individually by an ild review subcommittee , comprising external experts in the field of radiology , pulmonology and medical oncology . evaluation of ild - like events was performed using computed tomography ( ct ) and x - ray imaging , and by assessment of clinical information . completion of a specific registration form ( fig . 1 ) , which reported patient characteristics and treatment information , was mandatory before panitumumab treatment was initiated . the registration period of this post - marketing surveillance was june 2010 to november 2010 . patients were carefully observed , and information during their clinical courses for 10 months ( 42 weeks ) , or until discontinuation of panitumumab for any reason , was collected through the pre - specified case report forms ( crfs ) . at the time of registration , a recommendation letter to stop initiating treatment with panitumumab was sent from the vectibix appropriate use committee to each attending doctor if at least one item on the dark gray zone of the registration form was checked ( fig . 1 ) . subsequently , the following information relating to clinical course was collected using crfs : ( 1 ) patient background including prior chemotherapy and reason for its discontinuation / termination ; ( 2 ) administration of panitumumab including its dose and date , premedication to prevent infusion reaction , concurrent medications and combined therapy ; ( 3 ) laboratory tests including serum magnesium , calcium and potassium ; ( 4 ) adverse events including time to onset , grade according to national cancer institute common terminology criteria for adverse events ( ctcae ) ver . 4.0 , treatment , causality of panitumumab and recovery and ( 5 ) outcomes including disease progression , survival or death . \n figure 1.patient registration form excerpt . an adr was defined as an adverse event for which a causal relationship with panitumumab could not be ruled out . adrs were coded using medical dictionary for regulatory activities ( meddra ) version 15.0 . with regard to toxicity , skin disorders , ild , infusion reaction , electrolyte abnormalities and cardiac disorders were monitored with special interest . the effectiveness of panitumumab was assessed by overall survival , calculated using the kaplan meier method . overall survival time was defined as the time from the date of first administration of panitumumab to the date of death ( regardless of the cause of death ) or censored on the last date of survival ( date of confirmation of survival as recorded in the crf or , if there was no information regarding the date of confirmation of survival , the last date of panitumumab administration was used for calculation purposes ) . this survey was initiated in june 2010 and the data lock point of the final analysis was set at 12 september 2012 .", "during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients , the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig . 2 ) . \n figure 2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) . patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) . while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop , panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion . the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 . although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration . the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab . therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance . there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig . 1 ) . \n table 1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender male196563.7 female112036.3age ( years ) < 65152449.4 6574105834.3 7550316.3 median ( range ) ( years)65.0 ( 1890)kras status wild300397.3 mutant30.1 not determinable792.6primary tumor type ( duplicate counting ) colon186060.3 resected162152.5 unresected2397.8 rectal124440.3 resected105434.2 unresected1896.1 colorectal3085100.0 resected266186.3 unresected42313.7treatment line first - line31010.1 second - line54317.6 third - line or later223272.4ecog performance status 0187760.9 194230.5 22417.8 3220.7 430.1past treatment regimens no1735.6 yes ( duplicate counting)291194.4 folfox243979.1 folfiri190761.8 bevacizumab211368.5 cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration . although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients . treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration . although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) . figure 3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration , 1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy . combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment , the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan . combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half ( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months . panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) . the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) . the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table 2 ) . the most common classification , according to the meddra system organ class ( soc ) , was skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table 2 ) , followed by infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] , gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n table 2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8 skin and subcutaneous tissue disorders236476.639212.7 infections and infestations77125.01605.2 gastrointestinal disorders64220.81264.1 metabolism and nutrition disorders55217.91494.8 investigations2046.6742.4 general disorders and administration site conditions1113.6290.9 eye disorders832.780.3 nervous system disorders682.2180.6 respiratory , thoracic and mediastinal disorders632.0280.9 injury , poisoning and procedural complications521.750.2 blood and lymphatic system disorders331.1240.8 hepatobiliary disorders190.640.1 renal and urinary disorders120.470.2 musculoskeletal and connective tissue disorders100.320.1 vascular disorders90.320.1 cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) , 86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases . there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) . a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table 3 . \n table 3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4 ( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial no : 2005181 . summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases . trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 . the overall incidence rate of major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) . dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) . the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus . the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) . serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively . the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) . the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) . the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) . the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively . of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) . infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) . infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) . panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases . two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration . overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) . the time to onset of ild varied ( table 4 ) . as determined by ct imaging , diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal . the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n table 4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient , treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total , 1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these , the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) . meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n figure 4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy . overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy . the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases .", "during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients , the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig . 2 ) . \n figure 2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) . patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) . while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop , panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion . the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 . although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration . the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab . therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance . there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig . 1 ) . \n table 1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender male196563.7 female112036.3age ( years ) < 65152449.4 6574105834.3 7550316.3 median ( range ) ( years)65.0 ( 1890)kras status wild300397.3 mutant30.1 not determinable792.6primary tumor type ( duplicate counting ) colon186060.3 resected162152.5 unresected2397.8 rectal124440.3 resected105434.2 unresected1896.1 colorectal3085100.0 resected266186.3 unresected42313.7treatment line first - line31010.1 second - line54317.6 third - line or later223272.4ecog performance status 0187760.9 194230.5 22417.8 3220.7 430.1past treatment regimens no1735.6 yes ( duplicate counting)291194.4 folfox243979.1 folfiri190761.8 bevacizumab211368.5 cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration . although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients . treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration . although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) . figure 3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration , 1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy . combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment , the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan . combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half ( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n", "the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months . panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) . the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) .", "the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table 2 ) . the most common classification , according to the meddra system organ class ( soc ) , was skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table 2 ) , followed by infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] , gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n table 2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8 skin and subcutaneous tissue disorders236476.639212.7 infections and infestations77125.01605.2 gastrointestinal disorders64220.81264.1 metabolism and nutrition disorders55217.91494.8 investigations2046.6742.4 general disorders and administration site conditions1113.6290.9 eye disorders832.780.3 nervous system disorders682.2180.6 respiratory , thoracic and mediastinal disorders632.0280.9 injury , poisoning and procedural complications521.750.2 blood and lymphatic system disorders331.1240.8 hepatobiliary disorders190.640.1 renal and urinary disorders120.470.2 musculoskeletal and connective tissue disorders100.320.1 vascular disorders90.320.1 cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) , 86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases . there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) . a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table 3 . \n table 3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4 ( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases . trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 . the overall incidence rate of major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) . dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) . the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus . the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) . serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively . the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) . the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) . the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) . the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively . of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) . infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) . infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) . panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases . two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration . overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) . the time to onset of ild varied ( table 4 ) . as determined by ct imaging , diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal . the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n table 4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient , treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total , 1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these , the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "the overall incidence rate of major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) . dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) . the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus . the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively . the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) . the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) . the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) . the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively . of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) . no grade 4 cases , or deaths due to infusion reaction , infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) . panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases . two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) . of the total ild cases , 51.3% ( 20/39 ) the time to onset of ild varied ( table 4 ) . as determined by ct imaging , diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal . the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n table 4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild )", "the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient , treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) .", "in total , 1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these , the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n figure 4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy . overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy . the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases .", "the main objective of the current post - marketing surveillance study was to evaluate the safety of panitumumab in japanese patients with unresectable colorectal cancer ( and with the majority of patients possessing wild - type kras tumors ) in clinical practice . the overall incidence of adrs was 84.1% , with skin disorders representing the most common classification ( 78.4% ) . skin toxicity associated with panitumumab ( and other egfr antibodies ) is well known ( 20 ) . indeed , the development of skin disorders , particularly rash , is considered to be a predictive factor for the efficacy of egfr inhibitors ( 21,22 ) . as reported previously with egfr inhibitors ( 22,23 ) , dermatitis acneiform ( 69.9% ) was the most frequent skin disorder reported in the current study , occurring about 2 weeks after the initiation of panitumumab . in the current study , although major skin disorders such as dermatitis acneiform , paronychia , dry skin and pruritus occurred in 78.4% of patients , 93% of the patients who experienced skin disorders were able to continue panitumumab therapy . the egfr - signaling pathway potentially plays a pivotal role in regulating magnesium homeostasis , and , indeed , hypomagnesemia / magnesium has been shown in patients with colorectal cancer receiving egfr - targeting antibodies ( 24 ) . in the current study , 46.2% of patients were evaluated for serum magnesium , and grade 3 or higher hypomagnesemia occurred in 4.0% of patients , with a shorter median time to onset in patients treated with cetuximab immediately prior to panitumumab ( 39.5 and 71 days , respectively ) , indicating that the total duration of exposure to an egfr inhibitor is an important factor . from these findings , it is considered that the longer - term administration of anti - egfr treatment worsens the severity of hypomagnesemia ( 24 ) . however , in the present study , the incidence of hypomagnesemia and hypocalcemia peaked 3 and 4 months after the start of panitumumab administration , respectively . moreover , the incidence of hypomagnesemia observed in the current post - marketing surveillance study is similar to that reported in a phase ii clinical trial of panitumumab in japanese patients with metastatic colorectal cancer ( 13 ) . , almost all patients with prior experience of infusion reaction to cetuximab could receive panitumumab safely following premedication and with a reduced speed of infusion . some reports have shown that panitumumab can be administered successfully in patients after a severe infusion reaction to cetuximab ( 2529 ) . on the other hand , post - marketing fatalities due to infusion reaction with panitumumab have been reported in patients with a history of severe cetuximab - related infusion reaction ( 30 ) . therefore , administration of panitumumab to patients with a history of infusion reaction to cetuximab is not currently recommended . pulmonary toxicity including drug - induced ild represents an important adverse reaction that is associated with egfr - targeted cancer therapy ( 31 ) , with the potential to be fatal in some patients ( 32 ) . we prospectively established an ild subcommittee consisting of experts to accurately evaluate the incidence of ild . furthermore , in order to avoid panitumumab treatment , at the time of registration , a recommendation letter was issued to reconsider treatment with panitumumab if a patient had a history or complication of ild ( fig . 1 ) . in the current post - marketing surveillance study of panitumumab , the overall incidence rate of ild was 1.3% , with a mortality rate of 0.6%.the incidence of ild was similar to that reported in a post - marketing survey of cetuximab in japan ( 1.2% ) ( 23 ) . the ild subcommittee carried out a full evaluation of ild with panitumumab , concluding that the onset of ild can occur at any time after the start of panitumumab administration and that there were no findings specific to panitumumab , either clinically or in ct imaging . the dad type of ild occurred in some cases , as with cetuximab and the egfr tyrosine kinase inhibitors gefitinib and erlotinib , and may be fatal . at present , no major differences in the incidence of ild or in the rate of death due to ild were found in comparison with cetuximab ( 23 ) . thus , ild is one of the particular concerns with the use of panitumumab as well as other anti - egfr drugs . with regard to all panitumumab - related adrs , there were no differences between its use as monotherapy or in combination therapy with cytotoxic agents , and the profile , incidences , onset and outcomes of these adrs were very similar to those reported in previous clinical trials . in contrast to the combination setting with chemotherapy regimens , the monotherapy setting represents the efficacy of panitumumab in clinical practice more accurately ; therefore , patients treated with panitumumab monotherapy as a third - line or later therapy were selected for analysis of the effectiveness of panitumumab . the effectiveness of panitumumab , reported previously in clinical trials , was also confirmed in the current post - marketing surveillance study . in a pivotal , phase iii , randomized , controlled study ( 15 ) , the median overall survival time in the panitumumab group ( wild - type kras ) was 8.1 months ( 6.39.4 months ) ( 11 ) ; data obtained from the present surveillance study exhibit a median overall survival of 10.3 months ( 56.6% censored cases ) . whilst acknowledging that post - marketing surveillance studies have some inevitable limitations ( e.g. the lack of a control group ) that differ from those in well - controlled , prospective clinical trials , it is recognized that post - marketing surveillance studies provide a information based on the general population ( 33 ) . these factors result in greater external validity for post - marketing surveillance studies compared with well - controlled , randomized clinical trials ( 33 ) . despite these limitations , this large - scale , non - interventional study has some advantages . this study also provides health - care professionals with valuable information on the safety and effectiveness of panitumumab based on real - world data . in conclusion , the current post - marketing surveillance study in japanese patients with unresectable colorectal cancer confirmed the safety profile and effectiveness of panitumumab that has been reported previously in clinical trials . it is considered that the benefit / risk balance for the use of panitumumab in patients with unresectable colorectal cancer remains favorable also in japan .", "this work was supported by takeda pharmaceutical company limited . funding to pay the open access publication charges for this article was provided by takeda pharmaceutical company .", "has received a consultancy fee from takeda , speaker honoraria from takeda , taiho and yakult , and research funding from takeda , bristol - myers squibb , merck serono and chugai . has received consultancy fees from taiho , chugai , takeda and merck serono , speaker honoraria from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , kaken , johnson & johnson , toray , aska and bayer , a manuscript fee from chugai , and a donation from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , daiichi - sankyo , ono , eisai , kaken and tori . y.k . has received a consultancy fee , speaker honoraria and non - specific research support from takeda . k.m . has received a consultancy fee from takeda , speaker honoraria from takeda , taiho , yakult , chugai , merck serono and bristol - myers squibb , and research funding from taiho , pfizer and merck serono . t.h . has received a consultancy fee from takeda and speaker honoraria from takeda , chugai , taiho , pfizer , yakult and daiichi - sankyo . t.y . has received a consulting fee from takeda , speaker honoraria from chugai , takeda and merck serono , and research grants from taiho , daiichi - sankyo , lily , pfizer and yakult ." ]

[ "nevus lipomatosus cutaneous superficialis ( nlcs ) was first described by hoffman and zuhrelle in 1921.1 it s a rare idiopathic hamartomatous benign condition characterized by the presence of an ectopic mature adipose tissue within the dermis . it is classified into two clinical variants : the classical form , usually composed of multiple and grouped skin - colored , pedunculated and cerebriform nodules that often coalesce to form a plaque and a second more rare form , presenting as a solitary dome - shaped sessile papule or nodule.24 we report here eight cases of nlcs in order to assess the epidemiologic , clinical , and pathological features , as well as the management of this uncommon neoplasm .", "this study was a retrospective case series including all patients with histopathologically documented nlcs , who attended the dermatology department of charles nicolle hospital in tunisia during the last 14 years ; between january 1997 and december 2010 . for each patient , we recorded the following data : age , sex , duration of the lesions , their localizations , number , and size as well as their management and evolution . ethical approval and informed consent", "eight patients with nlcs were observed , three males and five females , aged between 7 and 41 years . in patients number 4 , 5 , 7 , and 8 , lesions had begun during childhood ( at 1 , 4 , 5 , and 6 years respectively ) . for the other four patients ( number 1 , 2 , 3 , and 6 ) lesions had begun in the third to fourth decade of life . clinical presentation was that of the classical form in seven patients and of the solitary form in one patient ( patient number 3 ) . in four patients it was one of the limbs which had the lesion , and in the other four patients it was the trunk ( figure 1 : patient 8 ; figure 2 : patient 5 ) . soft , skin - colored voluminous tumor ( 10 cm x 7 cm ) of the left buttock composed of multiple nodules with a cerebriform surface . histopathological features were typical of nlcs showing ectopic mature adipose tissue in the dermis ( figure 3 ) . table 1 summarizes the main epidemiologic , clinical and pathological features , and management of nlcs in all eight patients . \n table 1epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients.patientsexage ( years)durationlocationclinical aspecthistopathologytreatment1m381 yearleft buttockplaque of several soft skin - colored nodules of 1 cm to 2 cmmature adipose tissue in reticular dermis without connection to the hypodermissurgical resection2f383 monthsrigth thighmultiple soft yellowish papules coalescing into plaquesectopic mature adipocytes in reticular dermisabstention3f391 yearleft shoulderone soft skin - colored nodule of 1 cmlobular proliferation of fat tissue in the dermissurgical resection4m76 yearsright shoulderlarge ( 6 cm x 2 cm ) soft skin - colored verrucous plaque composed of nodules and papulesectopic mature adipocytes in reticular dermissurgical resection5m2420 yearslumbar area1 cm to 3 cm - grouped soft skin - colored papulesepidermal acanthosis , ectopic mature adipocytes in papillary dermissurgical resection6f3210 yearsthighmultiple soft skin - colored papules coalescing into a 5 cm plaqueepidermal acanthosis ; hyperkeratosis ; dermal proliferation of mature adipocytessurgical resection7f149 yearsflanklarge ( 6 cm x 2 cm ) soft skin - colored plaque composed of nodulesthinned epidermis , ectopic mature adipose tissue in reticular dermissurgical resection8f4135 yearsleft buttocksoft , skin - colored voluminous tumor ( 10 cm x 7 cm ) composed of multiple nodules with a cerebriform surfacenormal epidermis , mature fat cells in reticular dermis without hypodermis connectionsurgical resectionf = female ; m = male . epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients .", "nlcs is more commonly present from birth , but can appear later in life ; there is neither sex predilection nor familial trend in this disorder.1 in this study , there were five cases in which the lesion appeared in the first three decades , and in three cases nlcs appeared after 35 years . two clinical patterns of nlcs exist ; a multiple form or classical type , and the solitary form . in the classical type , lesions are congenital or they develop usually during the first two to three decades of life.5,6,7 it consists of multiple papules , skin colored or yellowish , coalescing into plaques with zosteriform , linear or segmental distribution . the lesions are slow - growing , with a smooth or cerebriform surface , and can reach a large size if left untreated . the largest size reported so far has been 40 cm x 28 cm.8 the most common sites are the pelvic girdle , the lower trunk , the gluteal region , and the thigh . the second clinical pattern of nlcs is a solitary papule or nodule mimicking skin tag,8 usually appearing later than the classical form , during the third to sixth decades of life.3,6,9 it can occur at any site , including unusual ones like the scalp , the eyelid , the nose , and the clitoris.1,1013 solitary form was also called pedonculated lipofibroma by mehregan et al . because of its distinctive clinicopathologic features in comparison with the multiple form.9,14 among the presented cases , there were seven cases with the classical form and one case with the solitary form ( case 3 ) , whilst in another tunisian study the solitary type was predominant : 11 cases out of 13.15 nlcs have an indolent and asymptomatic course . recently , a case of ulnar nerve entrapment accompanied by numbness and straining in the forearm , caused by nlcs , was reported and was treated by partial excision for symptomatic recovery.16 occasionally , nlcs may ulcerate after external trauma or ischemia . in two previously reported cases , surface of nevus was studded with multiple open comedons,2,17 and foul - smelling discharge may be associated.2 nlcs has been reported in association with other cutaneous disorders ; follicular papules and hypertrophic pilo - sebaceous units,18 angiokeratoma of fordyce,4 scattered leukoderma , caf - au - lait macules,2 and hemangioma.4 in our study , no associated skin abnormalities were present . to our knowledge , there are no systemic abnormalities or malignant transformation described with nlcs . nlcs should be clinically differenciated from nevus sebaceous , neurofibroma , lymphangioma , focal dermal hypoplasia , cylindroma , trichoepithelioma , and angiolipoma . histopathological evaluation is required for diagnosis showing ectopic mature adipocytes , intermingled with collagen bundles , and proliferating around the periadnexial adventitial dermis and the perivascular area . mature fat cells proliferate in reticular dermis and may extend to the papillary dermis.2,6,7 in the most characteristic feature of nlcs , there is usually no connection to the subcutaneous fat tissue ; for some authors this condition is necessary to establish diagnosis of nlcs.6 several studies documented increased vascularity in the subpapillary and papillary dermis ; epidermal changes have been reported , like mild to moderate acanthosis , basket weave hyperkeratosis , increased basal pigmentation , and focal elongation of rete pegs.3 adnexal structures may be reduced,3 and abnormal folliculosebaceous structures were reported in some cases such as sebaceous trichofolliculoma , folliculosebaceous cystic hamartoma , and dermoid cysts.7,19 in our study , diagnosis of nlcs was based on characteristic clinical aspects and confirmed by typical histopathological features . the genetic background of nlcs is still unknown , only one cytogenetic study was reported recently showing a 2p24 deletion.20 the pathogenesis of nlcs remains unknown . several theories have been proposed : hoffman and zuhrelle postulated that fat deposition in the dermis is secondary to degenerative changes ( metaplasia ) in the connective tissue.3 other authors hypothesized that adipocytes originate from the pericytes of dermal vessels.2,21 for others , fat cells represented a true nevus that resulted from the focal heterotopic development of adipose tissue.22 treatment is usually not necessary unless for cosmetic reasons . the treatment of choice is surgical excision , which is curative , and post surgical recurrence is rare . patients unwilling for surgery may undergo cryotherapy which yields partial but satisfactory results.8 recently , a case of classic nlcs successfully treated with co2 laser was reported with no recurrence during a follow - up period of 12 months.23 in this retrospective study , we did nt investigate chromosomal abnormalities to identify the genetic biomarkers of this congenital pathology , since cytogenetic analysis was reported in only one previous study.20", "physicians should be aware of this rare condition because early recognition enables more conservative resection of the tumor and less invasive reconstruction of the defect . nlcs is a rare skin malformation ; we report herein a large case series of eight patients where the classical form is predominant . our study may give additional data about epidemiological and clinical pattern of this hamartoma . further studies are needed , especially prospective studies , by using chromosomal analysis to reach conclusions about the role of the genetic abnormalities in the development of this hamartoma ." ]

[ "the use of chemotherapy in the treatment of cancer has opened new possibilities for improvement of the quality of life of cancer patients . despite its success , treatment with some of the most effective anticancer drugs , the mechanism of cisplatin ( cis - diamminedichloroplatinum ( ii ) or cddp ) induced nephrotoxicity has gradually been elucidated . studies have shown an increase in lipid peroxides in the renal tissue of cddp - administered animals , a decrease in reduced glutathione levels , and the induction of metallothionein , an antioxidant . these changes have been considered to result from the generation of reactive oxygen species ( ros ) . studies using chemiluminescence or electron spin resonance ( esr ) have shown that cddp generates oh radical [ 5 , 6 ] . nephrotoxicity involves kidney damage or dysfunction arising from direct or indirect exposure to drugs and industrial or environmental chemicals . cisplatin ( ( cis - diamminedichloroplatinum ( ii ) or cddp ) ) , an anti - neoplastic drug have been reported to induce nephrotoxicity . the kidney which is the major route of cisplatin excretion also accumulates it to a greater degree than other organs [ 6 , 8 ] . oxidative stress , inflammation , and apoptosis are some of the mechanisms already established to explain cisplatin induced acute kidney injury . a number of strategies have been proposed for the prevention / management of cisplatin induced nephrotoxicity , since there is no specific treatment , with the use of some synthetic drugs which have been popular . however , these drugs have some associated risks and side - effects , hence , the need for natural alternatives of plant origin ( plant foods / extracts ) with little or no side - effect . plants have limitless ability to synthesize aromatic substances such as polyphenols , mainly flavonoids , and phenolic acids , which exhibit antioxidant properties due to their hydrogen - donating and metal - chelating capacities . polyphenols are secondary metabolites of plants and are widely distributed in plant - derived foods , such as cereals , legumes , nuts , vegetables , and fruits , and in beverages such as green or black tea , and fruit juice . tannic and gallic acids are two commonly phenolic acids that are structurally related . tannic acid , a naturally occurring plant polyphenol , is composed of a central glucose molecule derivatized at its hydroxyl groups with one or more galloyl residues , whereas gallic acid is a trihydroxybenzoic acid , also known as 3,4,5-trihydroxybenzoic acid , which is widely distributed in green tea , red wine and grapes , witch hazel , sumac , oak bark , and other plants . considerable amounts of experimental data on the antioxidant activity of both tannic acid and gallic acids with emphasis on structure - function antioxidant activity have been reported . also , several authors have demonstrated that tannic acid and other polyphenols have antimutagenic and anticarcinogenic activities [ 1316 ] . extensive studies have been carried out on the protective effect of cotreatment and posttreatment of phenolic acids against cisplatin induced nephrotoxicity . hence , this study was carried out to investigate and compare the protective effect of administration of both tannic and gallic acids on normal and cisplatin induced nephrotoxicity in rats .", "male albino rats weighing 110185 g used for this experiment were purchased from a private animal colony , ikere - ekiti metropolis . the rats were maintained at 25c on a 12 hour light / dark cycle with free access to food and water . they were acclimatized under these conditions for two weeks prior to the commencement of the experiments . the experimental study was approved by the institutional animal ethical committee of the university of ado - ekiti , nigeria . chemicals such as tannic acid , gallic acid , oxidized and reduced glutathione , hydrogen peroxide ( h2o2 ) , dithionitrobenzene ( dtnb ) , thiobarbituric acid ( tba ) , and adrenaline were purchased from sigma chemical co. ( st . ethanol , acetic acid , h2so4 , sodium carbonate , sodium citrate , sodium azide ( nan3 ) , sodium chloride , potassium dichromate , tris - hcl buffer , sodium dodecyl sulphate ( sds ) , and ascorbic acid were sourced from bdh chemicals ltd . may - grnwald , giemsa , and hematoxylin and eosin ( h&e ) stains were purchased from hi - media labs , mumbai . all the kits used for bioassay were sourced from randox laboratories ltd . , crumlin , county antrim , uk . except stated otherwise , all other chemicals and reagents were of analytical grades and the water was glass - distilled . after two ( 2 ) weeks of acclimatization , 80 male rats were randomly divided into eight ( 8) groups of ten animals each : group i served as a normal control and received saline ( 0.85 w / v% ) orally for 7 consecutive days and on the 7th day , 1 hr after receiving the oral saline dose , the rats received a single i.p . group ii served as toxicant group and received saline ( 0.85% ) orally for 7 consecutive days . ga was orally administered at two doses , 20 and 40 mg / kg body weight ( bwt ) , to groups iii and iv , respectively , for 7 consecutive days . also , ta was orally administered at two doses , 20 and 40 mg / kg body weight ( bwt ) , to groups vi and vii , respectively , for 7 consecutive days . on the 7th day of pretreatment , a single i.p . dose of cisplatin ( 7.5 mg / kg bwt ) after oral treatment of ga and ta was given to the animals in groups ii , iii , iv , vi , and vii . groups v and viii received only higher dose ( 40 mg / kg ) of ga and ta orally for 7 consecutive days , respectively , and on the 7th day 1 hr ga and ta treatment received a single i.p . injection of saline ( 0.85% ) to ensure that groups v and viii received only the higher dose of ga and ta ; this was done in order to test that the higher dose did not produce any kind of toxic effects . the doses of ta and ga used were actually selected on the basis of preliminary dose escalation studies to determine the minimum dose of ta and ga required to produce an observable effect ( data not shown ) . uric acid , urea , and creatinine were determined using commercially available kits ( randox laboratories uk ) . tissue malondialdehyde ( mda ) content was determined as described by ohkawa et al . . tissue antioxidant parameters were also determined ; superoxide dismutase ( sod ) by the method of alia et al . , catalase ( cat ) by the method of sinha , reduced glutathione ( gsh ) by the method of ellman , and glutathione peroxidase ( gpx ) by the method of rotruck et al . . at the end of the experiment , whole blood of the sacrificed rats were collected into edta bottles and centrifuged at 800 g for 10 min to separate the plasma . the plasma was then decanted into plain sample bottle and stored in a refrigerator prior to analysis . the rat 's tissues ( kidney ) were rapidly isolated , placed on ice , and weighed . the tissue was rinsed in cold ( 0.9% w / v ) normal saline ( 1 : 3 , w / v ) and subsequently homogenized in sodium phosphate buffer ( ph 7.4 ) with ( 1 : 5 bt w / v ) using mortar and pestle as homogenizer and the homogenates were centrifuged at 4,000 g . the uric acid concentration was determined using colorimetric method as described by collin and diehl and morin and prox . briefly , 20 l of distilled water was added to 20 l of the sample which was mixed with 1 ml of hepes reagent ( 50 mm phosphate buffer , 4 mm 3,5-chloro-2-hydroxybenzenesulfonic acid ) and enzyme reagent ( 0.25 mm 4-aminophenazone , peroxidise , and uricase ) . thereafter , the mixture was incubated for 5 min at 37c and the absorbance at 520 nm was taken against reagent blank within 30 min . the urea concentration was determined using colorimetric method as described by searcy et al . . briefly , 10 l of sample was added to 0.1 ml of sodium nitroprusside 6 mm sodium nitroprusside , 1 g / l urease ) after which the mixture was incubated for 10 min at 37c . 2.5 ml of 120 mm diluted phenol and 2.5 ml of 27 mm sodium hypochlorite solution containing 0.14 n sodium hydroxide which was then added to the reaction mixture . thereafter , the mixture was incubated for 15 min at 37c and the absorbance at 546 nm was taken against reagent blank within 8 hours . the creatinine concentration was determined using colorimetric alkaline picrate method as described by jaffe method . briefly , 50 l of distilled water was added to 2 ml of working reagent ( 35 mm picric acid and 0.32 m sodium hydroxide ) before 50 l of sample was added . the absorbance at 492 nm was taken twice , firstly after 30 sec and secondly after 2 min . the creatinine concentration was subsequently calculated against the standard , using change in the sample absorbance ( absorbance ) . arginase activity was determined by the measurement of urea produced by the reaction of ehrlich 's reagent according to the modified method of kaysen and strecker . the reaction mixture contained 1.0 mm tris - hcl buffer , 1.0 mm mncl2 ( ph 9.5 ) , 0.1 m arginase solution and 500 ml of the enzyme preparation . the mixture was incubated for 10 mins at 37c . the reaction was terminated by the addition of 2.5 ml ehrlich reagent ( 2.0 g of p - dimethylaminobenzaldehyde in 20.0 ml of concentrated hydrochloric acid and made up to 100 ml with distilled water ) . superoxide dismutase ( sod ) was determined by the method of alia et al . . 50 l of supernatant was treated with 1000 l of 50 mm carbonate buffer ( ph 10.2 ) and 17 l of adrenaline ( 0.06 mg / ml ) . the absorbance was read at 480 nm in spectrophotometer for 2 minutes at 15-second intervals . sod activity was expressed as ui per 100 g protein ( 480 = 4.02 mmcm ) . the reaction mixture ( 1.5 ml ) contained 1.0 ml of 0.01 m phosphate buffer ( ph 7.0 ) , 0.1 ml of tissue homogenate , and 0.4 ml of 2 m h2o2 . the reaction was stopped by the addition of 2.0 ml of dichromate - acetic acid reagent ( 5% potassium dichromate and glacial acetic acid were mixed in 1 : 3 ratio ) . then , the absorbance was read at 620 nm : cat activity was expressed as moles of h2o2 consumed / min / g protein . . 1 ml of supernatant was treated with 500 l of ellman 's reagent ( 19.8 mg of 5,5dithiobisnitrobenzoic acid in 100 ml of 0.1% sodium citrate ) and 3.0 ml of 0.2 m phosphate buffer ( ph 8.0 ) . the activity of glutathione peroxidase ( gpx ) was assayed by the method of rotruck et al . . the reaction mixture containing 0.2 ml of edta ( 0.8 mm , ph 7.0 ) , 0.4 ml of phosphate buffer ( 10 mm ) , and 0.2 ml of tissue homogenate was incubated with 0.1 m of h2o2 and 0.2 ml of glutathione for 10 min . the activity of gpx was expressed as l mol glutathione oxidized / min / g protein . the lipid peroxidation assay was carried out using the modified method of ohkawa et al . . briefly , 300 l of tissue homogenate , 300 l of 8.1% sds ( sodium dodecyl sulphate ) , 500 l of acetic acid / hcl ( ph = 3.4 ) , and tba ( thiobarbituric acid ) were added , and the mixture was incubated at 100c for 1 thereafter , the thiobarbituric acid reactive species ( tbars ) produced was measured at 532 nm and calculated as malondialdehyde ( mda ) equivalent . one - way analysis of variance was used to analyze the results and duncan multiple tests were used for the post hoc analysis . statistical package for social science ( spss ) 10.0 for windows was used for the analysis .", "as evident from table 1 , administration of a single dose of cisplatin ( 7.5 mg / kg bwt ) caused a significant increase in the biomarkers of renal function ( creatinine , urea , and uric acid ) when compared with the control group that received only saline ( 0.85% w / v ) . however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40 mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement of renal function due to a decrease in the creatinine , urea , and uric acid levels when compared with the induced group ( group 2 ) . also , administration of a single i.p dose of cisplatin ( 7.5 mg / kg bwt ) caused a significant decrease in the biomarkers of oxidative stress [ superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) ] when compared with the control group that received only saline ( 0.85% w / v ) ( table 2 ) . however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40 mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement in the body 's antioxidant status by an increase in the activities of superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) when compared with the induced group ( table 2 ) . likewise , there was a significant ( p < 0.05 ) increase in the malondialdehyde ( mda ) content in rat kidney administered a single i.p dose of cisplatin ( 7.5 mg / kg bwt ) . however , both pretreatment with gallic and tannic acids orally at two doses , 20 and 40 mg / kg body weight , respectively , shows a significant ( p < 0.05 ) reduction of mda content in rat kidney when compared with the induced group ( figure 1 ) . \n figure 2 revealed that administration of a single i.p dose of cisplatin ( 7.5 mg / kg bwt ) caused a significant ( p < 0.05 ) increase in arginase activity when compared with the control group that received only saline ( 0.85% w / v ) . however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40 mg / kg body weight , respectively , shows a significant ( p < 0.05 ) decrease in arginase activity when compared with the induced group ( group 2 ) . photomicrographs of kidney sections from various treatment groups are shown in figures 3 , 4 , and 5 . histopathological examination of sections from rat kidney administered a single i.p dose of cisplatin ( 7.5 mg / kg bwt ) shows severe and generalized tubular epithelial cell necrosis associated with diffuse tubular lumina when compared with the control without cisplatin . however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40 mg / kg body weight , respectively , shows a marked improvement on kidney damage .", "about 25% of most commonly used drugs in intensive care units ( icus ) are potentially nephrotoxic and are recognized as considerable health and economic burden worldwide . among them , cisplatin , when used in cancer chemotherapy , induces renal impairment and acute renal failure by induction of reactive oxygen species , tubulointerstitial inflammation , and apoptosis . although various studies have been reported on the benefits of several agents in cisplatin induced renal toxicity , the basis of nephroprotection remains elusive . this makes the search for strategies to prevent nephrotoxicity constitute an active area of investigation . hence , it is reasonable to assume that a reinforcement of antioxidant defense of renal tissue by exogenous antioxidant such as phenolic acids should be a strategy to protect the kidney from the oxidative damage . in the present study , we compare the protective effect of administration of gallic and tannic acids ( two commonly phenolic acids that are structurally related ) of the same dosage against normal and cisplatin induced renal injury in rats for the first time by attenuating renal oxidative stress . the elevations of key kidney function biomarkers such as creatinine , uric acid , and urea have been suggested to be indicative of reduced renal functions [ 30 , 31 ] . thus , estimation of plasma creatinine and uric acid has been employed as key test to assess kidney function [ 30 , 31 ] . in the present study , the observed elevation in plasma creatinine , urea , and uric acid levels in induced rats this finding is consistent with that reported by earlier studies [ 32 , 33 ] . however , the restoration of the plasma creatinine , urea , and uric acid level in rats treated with both gallic and tannic acids ( table 1 ) suggests that both phenolic acids have the ability to prevent kidney damage and protect the kidney against nephrotoxicity . this , however , may be a function of their antioxidant properties and ability to inhibit arginase activity ( figure 2 ) . cisplatin increased arginase activity in the rat 's kidney , while the pretreatment with both gallic and tannic acids at two doses ( 20 and 40 mg / kg bwt ) , respectively , for 7 days resulted in a decrease in kidney arginase activity ( figure 2 ) . arginase is a hydrolytic enzyme responsible for the conversion of l - arginine to l - ornithine and urea . ornithine is an important biosynthetic precursor of polyamines which have been implicated to facilitate cell proliferation in certain cancer cells . another important enzyme , endothelium nitric oxide synthase ( enos ) , competes with arginase for the same substrate , arginine . the no produced plays an important role in both regulating renal hemodynamics and modulating inflammatory and proliferating response to various stimuli . therefore , inhibition of arginase activity slows the progression of renal failure in renal ablation . the increase in the kidney and plasma mda content ( figure 1 ) in the induced rats suggests lipid peroxidation . this agreed with earlier studies where administration of cisplatin caused inflammation and lipid peroxidation [ 6 , 7 ] . this could be as a result of increased hydrogen peroxide concentration produced in the kidney due to the depletion of antioxidant enzymes : superoxide dismutase ( sod ) , reduced glutathione ( gsh ) , and catalase ( cat ) activity ( table 2 ) . this also is consistent with earlier study where depletion of sod , cat , and gpx in rats resulted in increased mda concentration due to lipid peroxidation . the depletion of these antioxidants suggests that cisplatin induced nephrotoxicity could be a result of oxidative stress or suppression of the antioxidant enzymes , as previously reported by earlier studies [ 3537 ] . however , the reduced kidney and plasma mda content ( figure 1 ) and restoration of sod , gsh , and cat activities ( table 2 ) in pretreated rats suggest an improvement in the in vivo antioxidant status , which may be a function of the antioxidant properties of the phenolic acids ( gallic and tannic acids ) therefore , the observed decrease in the kidney gsh level ( table 2 ) in the induced rats suggests cisplatin induced nephrotoxicity , which is associated with a drastic reduction in kidney gsh content . this finding is consistent with earlier studies where gsh depletion was suggested to be due to the interaction of cisplatin with the molecules contain sulfhydryl groups [ 39 , 40 ] . however , restoration of gsh levels in the pretreated rats suggests the antioxidant and nephroprotective properties of the phenolic acids ( table 2 ) . furthermore , histopathology study revealed normal glomerulus and tubules with intact renal architecture in normal ( figure 3(a ) ) , gallic ( figure 5(a ) ) , and tannic acid ( figure 5(b ) ) group without cisplatin injection . degenerated tubular structures with vacuolization and loss of architecture were seen in cisplatin induced group ( figure 3(b ) ) . pretreatment with both gallic and tannic acids at two doses ( 20 and 40 mg / kg bwt ) , respectively , for 7 days resulted in excellent protection against nephrotoxicity induced by cisplatin and showed predominant normal kidney morphology ( figures 4(a ) , 4(b ) , 4(c ) , and 4(d ) ) .", "the results of the present study revealed that oxidative stress and apoptosis / necrosis play an important role in pathogenesis of cisplatin nephrotoxicity . gallic and tannic acids , two important pharmacologically active phenolic compounds , reduced cisplatin induced functional and histological renal damage . furthermore , they suppressed the generation of ros , lipid peroxidation , and oxidative stress in kidney tissues . these results indicated that both gallic and tannic acids exhibit nephroprotective effect and the possible mechanism of action by which they exert this effect could be due to their antioxidant properties and inhibition of arginase activity . however , tannic acid exhibited better nephroprotective potential than gallic acid which may be due to the glycosidation with a glucose moiety ." ]

[ "cavernous haemangioma is the most common primary orbital tumour in adults , accounting for 6% of all orbital lesions [ 13 ] . orbital schwannomas are benign peripheral nerve sheath tumours and constitute 1% of all orbital tumours [ 1 , 3 ] . orbital cavernous haemangiomas and schwannomas present as slow progressive enlargement and the most common presenting sign and symptom is painless proptosis [ 48 ] . although both orbital cavernous haemangiomas and schwannomas share many similar clinical and radiological characteristics [ 58 ] , it is important to distinguish between the tumours because they demonstrate important different features and the treatments of choice for the two are different [ 3 , 79 ] . magnetic resonance imaging , especially dynamic mr imaging , plays a crucial role in the differentiation of orbital cavernous haemangiomas and schwannomas [ 7 , 8 , 1014 ] . the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is considered the most important finding distinguishing the two tumours [ 1014 ] . however , the literature on mr imaging findings in the differentiation of these two tumours is based on small patient populations . consequently , this study aims to assess mr imaging findings distinguishing orbital cavernous haemangiomas from schwannomas and to elaborate on specific differences between the two tumours .", "between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol . mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients . most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus . informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data . magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15 ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images . a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288 256 matrix with two signals acquired were used . dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4 ms , a flip angle of 15 , one excitation , a matrix of 256 160 , a field of view of 220 220 mm , and a section thickness of 3.2 mm at 0 interval . a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2 ml / the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1 mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram . each mr sequence yielded 12 anatomical slices in 13 s ; this was repeated 12 times with an interval between each sequence of 12 s. total imaging time was 288 s ( 4 min 48 s ) . dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) . a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics . we chose the area that demonstrated the greatest degree of early enhancement so that the time intensity curve ( tic ) could be generated . the area of the roi typically was 8 mm . the contrast index ( ci ) was calculated from : ci = [ ( signal intensity post - contrast ) ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) . the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves . magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15 years experience ) . location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed . the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit . on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated . the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . progressive enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig . 1 ) . other patterns of enhancement included gradually heterogeneous enhancement and gradually homogeneous enhancement . \n fig . b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma . b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests . fisher s exact test was performed when the sample size in the subgroups was deemed too small .", "between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol . mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients . most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus . informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data .", "magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15 ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images . a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288 256 matrix with two signals acquired were used . dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4 ms , a flip angle of 15 , one excitation , a matrix of 256 160 , a field of view of 220 220 mm , and a section thickness of 3.2 mm at 0 interval . a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2 ml / s was used . the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1 mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram . each mr sequence yielded 12 anatomical slices in 13 s ; this was repeated 12 times with an interval between each sequence of 12 s. total imaging time was 288 s ( 4 min 48 s ) . dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) . a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics . we chose the area that demonstrated the greatest degree of early enhancement so that the time intensity curve ( tic ) could be generated . the contrast index ( ci ) was calculated from : ci = [ ( signal intensity post - contrast ) ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) . the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves .", "magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15 years experience ) . location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed . the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit . on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated . the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . progressive enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig . 1 ) . b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma . b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead )", "frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests . fisher s exact test was performed when the sample size in the subgroups was deemed too small .", "tables 1 and 2 describe the frequency distribution of non - enhanced mr imaging features ( table 1 ) and contrast - enhanced mr imaging features ( table 2 ) . there was a significant difference between cavernous haemangiomas and schwannomas regarding the location ( including anterior orbit , posterior orbit , or both anterior and posterior orbit ; extraconal or intraconal space ) , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of the contrast enhancement , enhancement pattern and type of time intensity curve ( tic ) ( p < 0.05 ) ( figs . 1 , 2 , 3 ) . \n b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) . f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead)fig . a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle . b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle . c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead)table 1frequency distribution of non - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . of patients594316anterior or posterior orbit0.004 anterior4779.63990.8850 posterior610.224.6425 both610.224.6425extraconal or intraconal space0.049 intraconal4474.63581.4956.2 extraconal1525.4818.6743.8nasal or temporal orbit0.250 nasal2644.11739.5956.2 temporal3355.92660.5743.8superior or inferior orbit0.937 superior2949.22148.8850 inferior3050.82251.2850configuration0.015 round610.249.3212.6 ovoid5084.73990.71168.7 irregular35.100318.7margin0.531 smooth4474.63376.71168.7 lobulated1525.41023.3531.3t1 signal intensity<0.001 hypointense711.900743.7 isointense5288.143100956.3t1 homogeneity<0.001 homogeneous5084.743100743.7 heterogeneous915.300956.3t2 signal intensity0.001 isointense58.500531.3 hyperintense5491.5431001168.7t2 homogeneity<0.001 homogeneous467843100318.7 heterogeneous1322001381.3signal intensity was compared with that of extraocular muscletable 2frequency distribution of contrast - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . of patients594316spread pattern of contrast enhancement<0.001 starting from a small point or portion4372.94310000 starting from a wide area1627.10016100enhancement pattern<0.001 homogeneous or heterogeneous1627.10016100 progressive4372.94310000ci1.8 0.61.4 0.30.003tic type<0.001 persistent1220.337956.2 plateau - shaped2440.71944.2531.3 wash - out23392148.8212.50 a 42-year - old man with a schwannoma . b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) . c e dynamic mr images obtained at 25 , 75 and 225 s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) . f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead ) a 47-year - old woman with schwannoma . a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle . b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle . c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead ) frequency distribution of non - enhanced mr imaging features signal intensity was compared with that of extraocular muscle frequency distribution of contrast - enhanced mr imaging features orbital cavernous haemangiomas were always a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging . schwannomas often demonstrated a heterogeneously isointense or mildly hyperintense mass in the intraconal or extraconal space . after gadolinium administration , all 43 cavernous haemangiomas showed enhancement starting from a small point or portion and contrast media filling up the tumour later , i.e. progressive enhancement ( fig . 1 ) . compared with the cavernous haemangiomas , all 16 schwannomas showed enhancement starting from a wide area and heterogeneous or homogeneous enhancement later ( fig . 2 ) .", "orbital cavernous haemangiomas , the most common primary orbital tumour in adults , demonstrate a predilection to affect middle - aged women ( 6070% ) , with a mean age of 43 to 48 years and range of 18 to 72 years based on several large series [ 4 , 7 , 9 ] . the most common presenting sign and symptom of the orbital cavernous haemangioma is painless proptosis . in the past , orbital cavernous haemangiomas were invariably resected because they could not be conclusively discerned from other tumours clinically or radiologically . however , orbital cavernous haemangiomas have recently been classified as type 3 low - flow arteriovenous malformations with a direct arterial in - flow and venous out - flow mechanism ( arterial low - flow type ) rather than as true neoplasms or proliferating hamartomas , and , to our knowledge , there is no evidence of malignant transformation in the literature . moreover , ct and mr imaging , especially mr imaging , have evolved to deliver high sensitivity and specificity in diagnosing orbital cavernous haemangiomas and distinguishing cavernous haemangiomas from other tumours , which have played a vital role in accurate diagnosis and correct treatment planning [ 5 , 7 , 12 , 14 , 16 ] . therefore , recent literature supports observation as an alternative to surgical excision for asymptomatic orbital cavernous haemangiomas and surgery can be withheld until vision is threatened or cosmetic appearance resulting from proptosis is unacceptable [ 3 , 9 ] . orbital schwannomas , constituting 1% of all orbital tumours , are benign peripheral nerve sheath tumours originating purely from schwann cells and usually seen in young to middle - aged adults and rarely in children . clinical findings of orbital schwannomas are generally similar to those of cavernous haemangiomas , but they are somewhat more likely to exhibit progressive growth and symptoms . thus , surgical excision is the treatment of choice for orbital schwannomas , different from orbital cavernous haemangiomas [ 3 , 79 ] . our results demonstrate that , in presence of a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging , one small point or portion of enhancement initially noted after administration of contrast medium and progressive enhancement on later enhancement images , favours the diagnosis of cavernous haemangioma instead of schwannoma . most orbital cavernous haemangiomas are identified in the intraconal space , predominantly in the lateral aspect of the intraconal space [ 7 , 9 ] . in our series , only 8 of the 43 orbital cavernous haemangiomas were in the extraconal space . however , orbital schwannomas more commonly arise from sensory nerves in the orbit , specifically the supraorbital , supratrochlear and lacrimal nerves , and thus are usually outside the orbital muscle cone . the nasociliary nerve is the only division of the ophthalmic nerve within the annular tendon . therefore , orbital schwannomas can be seen as an intraconal or extraconal mass . in our series , the frequency of the schwannoma being located in the extraconal or intraconal space is almost the same . orbital cavernous haemangiomas appear isointense to muscle on t1-weighted imaging and hyperintense to muscle on t2-weighted imaging [ 5 , 7 ] . although non - contrast mr imaging findings are non - specific , they may offer some advantage in the differentiation of orbital cavernous haemangiomas by demonstrating marked hyperintensity signal to muscle with internal septations on t2-weighted imaging , a hypointense circumferential rim corresponding to the fibrous pseudocapsule , and chemical shift artefact in the frequency - encoded direction secondary to high water content within the lesion surrounded by orbital fat [ 5 , 7 , 17 ] . orbital schwannoma is isointense with respect to the extraocular muscle on t1-weighted images and mildly hyperintense on t2-weighted images [ 6 , 18 ] . orbital schwannomas may undergo cavitary change , which appears as a cystic mass with straw - coloured fluid on gross pathological examination and looks homogeneously hypointense to muscle on t1-weighted images and hyperintense on t2-weighted images [ 8 , 19 ] . consequently , markedly homogeneous hyperintensity signal on t2-weighted imaging is one of the reliable findings distinguishing the two tumours . variable degrees of contrast enhancement observed in orbital cavernous haemangiomas were reported , probably related to variable imaging times after contrast medium infusion . however , multiphase dynamic contrast ct and mri demonstrated virtually pathognomonic imaging features of orbital cavernous haemangiomas [ 5 , 7 , 17 ] . on early imaging after the injection , one small point or portion of enhancement is initially noted because of its low - flow arterial supply , and the slow progressive accumulation of contrast medium continues within the dilated vascular spaces filling in centrally during the late venous phase . subsequently , homogeneous contrast enhancement occurs and persists in the delayed equilibrium phases [ 7 , 1216 ] . this enhancement pattern is referred to as a progressive enhancement pattern in the literature [ 10 , 11 , 13 ] . several multiphase dynamic contrast mr imaging protocols have been reported for studying orbital cavernous haemangiomas [ 7 , 11 , 12 , 14 ] . compared with orbital cavernous haemangiomas , no progressive enhancement pattern was found in orbital schwannomas [ 12 , 18 ] . therefore , our results and the literature showed that the progressive enhancement pattern will be the most reliable finding in distinguishing orbital cavernous haemangiomas from schwannomas . some reports in the literature indicated that orbital cavernous haemangiomas showed an initial central point or portion enhancement after administration of gadopentetate dimeglumine [ 7 , 1214 ] . however , our results show that enhancement starts from one peripheral point or portion of the tumour in most orbital cavernous haemangiomas . the initial enhancement point represents the connecting point of feeding vessels to the lesion , which is useful for estimating the connecting point of the feeding vessels . an unexpected result is that nearly half of orbital cavernous haemangiomas demonstrate a wash - out tic type frequently seen in the malignant tumour rather than a plateau - shaped or persistent tic type . this may be because contrast medium fills one portion of the tumour during an early phase after administration of contrast medium and contrast medium diffuses into adjacent spaces through the foramen of the fibrous interstitium between spaces at a later phase [ 1214 ] . thus it results in a decrease in concentration of contrast media in the portion of the tumour filled by contrast media during the early phase . there is a significant difference in tic type between orbital cavernous haemangiomas and schwannomas in contrast to a previous report that there is no significant difference in tic type between the two tumours . this is probably because we chose an roi covering the greatest degree of early enhancement rather than the whole tumour . recent literature on dynamic mr imaging in the breast , soft tissue and orbital masses indicates that an roi covering the greatest degree of early enhancement of the mass reflects haemodynamic alterations of the tumour better than an roi covering the whole tumour [ 10 , 21 , 22 ] . the multiphase dynamic contrast mr imaging findings of orbital cavernous haemangiomas have been described in the literature [ 1014 ] . however , our results provided additional information on dynamic contrast - enhanced mr imaging findings of orbital cavernous haemangiomas . first , short imaging time and interval of every phase on dynamic contrast mr imaging may accurately show one point or small portion of enhancement of the mass on early phase images after the injection of contrast medium and allow characterization of specific spread pattern of the contrast enhancement in small orbital cavernous haemangiomas , which can differentiate small orbital cavernous haemangiomas from small schwannomas and other tumours . second , tic type of orbital cavernous haemangioma is significantly different from that of schwannoma . previous literature showed small haemangiomas could not be differentiated from haemangiopericytomas as their enhancement appears early and strongly and calcification could be noticed in cavernous haemangiomas , contributing to the differential diagnosis [ 5 , 7 ] . however , calcification is occasionally seen in cavernous haemangiomas and plays a limited role in the differential diagnosis . our results revealed that strong enhancement starting from a wide area and rapid wash - out on dce mr imaging were seen in haemangiopericytomas that might be helpful in the differential diagnosis . capillary haemangiomas are a different sort of haemangiomatous tumour that are more common in children , often regress and have a different imaging appearance [ 5 , 7 ] .", "cavernous haemangiomas and schwannomas have different mr imaging features including the location , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of contrast enhancement , the enhancement pattern and tic type , which could be helpful in the differentiation of the two tumours . the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is the most reliable finding distinguishing cavernous haemangiomas and schwannomas . markedly homogeneous hyperintensity signal on t2-weighted imaging is another reliable finding distinguishing the two tumours . mr imaging , especially dynamic contrast - enhanced mr imaging , plays a vital role in the differential diagnosis of cavernous haemangiomas and schwannomas ." ]

[ "gingival enlargement is a common observation in clinical practice that may occur as a result of a response to varied stimuli and/or interactions with the host and the environment . these lesions could be either localized to certain aspect of the oral cavity or generalized affecting larger areas . the possible reasons for this condition may be related to plaque , hormonal imbalances , or systemic - induced manifestation . these excessive gingival distensions may adversely affect speech , mastication , tooth eruption , esthetics along with major hindrance to the maintenance of routine oral hygiene . such cases should be treated in a methodical manner , involving a detailed medical history followed by conventional nonsurgical therapy . however , in order to maintain successful therapeutic outcome , it is crucial to create patient awareness and motivation , along with a timely recall visits . this case series describes three different conditions of localized gingival overgrowths and its management with prime emphasis on the importance of patient compliance .", "neoplasms are characterized by progressive autonomous growth that can be either a benign or a malignant course , whereas nonneoplastic lesions are usually inflammatory or represent a reaction to some kind of irritation or low - grade injury . most of these lesions have similar clinical findings such as sessile or pedunculated nodule located at the interdental papilla with color variations from pale pink to erythematous . pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females . it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation . the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year . on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history . clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm 15 mm with an interproximal lingual extension [ figure 1a and b ] . the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . in general , early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network . there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity . an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 . hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned . a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation . oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr . hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval , . a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested . hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence . the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days . case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification . the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion . the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg . there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] . the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg the patient is still on follow - up since 6 months with no evidence of recurrence . however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias . peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about females are more commonly affected , and anterior maxilla is the most prevalent location of involvement . the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations . following the elimination of causative factors , excision of the lesion is the choice of treatment . the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back . intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] . a tentative diagnosis of fibroma was made and patient was informed about the treatment plan . the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr . india ) was advised twice daily so as to prevent further disease progression and was recalled after a week . the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas , stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] . thus , based on clinical and histopathological findings , the final diagnosis was determined as pof . case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period . however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] . gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group . it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers , gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months . the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene . the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years . intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] . the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations . a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm 15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] . case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits . she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker . a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling . the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] . the sessile growth revealed a reduction in size to approximately 7 mm 9 mm [ figure 3d ] . differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma . hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis . the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] . the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement . the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] . connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg . thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg . previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge .", "pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females . it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation . the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year . on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history . clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm 15 mm with an interproximal lingual extension [ figure 1a and b ] . the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . in general , early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network . there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity . an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 . hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned . a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation . oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr . hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval , . a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested . hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence . the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days . case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification . the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion . the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg . there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] . the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg the patient is still on follow - up since 6 months with no evidence of recurrence . however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias .", "peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about females are more commonly affected , and anterior maxilla is the most prevalent location of involvement . the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations . following the elimination of causative factors , excision of the lesion is the choice of treatment . the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back . intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] . a tentative diagnosis of fibroma was made and patient was informed about the treatment plan . the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr . india ) was advised twice daily so as to prevent further disease progression and was recalled after a week . the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas , stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] . thus , based on clinical and histopathological findings , the final diagnosis was determined as pof . case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period . however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] .", "gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group . it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers , gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months . the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene . the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years . intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] . the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations . a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm 15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] . case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . a provisional diagnosis of combined gingival enlargement patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits . she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker . a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling . the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] . the sessile growth revealed a reduction in size to approximately 7 mm 9 mm [ figure 3d ] . differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma . hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis . the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] . the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement . the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] . connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg . thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg . previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge .", "it is possible to misdiagnose reactive lesions arising from the gingiva . a detailed case history to determine the etiology and histopathological examination is essential for an accurate diagnosis and for proper management . clinical differential diagnosis for localized gingival overgrowths includes fibroma , peripheral giant cell granuloma , pg , peripheral odontogenic fibroma , and peripheral ossifying fibroma . a relatively healthy oral environment provided by the dentist and maintained by the patient will reduce local microflora that will help in eliminating the major focus of infection . a critical factor that assists successful therapeutic outcome the patient should be placed on a regular dental visit , in which a complete description of the existing dental condition should be informed . creating awareness and educating patients regarding the influence of poor oral hygiene and habits to these gingival lesions are essential to avoid further recurrence .", "", "" ]

[ "successful management and treatment of periodontal disease depends upon simple medical procedures such as health education , prophylaxis , scaling and root - planing , gingivectomy , gingival grafts , and different types of gingival flaps . periodontal pocket refers to the pathological increase in the depth of gingival sulcus , and is one of the main clinical features of periodontal disease . elimination of pathological changes of the pocket wall is among the objectives of surgical treatment for periodontal pockets , so that a stable and sustainable situation can be achieved . currently , several flap techniques have been applied for the treatment of pocket , among which modified widman flap is worth mentioning . this technique facilitates the use of equipment , in which pocket lining is removed with no effect on reducing the pocket depth . dental treatments are accompanied by patients hemodynamic changes ; for instance , increase in blood pressure and heart rate during dental treatment are influenced by various factors such as physical and physiological stress , painful stimuli , and activity of catecholamines present in local anesthetic solutions . heart rate , which is indicative of ventricular contraction , changes in different conditions , including pain , anxiety , stress , heart diseases , metabolic and endocrine disorders and heart muscle diseases , and is evaluated by peripheral pulse measurement or the electrocardiogram and pulse oximetry . for the measurement of blood pressure , different methods are applied , from which riva - rocci , korotokoff , dinamap , and finometer techniques can be enumerated . due to the risk of hypoxia in oral surgeries , pulse oximetry pulse oximeter is a reliable and sensitive apparatus in detecting small amounts of blood oxygen saturation , and in all patients , spo2 ( spo2 ) normal percentage is not less than 95% . a dental patient is exposed to stressors , such as physiological responses to emotional factors and/or pain . pain and anxiety , are important stimuli for the secretion of endogenous adrenaline , which plays a significant role in cardiovascular responses during dental treatment . local anesthetics mainly affect a limited area ; however , they will be absorbed from the injection site and exert general effects , especially on cardiovascular or central nervous system . it is obvious that more the concentration of epinephrine , the better the control of bleeding , but there will be more cardiovascular changes . in spite of the effect of local anesthetics on vital signs , their accurate application is able to dramatically reduce stress . given the above bodies of evidence and the importance of monitoring patients hemodynamic changes during oral surgery , the present study has been conducted to investigate these alterations during periodontal surgery outside the operating room with local anesthesia .", "this practical clinical trial study was carried out on patients with gingival disease referred to the clinic of dental faculty of babol university of medical sciences , and underwent modified widman flap surgery on anterior maxillary region in periodontology department . the equipments used in the study included ( 1 ) finger pulse oximeter ( soor afarinesh company ) , and ( 2 ) automatic sphygmomanometer ( omron company ) according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation . it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study . spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery . \n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period . data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level .", "according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation . it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study . spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery .", "\n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period .", "data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level .", "the present research has been implemented on 50 patients referring to periodontology department of dental faculty for periodontal surgery . among the study participants with the mean age of 42.58 7.14 years , ranging from 30 to 55 years old , there were 22 men and 28 women . in all cases , systolic and diastolic blood pressure , heart rate , and spo2 were evaluated during five stages ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation . the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied , there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] . comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage . statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] . comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage . significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] . comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] . comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery", "the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied , there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] . comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery", "the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage . statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] . comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery", "according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage . significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] . comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery", "similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] . comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery", "hemodynamic changes during dental treatment have long been one of the main concerns among practitioners and researchers . increase in blood pressure and heart rate during surgery is inevitable ; however , the effect of sympathetic nervous system is not well - identified on this augmentation . since the use of local anesthesia does not merely contribute to increased concentration of plasma noradrenaline without dental treatment , blood pressure response appears to be depending on dental treatment process itself . as mentioned earlier , elevated blood pressure during oral surgery is attributed to the sympathetic nervous system . due to possessing vasoconstrictors and anesthetic components , adrenaline is used as a vasoconstrictor along with lidocaine to increase the amount and duration of anesthesia . moreover , many researchers have pointed to changes probably effective on normal patients physiological stability . sedative drugs and local anesthetics can be used to control the autonomic nervous system responses and help to reduce the cardiovascular alterations . in addition , different people react differently to anxiety and pain , which could be significantly influential on their hemodynamic alterations . in the present study , blood pressure and heart rate were remarkably increased after injection of anesthesia ( p < 0.05 ) . according to figures 1 , 2 , and 3 , a meaningful difference has been found in the second step and following the anesthetic injection containing lidocaine and epinephrine ( 1:80000 ) ( p < 0.0001 ) in systolic and diastolic blood pressure and heart rate , on which the result was almost predictable . this can be associated with hemodynamic effects of adrenaline , as well as alpha - adrenergic effects to peripheral vasoconstriction and beta - adrenergic effects to increased cardiac contraction . furthermore , patient 's physiological stress before surgery and also painful stimulus following injection might lead to increased sympathetic activity and subsequently blood pressure and heart rate . thus , application of anxiety - reducing protocols such as psychological sedatives during treatment and minimizing patient 's waiting time can greatly control endogenous secretion of adrenaline . the study figures present a reduction in systolic and diastolic blood pressure following the second step and during the third , fourth , and fifth steps , indicating the elimination of stimulatory effect of adrenaline on blood pressure and heart rate and showing insignificant impact of other surgical steps on vital signs in cases of no anesthetics injection . furthermore , over time and acceptance of treatment processes by patient , the initial anxiety will be diminished and the person will reach to a relatively stable cardiovascular balance . since the peak effects of local anesthetics and alpha and beta epinephrine impact elimination occur in the third and the fourth steps , decrement in heart rate and blood pressure seems to be obvious in these stages . according to figure 2 , diastolic blood pressure has been slightly elevated in the fifth step , after stitching at the end of the operation , which is probably due to feeling of pain by patient and decrease in the effect of anesthetic . in a study by matsumura et al . investigating changes in blood pressure and heart rate during dental surgery , the results revealed an increase in both parameters after anesthesia with lidocaine ( 2% ) and epinephrine ( 1:80000 ) , which is compatible to findings of the present research . in faraco survey on the effect of anesthetics containing lidocaine and epinephrine on cardiovascular changes during dental implant surgery , blood pressure was evaluated using the automated noninvasive oscillometric technique during ten steps ; the results showed no significant difference in blood pressure and heart rate variability , which is not in accordance to the findings achieved in this research . in another study by gedik et al . on blood pressure , heart rate and temperature variability during periodontal surgery , patients were divided into four groups according to types of surgery , and blood pressure and heart rate were measured after anesthetic injection and the end of the operation ; blood pressure and heart rate were decreased in patients undergoing gingivectomy and periodontal flap , which is in consistence with the results obtained in the present research . however , no changes in heart rate and blood pressure have been observed following frenectomy and curettage , indicating that the difficulty in treatment process is an effective factor in blood pressure . as shown in figure 4 , no statistically significant difference has been found in the mean spo2 , measured by pulse oximetry , in different stages ( p > 0.05 ) . in other words , conventional periodontal surgery with technical and safety observations in an investigation by kaviani and birang , nausea , vomiting , decreased or increased blood pressure and other hypoxia complications following the use of nitrous oxide inhalation sedation during periodontal treatment have only been observed in 1 out of 32 cases , which was probably due to patient restlessness and movement during treatment . have addressed to pulse oximetry during oral surgery with and without intravenous sedation and found out a significant reduction in arterial blood oxygen levels in the group receiving midazolam . this study illustrates remarkable sedative effect of midazolam on anxious patient prior to the surgery ; nonetheless , this effect does not sustain during the operation . as mentioned , findings of various studies in terms of hemodynamic changes following dental treatments are contradictory , and such discrepancies could be related to different research approaches and different local anesthetics applied .", "different surgical stages will be accompanied by diverse effects on hemodynamic variability and pulse oximetry . according to the present study , blood pressure and heart rate are significantly increased after anesthetic injection and are decreased in the following stages by the elimination of adrenaline stimulatory effect . moreover , no significant alteration has been observed in spo2 during the operational steps in this research ." ]

[ "chronic kidney disease ( ckd ) is defined as a structural or functional kidney abnormality lasting for 3 or more months . the global prevalence of ckd is estimated to be more than 10% , and ckd has emerged as a public health problem . adverse outcomes of ckd such as kidney failure , cardiovascular disease ( cvd ) , and premature death can be prevented or delayed when treatment is initiated in the early stages of disease . as the earlier stages are often asymptomatic , ckd is usually detected during laboratory evaluation of comorbid conditions . glomerular filtration rate ( gfr ) is considered the index of kidney function , and for convenience is usually estimated using equations instead of by direct measurement . the modification of diet in renal disease ( mdrd ) study equation , which was replaced by the chronic kidney disease epidemiology collaboration 2009 equations ( ckd - epi equations ) , has been used to estimate gfr . however , this tended to underestimate the measured gfr and had less precision , especially at a gfr of 60 ml / min/1.73 m or more . the ckd - epi equations were reported as having less bias , improved precision , and greater accuracy than the mdrd study equation at a higher gfr of 60 ml / min/1.73 m or more . the ckd - epi equations led to a lower estimation of ckd prevalence , primarily due to a lower prevalence of gfr stage 3 , compared to the mdrd study equation . persistently increased protein excretion has usually been considered to be a marker of kidney damage , and screening with urine dipsticks has been acceptable for detecting proteinuria . recently , urinary albumin measurement was recommended as a marker of kidney damage , both for its standardization and also because albumin is the most important protein lost in the urine in most ckd . the albumin - to - creatinine ratio ( acr ) is the most preferred value among urinary albumin measurements . kidney disease : improving global outcomes ( kdigo ) has updated guidelines for defining , diagnosing , staging , and managing ckd since the establishment of the first uniform definitions and staging system for ckd in 2002 . more objective criteria , such as the ckd - epi equations for estimating gfr and classification of albuminuria , have been incorporated . moreover , the etiology of ckd was included in the definition ; hypertension and diabetes mellitus ( dm ) are systemic diseases known to cause ckd . consequently , kdigo guidelines provide relatively simple and precise methods for early diagnosis of ckd , and tend to yield fewer false positives , thereby avoiding incorrect diagnosis . the number of patients treated with renal replacement therapy ( rrt ) including dialysis and transplantation has increased dramatically in korea , from 28,046 in 2000 to 75,042 in 2013 . the prevalence of rrt was 1,446 patients per million population ( pmp ) , with an incidence of 234 pmp in 2013 . the cause of kidney failure was estimated to be dm in 48.0% , hypertension in 19.7% , and chronic glomerulonephritis in 8.3% of cases . the previously reported ckd prevalence calculated from the mdrd study equation the ckd prevalence calculated from the mdrd study equation alone was 8.8% in 2005 and 7.2% in 2007 in korea national health and nutrition examination survey ( knhanes ) subjects aged 20 ; the prevalence calculated using the mdrd study equation with the dipstick proteinuria method has tended to decrease since 2001 in men and since 2005 in women among knhanes i to iv ( 1998 to 2009 ) subjects aged 20 . since there are no reports on ckd prevalence using the ckd - epi equations to estimate gfr and acr to measure albuminuria for koreans , a new analysis of ckd prevalence is necessary . this study was therefore designed to investigate the prevalence of ckd in the korean population using knhanes samples , based on the definition and classification by kdigo 2012 guidelines , as well as the impact of changes through a comparison of the ckd - epi and mdrd study equations for the estimation of gfr , and the associations between ckd and major risk factors , including dm , hypertension , age , and gender .", "this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 . knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries . knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) . the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians . knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 . the inclusion criteria were ( 1 ) adults aged 19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results . individuals aged 19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 . the health examination was performed in a mobile health examination center . was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association . three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants . body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m . blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . a random urine sample ( first urine in the morning , if possible ) was obtained in a conical container . the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) . serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) . glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) . urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) . low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl . ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr . gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine . individuals with gfr < 60 ml / min/1.73 m or acr 30 mg / g were classified as ckd patients . hypertension was defined as having a systolic blood pressure ( sbp ) of 140 mmhg or a diastolic blood pressure ( dbp ) of 90 mmhg , or having received a physician s diagnosis . dm was defined as having a1c 6.5% , fbg of 126 mg / dl , or having received a physician s diagnosis . dyslipidemia was defined as having total cholesterol 240 mg / dl , ldl - c 160 mg / dl , hdl - c < 40 mg / dl , triglycerides 200 mg / dl , or having received a physician s diagnosis . analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations . the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias . in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design . the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables . the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables . the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage . a subpopulation was added to strata when performing the complex samples chi - square test . associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses .", "this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 . knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries . knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) . the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians . knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 . the inclusion criteria were ( 1 ) adults aged 19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results . individuals aged 19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 .", "blood pressure ( bp ) was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association . three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants . body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m . blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . a random urine sample ( first urine in the morning , if possible ) the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) . serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) . glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) . urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) . low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl .", "ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr . gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine . individuals with gfr < 60 ml / min/1.73 m or acr 30 mg / g were classified as ckd patients . hypertension was defined as having a systolic blood pressure ( sbp ) of 140 mmhg or a diastolic blood pressure ( dbp ) of 90 mmhg , or having received a physician s diagnosis . dm was defined as having a1c 6.5% , fbg of 126 mg / dl , or having received a physician s diagnosis . dyslipidemia was defined as having total cholesterol 240 mg / dl , ldl - c 160 mg / dl , hdl - c < 40 mg / dl , triglycerides 200 mg / dl , or having received a physician s diagnosis .", "analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations . the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias . in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design . the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables . the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables . the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage . a subpopulation was added to strata when performing the complex samples chi - square test . associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses . all statistical analyses were performed using ibm spss version 21.0 ( ibm corp . , armonk , ny , usa ) . the statistical significance level was set at p < 0.05 .", "among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig . the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively , the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) . the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) . mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) . mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) . mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) . mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) . mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively . the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) . the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively . the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 . the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively . the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr 30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) . the prevalence of acr 30 mg / g in individuals with gfr 60 ml / min/1.73 the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig . the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively . the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) . the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas or proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively . some interactions between demographic covariates , such as gender , age , and bmi , were present . the risk of bmi 25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) . the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and 68 . the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi 25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively . in addition , the composition of men significantly decreased in the 49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied . the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr 30 mg / g , women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) . the ors of gender to ckd , acr 30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p < 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively . the prevalence of bmi 25 , ckd , gfr < 60 ml / min/1.73 m , acr 30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 . the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr 30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 . hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) . the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and 3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women . diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) . the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and 2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women . dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) . increasing age and obesity ( bmi 25 ) showed a positive correlation with ckd .", "among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig . the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively , the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) . the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) . mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) . mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) . mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) . mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) . mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively .", "the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) . the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively . the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 . the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively . the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr 30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) . the prevalence of acr 30 mg / g in individuals with gfr 60 ml / min/1.73", "the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig . 2 . the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively . the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) .", "the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas or proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively .", "some interactions between demographic covariates , such as gender , age , and bmi , were present . the risk of bmi 25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) . the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and 68 . the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi 25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively . in addition , the composition of men significantly decreased in the 49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied . the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr 30 mg / g , women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) . the ors of gender to ckd , acr 30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p < 0.001 ) , 1.416 ( 95% ci , 1.193 to 1.682 ; p < 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively . the prevalence of bmi 25 , ckd , gfr < 60 ml / min/1.73 m , acr 30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 . the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr 30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 . hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) . the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and 3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women . diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) . the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and 2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women . dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) . increasing age and obesity ( bmi 25 ) showed a positive correlation with ckd .", "this study is the first to report ckd prevalence using gfr estimated by using ckd - epi equations together with acr for albuminuria in the korean population . the prevalence of ckd was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged 19 , and the ckd risk prognosis was reported in table 2 . the ckd risk prognosis from moderately increased to very high reported in this study was lower than that in the usa . gfr and albuminuria are independent and complementary predictors of important clinical outcomes , including ckd progression , end - stage renal disease , acute kidney injury , cardiovascular mortality , and all - cause mortality [ 16 - 19 ] . the frequencies of gfr < 60 ml / min/1.73 m showed sharp increases starting from around age 50 for men and 60 for women , whereas those of acr 30 mg / g increased relatively gradually , starting from earlier ages , as shown in table 4 and 5 . the rate of decline of measured gfr was reported to double in kidney transplantation donors aged > 45 years . the prevalence of estimated gfr < 60 ml / min/1.73 m using ckd - epi equations and the mdrd study equation was 2.2% and 2.0% in this study , but 2.6% and 3.2% , respectively in the previous study . the statistical method used in the current study was complex sample analysis incorporating sampling weight , in contrast to the previous study which did not consider weight in the analysis . the frequencies of gfr stage g1 and g2 by ckd - epi equations were 67.7% and 29.8% , whereas those by the mdrd study equation were 59.4% and 38.7% , respectively , in this study . these numbers are comparable to the previous study , considering the different analytical methods ; the frequencies of gfr stage g1 and g2 by ckd - epi equations were 64.5% and 28.9% , whereas those by the mdrd study equation were 47.6% and 49.2% , respectively . these results confirm the tendency to underestimate gfr by the mdrd study equation when gfr is 60 ml / min/1.73 m or greater . the previous report of lower prevalence of gfr stage g3 using ckd - epi equations than with the mdrd study equation was not observed in this study . among the population of the previous study , the frequencies of g3 by ckd - epi equations and the mdrd study equation ( 33.2% and 37.2% , respectively ) were far greater than those among the korean population ( 2.1% and 1.9% , respectively ) . the prevalence of acr 30 mg / g in the entire study population , and acr 30 mg / g in individuals with gfr 60 ml / min/1.73 the prevalence of acr 30 mg / g in the entire study population and acr 30 mg / g in individuals with gfr 60 ml / min/1.73 m aged 35 years was 10.2% and 8.7% , respectively , in the previous korean population . besides the different age criteria , the populations of the previous and current study are quite different ; the previous study was designed by the researcher group considering age , gender , and city factors not using the knhanes sample data . although the absolute values are different , the important point in common is that a large number of people have albuminuria before gfr decreases to < 60 ml / min/1.73 m ( 85.3% and 87.7% of the albuminuria cases did not have decreased gfr in the previous and current study , respectively ) . as shown in table 3 , test strips did not detect albuminuria in 81.5% of cases . at present , laboratory tests for albuminuria are not routinely performed during regular physical examinations in korea . the prevalence of ckd in men and women was 6.9% and 9.1% , respectively ( p < 0.001 ) . besides gender itself , age distribution , frequency of obesity ( bmi 25 ) , and concurrent chronic diseases , including dm , hypertension , and dyslipidemia , were different according to gender . thus , the risk of ckd is higher in women than men ( or , 1.354 ; p < 0.001 ) , and the difference seems to be caused by the susceptibility to albuminuria ( or , 1.416 ; p < 0.001 ) rather than decreased gfr ( or , 1.057 ; p = 0.683 ) . when gfr was estimated using the mdrd study equation , the increased risks of gfr < 60 ml / min/1.73 m in women were observed in previous studies : or , 3.16 ( p < 0.001 ) using knhanes iii and iv-1 ; frequencies of 3.4% , 9.7% , 10.2% , and 4.6% in women , and 1.0% , 5.4% , 3.1% , and 2.6% were seen in men through knhanes i - iv . there seemed to be no difference in acr according to gender in the previous study ( 10.1% and 10.3% in women and men , respectively ) . a similar observation was reported for proteinuria by gender ( frequencies of 3.3% , 2.3% , 1.4% , and 1.9% in women , and 3.1% , 3.0% , 2.8% , and 2.2% in men through knhanes direct comparison is difficult due to the difference in the designs of study populations and laboratory test methods using dipstick proteinuria and acr . increasing age and obesity ( bmi 25 ) correlated significantly with ckd , as shown in table 6 , which agreed with previous studies [ 11 - 13 ] . hypertension and dm are both major risk factors and consequences of ckd [ 21 - 23 ] . concurrent chronic diseases such as dm and hypertension increased the risk of ckd consistently [ 11 - 13 ] . diabetic patients are susceptible to glomerular disease and hypertensive patients to renal vascular disease . since the prevalence of dm was higher in men than women , with the opposite for prevalence of ckd , the or of dm to ckd in men was higher than women , as shown in table 6 . moreover , dm was correlated with increased acr rather than decreased gfr in this study . since increased acr appeared at an earlier age in women than men , and decreased gfr was preceded by increased acr , the difference between ors to gfr < 60 ml / min/1.73 m and acr 30 mg / g in hypertensive women was greater than hypertensive men . on the other hand , limited availability of direct ldl - c measurement , unstandardized laboratory assay methods for hdl - c , and lack of a uniform definition of dyslipidemia between the studies made it difficult to evaluate their associations . the major limitation of this study is the lack of the evaluation for chronicity assessment and use of single measurements for laboratory tests due to the cross - sectional data characteristics . according to the definition of ckd by the kdigo guideline the rationale for defining chronicity is to differentiate ckd from acute kidney diseases , which may require different interventions and have different etiologies and outcomes . in addition , there is still the discrepancy between measured and estimated gfr using ckd - epi equations . gfr estimation using equations is convenient , but measurement of gfr should be considered to diagnose a specific patient . finally , association analysis could not provide conclusive causal relationships due to the study characteristics . however , the representative features of the knhanes sample data can not be overlooked . in conclusion , the prevalence of ckd using estimated gfr by the ckd - epi equation and acr for albuminuria measurement was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged 19 . the prevalence of albuminuria ( acr 30 mg / g ) before decline in gfr ( gfr 60 ml / min/1.73 m ) was 5.7% . despite kdigo guidelines to prevent or delay progression of ckd in its early stages , implementation remains difficult due to practical limitations . the failure to recognize ckd in its early stage may result in complications such as premature rrt . thus , urinalysis to detect albumin should be strongly recommended for patients with higher risk of ckd and cvd . as there is a consensus about the risk of ckd with dm and hypertension , regular albuminuria testing for at least these patients to detect kidney disease at an early stage", "1 . the prevalence of chronic kidney disease ( ckd ) was estimated to be 7.9% in the korea national health and nutrition examination survey samples of 2011 and 2012 in korean adults aged 19 . early identification of ckd paired with appropriate management and earlier referral to specialty kidney services results in economic and clinical benefits . the majority of ckd patients have albuminuria prior to a decrease in glomerular filtration rate . 4 . regular laboratory tests for albuminuria in the high - risk group , especially for hypertensive or diabetes mellitus patients , should contribute to early detection of ckd ." ]

[ "while it is true that not all patients are able to dialyse themselves at home , it is a sad fact that many are never given the chance , despite the fact that patient choice has never been more discussed as an answer to almost any health care problem . although studies suggest that renal physicians and nurses regard home dialysis as the best treatment , it is in decline in most european countries , yet in - centre hospital - based dialysis is the most expensive and most disempowering option available . the practical barriers to home therapy vary from unit to unit but need to be identified and overcome if patients are to benefit from self - management . however , it can be difficult to convince both staff and patients if they have no direct experience or training in this aspect of renal replacement therapy . patient pathway to ( relative ) freedom a step which can be made much easier if staff are familiar with the stages of change model and have a basic understanding of motivational interviewing .", "pre - emptive living donor transplantation should always be promoted as the first - line treatment for kidney failure . however , where that is not possible , patients must receive timely , adequate and unbiased information and advice regarding the complete array of dialysis options available , including home - based peritoneal dialysis ( pd ) and haemodialysis ( hd ) . interestingly , a comparison of survival in canadian patients treated with nocturnal hd or deceased donor kidney transplantation showed no difference between the two treatments , suggesting that this intensive dialysis modality may be a bridge to transplantation , or even a suitable alternative , in the absence of an available living donor . traditionally around the world , medical and nursing staff , sometimes in conjunction with a social worker , will . a clear example of this was evident in the 1990s , when the uk had a large pd population simply because space in hospital hd facilities was limited at that time , and home hd had shrunk to the extent that most uk units no longer had viable programmes . since that time , in - centre hd facilities in the uk have expanded enormously , such that in some areas supply exceeds demand and the pd population has approximately halved in number . therefore , many patients currently are not able to choose home hd and patient selection presumably does not generally occur . staff are unlikely to be able to advocate a therapy of which they have no experience and are even less likely to advocate one which they perceive to be unavailable . therefore , health service managers and commissioners also have an enabling role and must embrace the value of self - care by providing the practical resources required . understandably , a patient may never be adequately informed about home hd or assisted automated peritoneal dialysis if these modalities are not operating in the unit concerned . many patients will assume that they are not suitable for a home therapy if it is not offered to them , without realizing why it has not been offered . ideally , every patient should learn about every dialysis modality , accepting that it is probably pointless to discuss pd with a patient who has previously had major abdominal surgery . if this were done , then some patients would inevitably demand the creation of home services where none currently exists , and in one or two uk units , this has already happened . experience within our own service suggests that where a dialysis unit enables and actively encourages self - management , patients will tend to select themselves , and if well motivated may overcome significant difficulties , such as needle phobia , housing problems and literacy issues , to exceed the expectations or predictions of even quite experienced dialysis staff . patients then become advocates themselves and can provide other patients with the necessary motivation to consider a home treatment , such that they approach staff , rather than vice versa .", "interestingly , the general consensus among nephrologists in canada , usa and uk in 2006 was that 45% of patients are suitable for a home therapy , and if you question the staff in your local hd unit , very few would wish themselves to be dialysed in - centre if the need arose . despite this , the reality is that pd numbers are in decline and home hd is virtually non - existent in many areas . selection to medical and nursing staff simply does not work and that many capable patients are not finding their way through there is much in our health care culture that expects care and treatment to be administered by caring and expert professionals , and this expectation is present in the providers of health care as much as it is in the recipients . there are of course individual differences in both staff and patients willingness to embrace self - care , but if we assume that cultures and individual preferences are not fixed , then we may find ways to move people along the self - care continuum . often , patients contemplating the need for renal replacement therapy are understandably and predictably hoping that dialysis will be done for them and will probably shy away from the thought of doing it at home. clearly , it is easier and quicker to point them in the direction of in - centre hd than to embark on a difficult and time - consuming discussion around self - care home dialysis and training . to undertake this task , the staff must first be themselves totally convinced that home treatment is in the patient s best interest if not , the patient will rapidly detect any hint of uncertainty and further discussion is probably futile . this also means that all members of staff with whom the patient comes into contact must be equally capable of espousing some or all of the benefits of home therapy , from nurse , to doctor , to social worker , to dietician and others . in other words , ethos must be that home therapy is not only viable but preferable and beneficial for those who are able to pursue it . for a unit s staff to be able to talk to patients with confidence requires direct experience of home dialysis , but obviously in many units which do not have a full range of home therapies , this may initially be impossible . under these circumstances , collaboration with another unit may enable staff to gain the training and experience required to be able to talk with authority and instill confidence in the patient . visiting patients in their home environment is an essential part of training for both medical and nursing staff .", "it is known that patients who start dialysis after adequate preparation tend to select a home therapy [ 5 , 6 ] . this is encouraging , but in some instances , education can default to telling the patient a series of facts , and once the full list of facts is ticked off , the patient is expected to make a choice . yet , we all know from our own educational experiences that this approach alone is insufficient . on the face of it , it seems plausible that suitable patients would accept the benefits of a home therapy , once they have been educated as to the reasons for the recommendation , and staff may be bemused when they do not do so . this aspect of everyday human behaviour has been most extensively studied in the field of smoking cessation , where it is recognized that someone who has accepted the need to quit is much more likely to be successful than someone who has not , despite both being aware of the future health benefits . the stages of change model suggests that , for most people , a change in behaviour occurs gradually , with the patient moving from being uninterested , unaware or unwilling to make a change ( pre - contemplation ) , to considering a change ( contemplation ) , to deciding and preparing to make a change ( preparation then action ) . therefore , before a patient is able to begin to engage in discussion about any dialysis therapy , they must have reached a point of acceptance that dialysis is necessary . if they are not at this point , then any attempt at education will be largely futile , especially if it is simply repeated when it may be counter productive and alienating . even once they have accepted , the need to change a patient may still not be able to engage in the process of change due to real and significant hindrances , such as needle phobia , general anxiety , low mood or fatigue . 5the goal for chronic kidney disease patients at the pre - contemplation stage is to begin to think about the likely 5need for dialysis in the future and where the dialysis should take place . the task for physicians is to empathetically engage the patient in contemplating this change to their life . during this stage , patients may appear argumentative , hopeless or in denial , and the natural tendency is for physicians to try to convince them with more facts , which usually engenders resistance . \n figure 1 describes the stages of change which anyone confronting a life changing event , such as the need to start dialysis , must pass through in order to come to terms with their new situation . asking the patient to indicate which rung of they ladder they feel they are on can be a helpful first step . bringing a patient to the stage of taking action in a defined timescale is of great importance since progressive renal disease will not wait , and it is clear that those who start dialysis in an unplanned fashion fare less well . on the other hand , a patient who has reached the action stage may be able to select themselves for either home or hospital therapy as they begin to understand what is , and is not , desirable and possible given their specific circumstances .", "the uk national institute for health and clinical excellence ( nice ) produced guidance on home compared to hospital hd for patients with end - stage renal failure in 2002 . suitable for home hd should be offered the choice of having hd in the home or in a renal unit . the definition of practical characteristics of patients suitable for home hd according to nice guidance 2002 although these guidelines are entirely clear and sensible , the manchester experience is that the definition of enthusiastic and determined patients who might initially have been deemed unsuitable are found to be not only capable but successful home dialysers . indeed , the current feeling is that very few physical disabilities preclude home treatment if the patient is keen to proceed , and level of enthusiasm is a more reliable guide .", "once a patient has arrived at the point of choosing a home therapy , the pathway to their first dialysis at home must be as smooth and pot - hole free as possible . this requires some resources and infrastructure to be already in place , and if they are not , then all but the most determined patient is likely to be put off . many patients commitment at this stage is fragile and if the path is too rough or steep , or not well signposted , they may understandably decide to turn back . therefore , from this point on , communication , planning and action must be slick and efficient in order to build the patient s confidence . the experience and confidence of the staff is crucial so that even if the patient is unsure of the process , they feel fully able to trust the team around them and to know who to turn to for reassurance or answers . it requires staff to engage with patients in conversations about what is important to them and what they value in more general terms ( e.g. freedom , independence , safety , quality and quantity of life ) and about what the complex hindrances to self - care may be so that an individualized response to these sorts of questions can move patients along the self - care continuum .", "when faced with the spectre of dialysis treatment , most of us would not naturally opt for a home therapy . traditional methods of patient selection have not prevented a steady decline in home therapies over the past two decades , yet most dialysis unit staff say they would opt for a home therapy for themselves . the stages of change model coupled with basic motivational interviewing techniques can enable staff and patients to engage with each other in a more meaningful way such that many patients will be better able to understand the benefits of self - management and treatment at home . practical infrastructure barriers must also be tackled in order to smooth the pathway to home dialysis ." ]

[ "the epstein barr virus ( ebv ) was described by michael anthony epstein and yvonne barr , who isolated ebv virus particles from endemic burkitt lymphoma specimens . ebv is a double stranded dna virus of the herpes virus family and is the most common virus affecting humans . the infection occurs through oral transmission via saliva . by the age of 40 , over 90 % of the world population has experienced an ebv infection . ebv also plays a pathogenic role , mainly in the development of several t cell and b cell lymphoproliferations ( see below ) but is also associated with different types of rare epithelial and mesenchymal neoplasms . ebv is mainly a b - lymphotropic and epitheliotropic virus and rarely infects t cells and nk cells . the life cycle of ebv is composed of a lytic state and a latency state , the latter allowing lifelong persistence of the virus in the host . the replication cycle of ebv is composed of three phases : ( i ) entry into either epithelial cells or naive b cells after oropharyngeal transmission , ( ii ) lytic replication , and ( iii ) latency . once a cell has become infected , the viral capsid dissolves and the dna is transported into the cell nucleus . in epithelial cells , the entry of ebv into the cell is usually directly followed by the lytic replication of the virus , which leads to production of infectious virions using the virus s own replication machinery . ebv persists in b cells throughout the differentiation from naive b cells to germinal center cells to long - lived memory b cells . in memory b cells , dependent on the expression of specific ebv - associated proteins and rnas , three different viral latency types have been described , which can be reproduced to a certain extent by immunohistochemical markers ( table 1).table 1ebv - latency types and expressed antigenslatency typeebv - associated antigens0/ieber , ebna1ii ( default program)eber , ebna1 , lmp1 , lmp2biii ( growth program)eber , ebna1 - 6 , lmp1 , lmp2a + blyticall lytic antigens ( e.g. , zebra ) ebv - latency types and expressed antigens", "most humans become infected with ebv during childhood , when the infection usually proceeds asymptomatically or with mild symptoms that are undistinguishable from other viral infections . if the infection occurs in young adults and adolescents , it can manifest as mononucleosis ( glandular fever ) . ebv tonsillitis differs from unspecific tonsillitis related to other causes in its histologically detectable polymorphic lymphoid infiltration with abundant plasmablasts and occasional hodgkin - like cells expanding in the interfollicular area of the tonsils . in affected lymph nodes , an extension of the interfollicular areas with a similarly polymorphic infiltration to that in tonsils can be observed . within the peripheral blood and bone marrow , cd8 positive lymphoblasts ( pfeiffer cells ) might be observed . the histomorphological picture of primary ebv infection shares histomorphological features with other ebv - driven lymphoproliferative diseases ( lpd , table 2 ) . therefore , the definite diagnosis of infectious mononucleosis is based on the following features : ( i ) young patient age , ( ii ) sole or main manifestation in the tonsils , ( iii ) absence of immunosuppression in the patient , and most importantly ( iv ) a serological test indicating ebv primary infection ( positive igm titer).table 2spectrum of polymorphic ebv driven lpd with variable neoplastic potential spectrum of polymorphic ebv driven lpd with variable neoplastic potential following the primary infection , patients become asymptomatic virus carriers with a very limited number of ebv - positive b cells . a tissue correlate of this phenomenon is the occasional presence of single small ebv - encoded rna ( eber)-positive b cells , so called bystander cells , within lymph nodes , mucosa - associated lymphatic tissue , or lungs . rare complications of primary ebv infection , such as the chronic active ebv ( caebv ) disease and/or ebv - associated hemophagocytic syndrome have recently been reviewed in this journal .", "since the vast majority of humans have been infected by ebv in childhood and adolescence , ebv - associated lpds in adults usually arise from reactivation of the virus from ebv - infected memory cells . such reactivation is generally considered to consist in an escape from the normal immunosurveillance and preferentially occurs at sites representing the physiological reservoir of ebv - infected b cells , such as the lymphatic tissues and mucosa . in these patients , age - related decline in immunocompetence is assumed to be the cause of ebv - lpd [ 4 , 5 ] . however , the diagnostic pathologist should stress that a clinical work - up of the immune status is warranted once an ebv reactivation is detected . ebv reactivation in adults occurs primarily in the form of ebv - lpd presenting with a polymorphic histological and clinical picture . dependent on the clinical scenario and the preferential site of involvement , different types of polymorphic ebv - lpd can be distinguished . they are outlined in table 2 and include polymorphic posttransplantation lymphoproliferations ( ptld ) , ebv - positive mucocutaneous ulceration ( ebvmcu ) , and lymphomatoid granulomatosis . depending on the clinical scenario and the extent of tissue involvement , these conditions cause variable clinical symptoms . polymorphic ebv - lpd regress in most patients if immunocompetence can be reestablished , e.g. , by withholding immunosuppressive therapy . nevertheless , a subgroup of polymorphic ebv - lpd progress to more aggressive diseases , usually accompanied by a transformation to the morphology of a fully developed lymphoma ( designated as monomorphic polymorphic ebv - associated lymphoproliferations share histomorphological and immunophenotypical features , which are certainly not specific but should alert the histopathologist to an ebv - driven disease when analyzing lymphatic proliferations . these features include the following : tissue necrosisepithelial ulcerationangiotropic vasculitis - like lymphoid infiltratespolymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cellsintermingled large t cells ( often cd8 positive)b blasts , with weak cd20 expression and expression of cd30 epithelial ulceration angiotropic vasculitis - like lymphoid infiltrates polymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cells intermingled large t cells ( often cd8 positive ) b blasts , with weak cd20 expression and expression of cd30 if the clinical scenario ( immunosuppression ) or the histological features are suggestive of an ebv - lpd , testing by eber in situ hybridization should be initiated ( see below ) . when the diagnosis of an ebv - positive polymorphic lpd has been confirmed by detection of ebv in the tissue , the nomenclature ( classification ) of the disease is dependent on ( i ) the clinical context and ( ii ) the involved site ( table 2 ) . ebv - lpd histologically resembling a lymphomatoid granulomatosis might occur after organ transplantation , in which case the disorder is named according to both the clinical scenario and the histopathological picture as ebv - positive polymorphic ptld under the histopathological picture of a lymphomatoid granulomatosis . the most important diagnostic message from the pathologist to the treating clinican should be ( i ) the ebv association of the lesion and ( ii ) the clear distinction of polymorphic ebv - lpd from fully developed lymphomas.fig . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) f 400 histomorphology of ebv - associated lymphoproliferative diseases . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) are indicated .", "ebv - positive lymphomas occur in immunocompetent patients , but are much more frequent in immunocompromised patients . in the latter patient group , the latency type of ebv detectable in the lymphoma cells frequently but not exclusively indicates higher viral activity with expression of more ebv encoded proteins ( latency type 3 ) and sometimes active viral replication . lymphomas occurring in immunocompetent patients , such as hodgkin lymphoma , usually display latency types 0/1 or 2 with a more restricted pattern of ebv - associated proteins and no viral replication ( table 3 ) . the diagnostic criteria for these lymphomas have been described elsewhere and do not necessarily require the detection of ebv because of the presence of ebv in the lymphoma cells . in rare cases , detection of ebv is a mandatory diagnostic feature ( e.g. , in ebv - positive lpd of childhood ) . nevertheless , in certain lymphoma entities , e.g. , nasal and extranasal nk t cell lymphoma , angioimmunoblastic t cell lymphoma , primary effusion lymphoma , and plasmablastic lymphoma , ebv association remains an important clue towards the correct diagnosis . of further relevance might be the identification of ebv - positive diffuse large b cell lymphoma ( dlbcl ) of the elderly . histological features that are useful for identifying dlbcl candidates for screening for ebv have been described as angiotropic growth , necrosis , and polymorphic , plasmablastic or hodgkin - like differentiation and expression of cd30 .table 3ebv - positive lymphomas and latency types and their association with immunocompromised statelineageimmunocompetence of patientsentitylatency typeb cellcompetentclassical hodgkin lymphoma2endemic burkitt lymphoma0/1sporadic burkitt lymphoma0/1ebv + dlbcl of the elderlyvariableebv + dlbcl associated with chronic inflammation ( pyothorax lymphoma)predominantly 3compromisedprimary effusion lymphoma0/1plasmablastic lymphoma0/1lymphomatoid granulomatosis grade 3 and dlbcl arising from the former3monomorphic ptld \n variablelymphomas associated with hiv infection \n variablelymphoproliferative disease associated with primary immune disorders \n variableother iatrogenic immunodeficiency - associated lymphoproliferative disorders \n 3 t cellcompetentangioimmunoblastic t cell lymphoma0/1 or 2extranodal nk / t cell lymphoma2compromisedebv - positive t cell lymphoproliferative disease of childhood and young adults \n variable \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease ebv - positive lymphomas and latency types and their association with immunocompromised state \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease", "the diagnostic criteria for ebv - positive lymphomas are described in the current who classification . however , distinguishing polymorphic ebv - lpd and a fully developed ebv - positive lymphoma can be challenging . the most important feature , large sheets of ebv - positive blasts and absence of a polymorphic cellular picture , should raise suspicion of an ebv - positive lymphoma , especially a dlbcl . a specifically difficult task is the differentiation of a polymorphic ebv - lpd from a fully developed classical hodgkin lymphoma , because the latter may be composed of a similarly polymorphic cellular infiltrate to that of the polymorphic ebv - lpd . pathologists should be aware of the fact that proteins expressed in hodgkin lymphoma , such as cd15 , can also be observed in polymorphic ebv - lpd , like ebvmcu , and therefore the final diagnosis will depend not only on the histopathological features but also on the clinical context , including the site of involvement . for example , hodgkin lymphomas rarely involve epithelial barrier organs , but ebvmcu by definition always do ( table 2 ) . in our experience , clonality testing is not helpful in differentiating ebv - positive lymphoma from polymorphic lymphoproliferations , as viral - driven lpd can also result in a clonal expansion of t and b cells . testing for ebna2 expression is useful in some cases , since expression of ebna2 strongly argues against hodgkin lymphoma ( table 2).table 4differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphomafeaturehodgkin lymphomapolymorphic ebv + lpdhistomorphologymixed cellularity infiltration with hodgkin ( like ) cells , necrosispredominant epitheloid cells and eosinophilspredominant plasmablasts , plasmacells , angiotropismimmunophenotype of large cellscd30 > cd20cd20 > cd30expression of ebv antigens eber + cells equal to lmp1 + cells eber + large cells only ebna2 negative eber + cells more than lmp1 + cells eber + small and large cells ebna2 + /clinical scenariopredominant immunocompetentpredominant immunocompromisedsite mediastinum nodal > > extranodal extranodal > > nodal mucosa differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphoma", "the question as to which specimen should be tested for ebv depends on the histopathological findings on the one hand and on the clinical context on the other hand . table 3 summarizes our criteria and favored method of testing for ebv , fig . if pathologists are confronted with lymphoid tissue histologically resembling inflammatory ( reactive ) tissue in immunocompetent patients , the decision whether or not to test for ebv is primarily based on histological features ( table 3 ) . in immunocompromised patients , the method of testing should be eber in situ hybridization , which is most sensitive for detecting ebv , especially in lesions that can not necessarily be expected to express other viral antigens such as lmp1 ( table 3 ) . since indolent ( low grade ) b cell lymphomas are virtually never associated with ebv , testing can not be recommended . for other entities , which are listed in tables 3 and 5 , testing should be part of the standard diagnostic work - up . immunohistochemical staining for lmp1 is sufficient for hodgkin lymphoma , since these lymphomas express the latency type 2 . however , other lymphomas listed in table 5 , such as diffuse large b cell lymphomas , require testing by eber because lmp1 is frequently not detectable.fig . 2an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) . eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . d 400table 5recommendations for selecting tissue specimen and methods for ebv testinglymphomanodal and extranodal lymphatic tissue not presenting overt lymphoma ( polymorphic lpd)histopathological featuresclinical contexttesting by ebertesting by ebertesting by eberb cell lymphomas endemic burkitt lymhoma hodgkin - like large cells and hodgkin or reed - sternberg cells inherited immunodeficiency plasmablastic lymphoma primary effusion lymphoma posttransplantation pyothorax associated lymphoma dlbcl with histological features suspicious for ebv association necrosis drug induced immunocompromised state angiotropism of blastic cells any monomorphic ptld lymphomatoid granulomatosis plasmablastic infiltrate hiv infectiont cell lymphomas ailt extranodal nk / t cell lymphoma ebv - positive t cell lymphoproliferative disease of childhood and young adultstesting by eber or lmp hodgkin lymphomatesting by eber , lmp1 and ebna2 differential diagnosis between hodgkin lymphoma and ptld \n ptld posttransplantation lymphoproliferative disease , aitl angioimmunoblastic t cell lymphoma an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) . eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . original magnification a d 400 recommendations for selecting tissue specimen and methods for ebv testing \n ptld posttransplantation lymphoproliferative disease , aitl angioimmunoblastic t cell lymphoma", "although epidemiologic data are lacking , ebv - lpd distinct from overt lymphoma will be increasingly recognized . this is due on the one hand to the aging of many populations , with an increase in age - associated ebv reactivation . on the other hand , widespread access to commercial assays and automatization leads to an increasing use of in situ hybridization for eber by diagnostic pathologists . additionally , newly recognized subgroups of lymphomas , such as the ebv - positive dlbcl of the elderly , require the detection of ebv in more lymphoma entities as part of the diagnostic process ." ]

[ "echinococcosis or cystic hydatid disease is an endemic parasitic disease in human populations in iran and some parts of the world . it is caused by larvae stage of echinococcus granulosus ( 1 ) . in the life cycle of e. granulosus , clinical treatment of cysts includes albendazole ( abz ) or mebendazole ( mbz ) therapy in combination with either surgical resection ( 2 ) . the role of enzymes in living organism is clear and remarkable and the worms are seriously dependent on these activities . parasite components such as enzymes have specific biological functions , which are necessary for parasite survival and are supposed to have an important role in host - parasite interactions and disease progress ( 3 ) . parasite s enzymes are attractive purposes that are be explored for the development of diagnostic method and vaccines . they mediate processes like tissue invasion , feeding , evasion ( escape the immune system ) of host immune response etc . glutathione - s - transferase ( gst ) is an enzyme , which has a significant role in the detoxification of parasite metabolites ( endogeous ) , host metabolites ( xenobiotics ) and drugs through their conjugation to glutathione ( 5 ) . gst activity in e. granulosus has been described in the cytosolic portion of protoscoleces obtained from sheep cysts and activated by pre - treatment of protoscoleces with gst inducers ( 6 ) . alkaline phosphates ( alp ) is an enzyme that plays an important role in dampening host immune responses and also plays a role in feeding parasites ( 7 ) . most phosphatases have been found in the absorption system of cestodes , excretory system in trematodes and intestinal cells of the nematode ( 8) . proteolytic enzymes of parasites have been given more attention than other enzymes , because they play a vital role in parasite survival and are involved in many fundamental physiologic processes ( 3 ) . the activities of protease described in e. granulosus ( 9 ) , has been detected in hydatid cyst fluid , cyst wall and in protoscoleces . this enzyme is responsible for breakdown of proteins in all living tissues in order to be used by the cells ( 10 ) . in addition to their known role in the catabolism , they have a part in protein processing in evasion from immune system , leaving the cyst , molting of the parasites and in diagnosis , especially cysteine proteases as serological markers . proteases have generally been identified as potential drug targets in parasites ( 11 , 12 ) . the purpose of this study was to determine the effects of abz and mbz on the activity of the gst , alp and proteases in the protoscoleces of hydatid cyst and to evaluate their inhibitory effects on enzyme activity .", "protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) . protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) . viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) . mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company . no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1 g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) . dmso , and five culture medium the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) . the precipitates were discarded and the total protein and enzyme activities were measured as follows : the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure the protein concentration , standard solutions ( bovine serum albumin ) and e / s products were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added . the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature . a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) . in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube . to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well . the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) . eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed . then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure . sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min . the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) . e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c . the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c . finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm . solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) . folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color . in order to determine the statistically significant difference between protein concentrations , gst , alp and protease activities of e / s samples of treated and control groups , t - test was used . a duplicate set of samples were taken for each test and at the end , their average was taken in to account .", "protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) . protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) . viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) . mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company .", "no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1 g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) .", "the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) . the precipitates were discarded and the total protein and enzyme activities were measured as follows :", "the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure the protein concentration , standard solutions ( bovine serum albumin ) and e / s products were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added . the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature . a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) .", "in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube . to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well . the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) .", "eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed . then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure .", "sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min . the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) . e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c . the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c . finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm . solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) . folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color .", "in order to determine the statistically significant difference between protein concentrations , gst , alp and protease activities of e / s samples of treated and control groups , t - test was used . a duplicate set of samples were taken for each test and at the end , their average was taken in to account .", "the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively . the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n the results of gst , alp and protease activity are shown in table 2 . gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml . alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively . alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml . protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml . gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst activity = od / min0.0096total voluesample voluedilution factor \n alp activity = od 2764 \n protease activity=(umole tyrosine equivalents released)(total volume of assay)volume of enzymetime of assayvolume of filtratespecific activity = units / ml enzymemg solid \n statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) . a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) . meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )", "the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively . the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n", "gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml . alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively . alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml . protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml . gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst activity = od / min0.0096total voluesample voluedilution factor \n alp activity = od 2764 \n protease activity=(umole tyrosine equivalents released)(total volume of assay)volume of enzymetime of assayvolume of filtratespecific activity = units / ml enzymemg solid \n", "statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) . a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) . meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )", "glutathione transferases ( gst ) are multifunctional enzyme present in both animal and plant kingdoms . the enzymes are regarded as parts of the phase ii detoxification system that catalyse glutathione ( gsh ) conjugation of a multitude of exogenous and endogenous toxic compounds ( 18 ) . gst is one of the major detoxification system component found in helminthes , particularly at high levels in cestodes and digeneas . in the tissue extracts of parasites gst activity gst activity exists in e / s products and in the surface of digenea worms such as schistosoma , fasciola and hookworm necator . gst in e / s products act as an anti - inflammatory agent and neutral lipid peroxidation products in the mucous membranes ( 20 , 21 ) . researches related to chemotherapy and immunotherapy , have identified glutathione of parasitic worms independently as a potential target for treatment ( 22 ) . these acts include the digestion of host s tissue in order to provide food for parasites , preventing blood coagulation , facilitating entry into the host s immune system , and disrupting it ( 23 ) . alp is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules , including nucleotides and proteins ( 24 ) . the phosphatases have been found frequently in the structure of absorption like tegument of cestodes , intestinal cell of the nematodes and excretory system of trematodes . while alkaline phosphatases have been found in adult cestode , the larval forms have a predominance of acid phosphates . this change in these enzymes may be associated with the growth and development of the reproductive system in adult worms ( 8) . alp as protein plays a role in immune evasion by generating the potent immunosuppressant adenosine . since adenosine is also an important nutrient , alp may also play a role in parasite feeding ( 7 ) . several studies have been carried out on characterization of hydatid cyst protoscoleces e / s products ( 25 , 26 ) . in the present study , the effect of abz and mbz on protein content and some enzymatic activities of e / s products were examined . protein concentration in treated groups e / s products were less than that of the control group e / s sample . abz and mbz cause degenerative changes in intestinal cells of the worm by binding to the colchicine - sensitive site of tubulin , therefore blocks its polymerization into microtubules and inhibits the cell proliferation in metaphase stage ( 27 , 28 ) . based on these findings we can expect the decreasing of total protein in treated parasite culture media , which correlates our study that shows an average protein of test group is less than control group . in this study , a significant decrease observed in the activity of gst , alp and protease enzymes of the e / s products of treated groups in comparison with the control group . the inhibition of enzymes activity induced by mbz and abz might damage the defensive system , metabolism and nutrition of the parasite . the inhibition of gst activity induced by anti - helmintics may contribute to passive the detoxification mechanism ( 29 ) . the abz and mbz cause reduction in protease enzymes activity , and effect on the parasite activities including assault , migration , feeding , and particularly survival in the host . since protease enzymes are proteins , the reduction of their activity could be attributed to interference of benzimidazole with protein synthesis ( 30 ) . alp is an important enzyme of helminthes parasites associated with absorption and/or digestion of food materials . it has a role in modulating the host immune response ( 7 , 31 ) . higher concentrations of alp found in the some areas of intestine and sub - cuticular layers of the worm associated with protein transport processes ( 32 ) . various commercial drugs ( anthelmintic ) and chemical compounds are shown to bring about changes in the activity of alp in various soft - bodied helminth parasites like cestodes ( 33 ) . when taenia solium cysts was treated with praziquantel and abz , alp secretion in the culture medium based on the results obtained in this study , the reduction or inhibition of the enzymes ( gst and alp ) caused by abz and mbz could lead to alteration of metabolism in helminthes parasites . therefore , the study indicates that both anti hydatid benzimidazoles ( abz and mbz ) have an inhibitory effect on the activity of gst , alp and protease enzymes in e / s products of e. granulosus protoscoleces in vitro . protein concentration in e / s products of mbz groups was slightly higher than that of the abz groups but this difference was not significant . the results showed that the effect of abz on the activity of gst and alp is more than mbz and this may be due to the different structures of these drugs . in addition , the above - mentioned results might account for at least in part , difference in antihydatid mechanism of the both benzimidazole ( 5 ) .", "both drugs have an inhibitory effect on the activity of gst , alp and protease enzymes . the results of the two drugs indicated that abz is more effective on the enzymes activities ( gst and alp ) as compared with mbz . this may by attrirbuted tothe different structures of the two drug and might account for at least in part , differencein antihydatid mechanism of these benzimidazole derivation ." ]

[ " anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease. an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia. phased - array mri is recommended as the preferred imaging modality for regional staging. imaging allows detection of infectious complications , planning of radiotherapy or salvage surgery. follow - up mri allows reliable assessment of therapeutic response after chemo - radiotherapy . anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease . an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia . phased - array mri is recommended as the preferred imaging modality for regional staging . ", "an uncommon malignancy in the general population , squamocellular anal carcinoma ( scac ) accounts for approximately 1 % of all gastrointestinal neoplasms and less than 5 % of anorectal tumours . in past decades , scac was usually diagnosed at a relatively advanced age with a significant female predominance , and believed to be an indolent disease secondary to chronic irritation . in recent years , similarly to uterine cervix dysplastic changes , oncogenic human papillomavirus ( hpv ) has been detected in the vast majority ( up to 90 % ) of invasive scacs , and linked to the development of low- and high - grade premalignant anal intra - epithelial neoplasms ( ain ) , particularly with high - risk or multiple hpv serotypes infection [ 1 , 2 ] . furthermore , the incidence of scac is steadily increasing , particularly in patients with risk factors such as human immunodeficiency virus ( hiv ) infection , history of anoreceptive intercourse , coexistent cervical dysplasia or cancer , immunosuppression , inflammatory bowel diseases ( ibd ) and cigarette smoking . currently , at least half of scacs occur in relatively young ( 4060 years ) hiv - positive individuals , most often men who have sex with men ( msm ) [ 35 ] .", "the surgical anus is about 4 cm long from the anorectal junction to the perianal skin on the external anal margin ( verge ) . the internal anal sphincter consisting of smooth muscle is separated from the external , striated muscle sphincter by the fatty intersphincteric space . along with the puborectalis and levator ani muscles , the external anal sphincter forms the sphincter complex . located approximately halfway along the anus , the dentate line marks the transition from the squamous epithelium to the intestinal mucosa . thus , histologically scacs can be either keratinising or non - keratinising according to their origin below or above the dentate line , although with similar biological behaviour [ 6 , 7 ] . anal margin and anal canal scac originating distal to the dentate line drain to the inguinal and femoral lymph nodes . when the primary tumour arises above the dentate line , regional lymph nodes include the inguinal , internal iliac and perirectal nodes , whereas the external , common iliac and para - aortic nodes are considered non - regional [ 68 ] . because of its anatomical location , in most cases scac is diagnosed clinically in patients with rectal bleeding , pain , discharge or palpable masses . alternatively , lesions may be detected during follow - up of high - risk individuals . following physical examination including digital rectal and vaginal examination , ano - proctoscopy and biopsy , imaging is required to evaluate the local extent of the lesion , lymph node involvement , possible invasion of adjacent organs and distant metastases [ 6 , 9 ] . imaging the anal canal and perianal structures may prove technically challenging to perform and interpret . in past years , trans - anal ultrasound ( trus ) and magnetic resonance imaging ( mri ) techniques allowed an accurate assessment of tumour size and depth of mural invasion [ 7 , 10 , 11 ] . unfortunately , in patients with anal lesions , positioning of endoanal sonography probes and mri coils is hampered by pain and stricture . trans - anal imaging combines an excellent spatial detail with a limited field - of - view that prevents panoramic assessment of entire ischiorectal spaces and of regional lymph nodes . furthermore , trus has limited specificity for differentiation of residual tumour versus post - treatment fibrosis [ 1113 ] . currently , mri performed using external phased - array coils on high - magnetic - field scanners is the imaging modality of choice to investigate the anal region . significant advantages of mri include its native multiplanar capability , superior soft - tissue differentiation , biological non - invasiveness and optimal safety profile of gadolinium - based contrast agents . acquisition protocols heavily rely on high - resolution t2-weighted sequences along three planes , with coronal and axial scans planned slightly oblique , respectively parallel and perpendicular to the long axis of the anal canal . despite the increased tumour conspicuity provided by background fat suppression , short - tau inversion recovery ( stir ) sequences are less useful because of limited spatial detail and difficulty to delineate anatomic landmarks . at our centre , t1-weighted sequences including fat suppression in at least one plane are routinely acquired following standard - dose intravenous gadolinium contrast , to allow detection of lesion enhancement . conversely , other authors discourage post - contrast mri acquisitions by stating that enhanced images do not offer additional information to the high soft tissue contrast intrinsic to t2-weighted imaging [ 7 , 10 , 1315 ] . although with limited contrast resolution compared with mri , volumetric multidetector computed tomography ( mdct ) acquisitions including image reformations along arbitrary planes allow visualisation of anorectal abnormalities in their cranio - caudal extent with relationship to key anatomical landmarks such as the sphincter complex [ 1618 ] .", "although the dentate line is not directly recognisable , its position can be inferred as it corresponds approximately to the upper portion of external sphincter muscles . sensitivity of trus and mri for the identification of scac has been reported to approach 90100 % , with high concordance regarding tumour size , although on a limited number of patients , and more precise results with ultrasound for smaller , superficial tumours . neoplastic tissue in the anal canal has low - to - intermediate t1 signal intensity and positive enhancement after intravenous gadolinium contrast . on t2-weighted and stir sequences , untreated neoplasms display intermediate signal intensity , lower to that of normal ischioanal fat and almost always superior to the internal reference standard represented by uninvolved anal sphincters and gluteal muscles ( figs . 1 , 2 ) . although with limited sensitivity compared with mri , on ct images scac may be detected as solid , enhancing nodules or masses within the anus ( figs . 2 , 3 ) . axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads)fig axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads)fig . post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scacfig . 4an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri . sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) a 40-year - old msm with bioptic diagnosis of scac . axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads ) a 62-year - old female with biopsy - proven scac . axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads ) a 57-year - old woman undergoing abdomino - pelvic mdct for unrelated reasons . post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scac an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri . sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) staging is essential for both prognostic information and correct therapeutic planning , and is performed according to the uicc / ajcc tumour - node - metastasis ( tnm ) system ( table 1 ) , including local lesion extent , lymph node status and distant metastatic spread . in 2010 the european society for medical oncology ( esmo ) recommended mri as the primary imaging modality to accurately stage scac , taking into account the maximum tumour diameter , possible invasion of adjacent organs and nodal involvement [ 6 , 13].table 1tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of originanal canalanal marginprimary tumour ( t)txprimary tumour can not be assessedt0no evidence of primary tumourt1tumour 2 cm in greatest dimensiont2tumour 25 cm in greatest dimensiont3tumour > 5 cm in greatest dimensiont4tumour of any size invading adjacent organ(s ) e.g. vagina , urethra , bladdertumour invading deeper structures ( skeletal muscle , cartilage)regional lymph nodes ( n)nxregional lymph nodes can not be assessedn0no regional lymph node metastasisn1metastasis in perirectal lymph node(s)regional lymph node metastasisn2metastasis in unilateral internal iliac and/or inguinal lymph node(s)n3metastasis in internal iliac and perirectal lymph nodes and/or bilateral internal iliac and/or bilateral inguinal lymph nodesdistant metastasis ( m)m0no distant metastasism1distant metastasis tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of origin particular care should be applied to choosing the longest lesion diameter on t2-weighted images , since correct t parameter staging relies on this measure being below 2 cm , over 5 cm or intermediate ( fig . 1 ) mri demonstrates good correlation with physical findings concerning t stage , whereas infiltration of adjacent organs and sometimes tumour size are clinically underestimated . extramural neoplastic spread may involve the sphincter complex muscles ( external sphincter , levator ani and puborectalis ) and most commonly occurs towards the anterior urogenital triangle with possible vaginal , urethral or bladder involvement . sometimes , the tumour may also extend laterally with invasion of the ischioanal fossa ( fig . 2 ) , superiorly to the rectum and mesorectal compartment , or inferiorly to the skin and subcutaneous planes of the perianal region . in such instances , t2-hyperintense solid tissue is seen infiltrating the even more hyperintense fat in the ischiorectal ( fig . 2 ) and subcutaneous spaces , encasing the lower signal intensity skeletal muscles , or isointense structures such as the vagina ( fig . 5 ) , prostate and urethra ( fig . notably , radiologists should remember that anal canal carcinoma directly invading the rectal wall , perianal skin , subcutaneous or the sphincter muscle does not imply assessing the tumour stage as t4 . for anal margin scac , a t4 lesion is defined by invasion of deeper structures such as the skeletal muscle or cartilage [ 9 , 10 , 13].fig . axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement . biopsy diagnosed scac with superimposed infection a 32-year - old hiv - positive woman with clinical diagnosis of anovaginal fistula . axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement . biopsy diagnosed scac with superimposed infection the incidence of regional nodal involvement increases with primary tumour size . lymph node metastases may be present ( in 25 % of cases ) even with superficial ( up to t2 ) scacs , and are unreliably assessed clinically . nodal staging evaluation relies on the distance from the primary tumour rather than on the number of involved nodes . mri is highly helpful to assess lymph node metastatic involvement , although the mere size criterion is far from accurate and associated with both false - positive and false - negative results . short - axis threshold values of 8 mm , 5 mm and 10 mm have been suggested for pelvic , perirectal and inguinal lymph nodes , respectively . additional helpful features to increase specificity include loss of the normal bean - shaped morphology and fatty hilum , internal t1 and t2 signal heterogeneity with central necrosis , and inhomogeneous enhancement ( figs . 6a 65-year - old woman with history of previously treated small scac 3 years earlier . axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure . surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scacfig . axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) a 65-year - old woman with history of previously treated small scac 3 years earlier . axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure . surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scac the same patient as in fig . 2 . axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) as suggested by ecco guidelines , search for distant spread is usually performed by means of contrast - enhanced body mdct , with conventional imaging appearances of liver and lung metastases . dissemination is very uncommon ( less than 5 % of patients at initial diagnosis , and is usually encountered in association with post - treatment recurrence . alternatively , in immunocompetent patients f - fluorodeoxygluocose positron emission tomography ( fdg - pet / ct ) has high specificity for nodal and visceral dissemination . at diagnosis , fdg - pet / ct may alter staging of anal scac in 20 % of patients , leading to inclusion of involved pelvic or inguinal lymph nodes in the radiation field [ 6 , 7 ] .", "in our experience , not infrequently anal tumours coexist with inflammatory conditions such as proctitis and abscesses . in such instances , mdct and mri provide confident detection of perirectal inflammatory changes and purulent collections that are differentiated from solid neoplastic tissue , thus allowing a correct therapeutic choice including surgical drainage as necessary ( figs . 5 , 8 , 9 , 10 ) . resolution of associated inflammatory changes during treatment is easily monitored by cross - sectional imaging ( figs . 8a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass . axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) . axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space . surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracksfig . staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) . incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) . subtotal regression of changes after treatment is seen on follow - up mri ( d ) a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass . axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) . biopsy confirmed superinfected scac a 53-year - old man with purulent drainage and clinical diagnosis of perianal inflammation . axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space . surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracks a 39-year - old hiv - infected man with biopsy - proven scac . staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) . incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) . subtotal regression of changes after treatment is seen on follow - up mri ( d ) furthermore , cross - sectional imaging particularly with mri also proves useful to differentiate anal carcinoma from other causes of local pain and perineal masses , such as pilonidal sinus diseases , gartner duct or bartolini gland cysts , tailgut cysts , uncommon soft - tissue neoplasms , urethral cancer , lymphoma or metastases [ 14 , 22 ] .", "in the past , scac was treated with abdomino - perineal resection and permanent colostomy . currently , anal margin and small canal tumours without evidence of nodal spread may be successfully excised . in all other cases , the standard treatment includes radiation combined with mitomycin - c plus infusional 5-fu chemotherapy , yielding an 80 % 5-year survival rate with preservation of sphincter function . advanced t3/t4 tumours have worse outcomes with a 4068 % 3-year disease - free survival . salvage surgery with abdomino - perineal resection is reserved for persistent or recurrent tumours [ 3 , 6 , 2325 ] . after radio - chemotherapy , imaging follow - up with mri represents a useful complement to clinical evaluation in the assessment of therapeutic response . shortly after treatment completion , interpretation of mri is usually challenging due to the superimposition of inflammatory changes resulting from radiotherapy . performed at least 68 weeks after treatment completion , mri provides confident , reproducible assessment of post - treatment modifications . findings indicative of a positive response include size reduction and diminished t2 signal intensity of the treated tumour and associated adenopathies ( figs . the appearance of t2-hypointense signal is consistent with fibrosis , although it does not allow excluding minor residual neoplastic foci for sure ( fig . stability in size and signal intensity of any residual abnormality visible at mri in the site of the treated lesion 1 year after therapy has been reported to be strongly associated with a favourable outcome . locoregional and/or distant recurrence occurs in up to 35 % of treated patients , and is strongly associated with advanced ( t3t4 ) stage and nodal involvement at presentation [ 7 , 20 ] . persistent and locally recurrent tumours often display an aggressive behaviour , with possible extensive invasion of the adjacent organs and pelvic bony structures , and a tendency for lymphatic dissemination ( figs . initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) . complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapyfig . mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features . after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathiesfig . initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) . after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumourfig . 14an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities . initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6 cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion . after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d ) an 83-year - old man with known scac . initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) . complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapy a 46-year - old female patient with biopsy - proven scac . mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features . after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathies a 46-year - old hiv - infected man with biopsy - proven scac . initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) . after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumour an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities . initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6 cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion . after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d )", "after the introduction of highly active anti - retroviral treatment ( haart ) , people with hiv infection or acquired immunodeficiency syndrome ( aids ) gained a greatly improved life expectancy with better immune conditions , at the price of an increased tendency to develop tumours . although it is considered a non - aids - defining malignancy , currently scac ranks third ( 8.2 % ) among neoplasms observed in hiv / aids populations , with a substantially higher incidence in msms and in long - standing infected people [ 4 , 26 ] . dysplastic intraepithelial lesions are highly prevalent in hiv - infected people , and hiv represents a marker for coinfection with other sexually transmitted diseases such as hpv . since the risk of developing scac increases with the total time elapsed with cd4 + count below 200 cells/l , it has been hypothesised that hiv - related immune suppression acts as a cofactor to hpv in the development of anal dysplasia and progression to overt carcinoma [ 1 , 3 , 4 ] . screening procedures including high - resolution anoscopy and cytology smears are increasingly adopted at hiv care centres , to allow detection of scac precursors and early - stage tumours amenable to limited excision and topical therapies . prevention should limit the occurrence of advanced stages at diagnosis in the future [ 4 , 2629 ] . a high prevalence ( at least 30 % ) of anorectal complaints is characteristic of hiv - positive patients , particularly those practicing anoreceptive intercourse . differential diagnosis encompasses a wide spectrum of abnormalities , including non - specific anal diseases such as haemorrhoids , fissures , fistulas and abscesses , along with venereal infections , viral ulcers , and a non - negligible ( 7 % ) rate of neoplasms such as scac , lymphoma and kaposi s sarcoma . therefore , when performing cross - sectional imaging procedures on hiv - infected patients , even for unrelated complaints , special attention should be paid to the anal region , with a focus on the possible identification of solid , enhancing tissue consistent with tumour that indicates need for biopsy ( figs . 5 , 8 , 10 , 13 ) [ 21 , 30 , 31 ] . despite initial discouraging reports , in the haart era hiv - positive patients are likewise treated with standard chemo - radiation regimens , reaching satisfactory results in terms of local control and survival rates , although with increased toxicity and frequent local recurrences [ 4 , 5 , 26 , 27 , 32 ] .", "some literature reports have highlighted the increased risk of anus and lower rectum carcinomas associated with long - standing , severe perianal fistulising crohn s disease ( cd ) ( fig . , scac reaches a 14 % proportion among all colorectal cancers , which is ten - times higher than the usual figure . furthermore , cd patients develop anorectal carcinomas at a younger age ( 20 years earlier ) than the general population . according to the hypothesised pathogenesis , fistulas probably allow hpv an easier access to the epithelial layers , and chronic mucosal regeneration may ultimately lead to neoplastic changes . however , anal tumours may occasionally develop in patients with ulcerative colitis ( uc)-related ( fig . 15a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton . contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) . initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) . shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) . mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) . after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton . contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) . abdomino - perineal resection for scac was performed a 39-year - old woman with history of ulcerative colitis and perianal inflammation . initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) . shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) . mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) . after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) diagnosis is often unsuspected or delayed because of pre - existent , unspecific complaints and clinical assessment is hampered by complex inflammation with stricture and local pain . as a result , ibd - associated anal cancers are often advanced at presentation , may require extensive surgery plus chemotherapy and radiotherapy , and are associated with a severe prognosis . therefore , patients with early - onset or long - standing perianal cd should undergo clinical and imaging surveillance , particularly when new or changed symptoms develop . radiologists should be aware of the increased risk for anorectal cancer in middle - aged ibd patients , and clearly report any solid tissue as suspicious for neoplasm and suggest biopsy ( figs . ", "the established association with hpv infection and premalignant intra - epithelial dysplastic changes provides insight into the pathogenesis of hiv- and ibd - related anal cancers , and the possibility of prevention or early diagnosis through screening of high - risk individuals [ 1 , 2 ] . state - of - the - art cross - sectional imaging with high - resolution mri using external phased - array coils and multiplanar mdct allow detailed , comprehensive visualisation of abnormalities involving the anus and perineal region . currently , mri represents the modality of choice for primary regional staging of scac , assessment of complications , of therapeutic response following chemo - radiotherapy , and of possible recurrences [ 9 , 10 , 13 ] ." ]

[ "black yeasts from the family herpotrichiellaceae ( order chaetothyriales ) are fungi with a remarkable dual ecology . on the one hand , they have a unique ability to adapt to extreme environments ( exposure to toxic chemicals , high temperature , scarcity of nutrients , acidic , and/or dry conditions ) , while on the other hand , they exhibit a significant human pathogenic potential . unlike common opportunistic fungi , herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease and only occasionally in immunocompromised patients . also , more often than any other fungal group , these organisms have been reported from environments that are rich in aromatic compounds . the first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote , while their occurrence on untreated wood was comparatively low , indicating that accumulation of aromatic compounds might promote the growth of these fungi . the concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis , isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [ 26 , 27 ] . it has been hypothesized that wild berries constitute a natural niche of e. dermatitidis , which might then be ingested by birds and humans and sporadically resulting in mycoses . deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus . the related species exophiala bergeri , exophiala heteromorpha , exophiala oligosperma , and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [ 57 , 11 , 27 ] . however , the connection between route of infection and natural occurrence is less evident with these species . the tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . besides hydrocarbon exposure , environmental conditions in biofilters are characterized by a relative low water activity and acidification of the filter bed , which might lead to biomass inhibition problems . in this respect , fungal colonization in air biofilters has generally been related to an improved bioreactor performance . detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [ 3 , 20 , 29 ] , which appears to be quite an uncommon metabolic feature in the eukaryotes . molecular phylogenetic characterization of these biofilter fungi has shown a predominant affiliation to the genera exophiala and cladophialophora ( fam . herpotrichiellaceae ) , particularly to the species exophiala lecanii - corni , e. oligosperma , and the recently described e. xenobiotica , cladophialophora saturnica , and cladophialophora inmunda [ 1 , 6 ] . despite the medical and environmental relevance of herpotrichiellaceous black yeasts , little is understood on their biodiversity and natural occurrence . in order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission , the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons . different environmental samples related to the life cycle of e. dermatitidis were used as inocula .", "the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons . environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14 days . the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n table 1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95 n , 51045.96 ea2oak railway tie , between railsnear station , hollandsche rading521041.95 n , 51045.96 ea3oak railway tie , outside railsforest area , hilversum521220.68 n , 5119.66 ea4oak railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ea5concrete railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ec1berry , sorbus aucupariaroadside , de bilt52 717.18 n , 5 948.13 ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19 n , 5 931.42 ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec4berry , sorbus aucuparialapersveld park , hilversum521254.74 n , 51110.25 ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52 n , 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed4fresh goose feceslapersveld park , hilversum521254.74 n , 51110.25 ed5old goose feceslapersveld park , hilversum521254.74 n , 51110.25 e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . serum flasks of 100 ml were filled with approximately 25 ml of perlite granules , saturated with mineral medium . each environmental sample ( 13 g ) was washed with phosphate - buffered saline , and the suspension ( 40 ml ) was used to inoculate four different batches ( 10 ml each batch ) . as a control , these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth . the inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene . this gas phase was generated by placing 10 ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate . a solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3 months . after this time , the perlite granules in each flask were washed with 5060 ml sterile water . one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters . a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions . the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) . pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50 l volumes containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp and 2.0 u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) . amplification was performed with cycles of 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) and 72c ( 120 s ) , with a final 7-min extension step at 72c . amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) . sequence pcr was performed as follows : 95c for 1 min , followed by 30 cycles consisting of 95c for 10 s , 50c for 5 s , and 60c for 2 min . reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) . sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) . phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .", "the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons . environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14 days . the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n table 1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95 n , 51045.96 ea2oak railway tie , between railsnear station , hollandsche rading521041.95 n , 51045.96 ea3oak railway tie , outside railsforest area , hilversum521220.68 n , 5119.66 ea4oak railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ea5concrete railway tie , between railsforest area , hilversum521220.68 n , 5119.66 ec1berry , sorbus aucupariaroadside , de bilt52 717.18 n , 5 948.13 ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19 n , 5 931.42 ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec4berry , sorbus aucuparialapersveld park , hilversum521254.74 n , 51110.25 ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70 n , 5 951.98 ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52 n , 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74 n , 51110.25 ed4fresh goose feceslapersveld park , hilversum521254.74 n , 51110.25 ed5old goose feceslapersveld park , hilversum521254.74 n , 51110.25 e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons", "the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . serum flasks of 100 ml were filled with approximately 25 ml of perlite granules , saturated with mineral medium . each environmental sample ( 13 g ) was washed with phosphate - buffered saline , and the suspension ( 40 ml ) was used to inoculate four different batches ( 10 ml each batch ) . as a control , these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth . the inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene . this gas phase was generated by placing 10 ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate . a solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3 months . after this time , one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .", "a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions . the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) . pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50 l volumes containing 25 ng of template dna , 5 l reaction buffer ( 0.1 m tris hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2 mm of each dntp and 2.0 u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) . amplification was performed with cycles of 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) and 72c ( 120 s ) , with a final 7-min extension step at 72c . amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) . sequence pcr was performed as follows : 95c for 1 min , followed by 30 cycles consisting of 95c for 10 s , 50c for 5 s , and 60c for 2 min . reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) . sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) . phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .", "a total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons ( table 2 ) . of those , a single mold ( aspergillus fumigatus ) other than black yeasts was isolated from berries of sorbus aucuparia ( sample c1 ) . the highest isolation rates were achieved with samples taken from creosoted wooden railway ties , particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate ( 39 isolates , samples a1 and a3 ) . these samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates ( 17 and 10 strains , respectively ) , but not a single fungus was isolated when naphthalene was supplied as the sole carbon source . though in lower numbers , black yeasts were also isolated from the tie section located between the rails ( 14 strains , samples a2 and a4 ) . besides creosote , these samples were likely to contain fecal and mineral oil contamination as well . table 2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments , black yeasts were isolated in two out of four berries from s. aucuparia ( 12 isolates , samples c1 and c2 ) , but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna ( samples c5 and c6 , respectively ) . soil mixed with bird feces ( guano ) also yielded melanized fungi in one out of five tested samples ( six isolates , sample d2 ) . no black yeasts were isolated when suspensions of the samples described above ( table 1 ) were directly plated onto sga plates , without the enrichment step on volatile aromatic hydrocarbons , while several common and heavily sporulating fungal species were encountered . phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups ( fig . 1 ) . sequences from reference strains deposited at the cbs collection ( utrecht , the netherlands ) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis . those encompassed the type strains of e. xenobiotica , e. bergeri , exophiala spinifera , e. oligosperma , e. heteromorpha , e. dermatitidis , e. lecanii - corni , and phialophora sessilis , as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons : cladophialophora immunda , c. saturnica , and cladophialophora psammophila sp . nov . , the latter species being in the process of description ( badali et al . , submitted ) . strains of e. xenobiotica , e. bergeri , and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . \n figure 1neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate . sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches were also included in the analysis ( genbank sequence codes are given between square brackets ) neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate . sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches were also included in the analysis ( genbank sequence codes are given between square brackets ) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica ( 32 strains ) . the majority was highly homologous ( 99% ) to the ex - type strain , and some were slightly deviating ( 97% homology ) . a second group of isolates ( nine strains ) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain . the cluster encompassing e. bergeri also included a major group ( 24 strains ) , the sequence of which deviated significantly from the ex - type strain ( 94% sequence homology ) , so that its affiliation to e. bergeri might be put into question . blast searches on this latter group into the genbank genomic database revealed a close match ( 98% homology ) with an unidentified exophiala strain isolated from a rock surface . a minor e. bergeri sibling group was composed of two strains for which no known sequence match was found . a third group ( three strains ) did not match any known reference type strain , but genbank searches revealed a high sequence homology ( 98% ) to the sequence of an uncultivated fungus from a municipal composting plant . except for a single isolate ( dh18150 ) obtained from a s. aucuparia berry ( sample c1 ) , the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails ( samples a1 and a3 ) . within this group , three strains were obtained under benzene enrichment , while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates . besides e. xenobiotica , sample a3 also yielded five isolates upon benzene enrichment , which were identified as e. bergeri . the related between - the - rails section from that same tie ( sample a4 ) and the guano - rich soil ( sample d2 ) yielded 1 and 3 strains of e. bergeri , respectively , using toluene as the enrichment substrate .", "the occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . under common laboratory culture conditions , numerous protocols for the selective isolation of black yeasts have been developed during the last decades ( an overview is presented in table 3 ) , which have yielded new data on the presence of these fungi in a wide range of artificial environments , sometimes with no obvious counterpart in nature . several methods are based on osmotolerant and oligotrophic abilities of target fungi . with these regimens , the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales , although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales . applying enrichment methods based on the inoculation of rodents , exposure to aromatic hydrocarbons , assimilation of rare sugars , and/or incubation at high temperature , most isolated black yeast - like fungi are members of the chaetothyriales . it should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi , and they are therefore classified with difficulty in any of the known ecological categories . \n table 3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala , fonsecaea , cladophialophorachaetothyrialesopportunists[2 , 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala , fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora , exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium , hortaeadothidealeshalophiles[31 , 32]suspendcryomyces , friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family . for example , under hydrocarbon atmospheres , the berry samples were free from aureobasidium pullulans , which is a member of the dothideales , otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries . this species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment . the presence of heavily sporulating airborne fungi , frequently found in soil and litter , was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus . interestingly , the dothidiaceous species amorphoteca resinae , known as creosote fungus for its common isolation from creosoted wood , was found neither in this study nor in previous fungal isolation surveys from creosoted wood [ 26 , 27 ] . consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples , such as wild berries , only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . in particular , the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments . with advanced analytical techniques , it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature , though at very low concentrations . toluene , for example , is produced biologically in different natural environments including maturing berries . on the other hand , as extremophilic and slow - growing microorganisms , incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts , when compared to traditional liquid enrichment cultures . regarding both the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions , wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts . creosote is a distillate derived entirely from tar , which is rich in a wide variety of polycyclic aromatic hydrocarbons , phenols , and cresols , and has widely been used as wood preservative against microbial decay . due to its carcinogenic character , the use of creosote has been banned in the european union . yet , creosoted wood utilities are still widely present in the open environment , such as telephone poles , railway cross ties , switch ties , and bridge timbers . also , creosote is an important soil contaminant in former wood creosoting plants . in a previous study , e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c , this protocol being quite selective for e. dermatitidis . it was hypothesized that the life cycle of this species involved the occurrence on wild berries , ingestion and passage through the human intestinal tract , and feces deposition on railway ties . subsequent enrichment on creosoted wood might then be related not only to the oligotrophy , thermotolerance ( particularly under tropical conditions ) , acidotolerance , and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds . the temperate wild berry sampling locations that were the subject of our study ( table 1 ) had previously been analyzed for the occurrence of black yeasts [ 18 , 26 ] . collected berries were homogenized , diluted , and used for spread plates or were subjected to the raulin s pre - incubation protocol . in these earlier studies involving several hundreds of samples , only a single strain of e. dermatitidis the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics . this hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil , using the mineral oil - flotation protocol [ 15 , 24 ] . from the present enrichment program on volatile alkylbenzenes , it appears that exophiala species , though other than the thermophilic e. dermatitidis , are regularly present on substrates that are characteristic from the latter species ( berries , bird feces , and creosoted railway ties ) . it is known to be associated with mild cutaneous infections in humans and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [ 6 , 24 , 26 ] . , it was regarded to be an extremely rare opportunist , although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . using the selective oil - flotation method , e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives . a distinct cluster was identified within the e. bergeri group , which might belong to a yet - undescribed species . strains from this group were predominantly isolated from the section between the rails of sampled wooden ties , which were partly contaminated by machine oil and human feces . the remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails , where this contaminating effect was less marked . three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere . no close sequence homology against reference strains was found for these fungi , indicating that a yet - undescribed species is concerned . the phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study . the extremophilic nature of black yeast , in combination with their capacity to metabolize aromatic pollutants , makes them ideal candidates for specific bioremediation purposes . prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard , but , as phylogenetic data on these organisms accumulates , it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings ( de hoog , unpublished ) . indeed , enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories , such as cladophialophora bantiana or e. dermatitidis . this suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . yet , more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches . the application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons ." ]

[ "the metabolic syndrome ( mets ) is a clustering of metabolic and cardiovascular risk factors that have been widely discussed for at least 20 years . although some have questioned the clinical utility of metabolic syndrome , there are many reasons to believe that this entity is useful . identification of metabolic syndrome is a simple measure of finding people with a clustering of risk factors that put them at increased risk of diabetes and cardiovascular disease ( cvd ) . furthermore , such individuals need more intensive lifestyle interventions at an early stage to delay the disease progression to a still higher - risk category . also , the identification of metabolic syndrome will attract attention to various other related conditions such as fatty liver , polycystic ovary syndrome , and obstructive sleep apnea . since the first official definition of the metabolic syndrome put forward by a working group of the world health organization ( who ) in 1998 , a number of different definitions have been proposed . the latest definition given by international diabetes federation ( idf ) takes into account the evidence that abdominal obesity is an important component of the metabolic syndrome and proposes gender- and race - specific cut - offs for waist circumference ( wc ) . although the need for different wc is attributed to ethnic variation , it was observed that even within the same population , people with identical wc but different heights have different risks for metabolic syndrome . several studies from asia indicate that waist - to - height ratio ( whtr ) is more strongly associated with cvd risk factors than other anthropometric measures such as wc , body mass index ( bmi ) , and waist hip ratio ( whr ) . the aim of our study was to perform a comparative validation of wc , bmi , whr , and whtr for defining the metabolic syndrome in indian population living in the urban and rural areas of rohtak district , haryana , india . we used the idf definition of metabolic syndrome , excluding the measure of obesity , to determine which obesity measure among wc , bmi , whr , and whtr , and what appropriate cut - off value are most closely predictive of the non - adipose components of the idf 's definition of metabolic syndrome .", "informed consent was obtained after explaining the details of the procedure to all subjects . in rural population of these two rural blocks , 85 anganwadi centers ( awc ) were selected by the random selection method . from the population survey register of awc , male and female patients were randomly selected and called at the awc on a specified date and time after an overnight fast . in urban areas , two out of total six urban health centers were selected randomly , and from the survey registers of these two health centers , male and female patients were selected randomly . a day before the study , all subjects were advised to observe overnight fasting ( at least 8 hours ) and called at the nearest health center / awc in the morning . initial evaluation included detailed history and clinical examination of the subjects to exclude any systemic diseases . anthropometric indices including height ( without shoes and socks ) , weight , wc , and hip circumference were recorded for the subjects . height was measured to the nearest 0.1 cm using a portable stadiometer and weight measured to the nearest 0.1 kg using calibrated platform scales . waist circumference was measured to the nearest 0.1 cm at the midpoint between the subcostal margin and the margin of the supracristal plane according to the idf diagnostic criteria . hip circumference was measured to the nearest 0.1 cm around the thighs , at the height of the greater trochanter , with the patients in the standing position . blood pressure was recorded after patients were made to sit and rest for at least half an hour . blood pressure was recorded thrice at 5-minute intervals in a sitting position in the non - dominant arm , with the value to the nearest 2 mm hg , using a standard adult mercury sphygmomanometer . patients suffering from chronic renal , pancreatic or other severe illness , pregnant women , and women who delivered 2 months or less preceding the study , patients on lipid lowering agents , steroids , nicotinic acid , or other medications likely to cause dysglycemia , were excluded from the study . in the morning after an overnight fasting period , blood samples were obtained from the antecubital vein and transfused into vacuum tubes containing edta . all patients were allowed to sit and rest for at least half an hour before the blood samples were taken . plasma glucose , total cholesterol , triglycerides , and hdl - cholesterol were measured . according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome , he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes . all analyses were done separately for men and women and according to their place of residence . continuous data were expressed as mean sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity . the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor . it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker .", "according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome , he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes . all analyses were done separately for men and women and according to their place of residence . continuous data were expressed as mean sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity . the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor . it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker . the statistical program for social sciences , version 17.0 ( spss inc . , chicago il ) , was used for all statistical analyses .", "in the present study , 3,042 individuals were screened for the prevalence of metabolic syndrome and their baseline characteristics , as shown in table 1 . out of the 3,042 individuals selected , 1,693 were from rural areas ( male , 814 ; and female , 879 ) and 1,349 from urban areas ( male , 704 ; and female , 645 ) . in our study participants , the prevalence of metabolic syndrome according to the idf criteria was 23.8% and 42.6% in urban men and women , respectively , while it was 14.9% and 36.3% in rural men and women , respectively . in males , high blood pressure was the most common abnormality , followed by low hdl and hypertriglyceridemia . in females , impaired fasting glucose and diabetes mellitus were seen in 23.2% and 10.7% of the men , respectively , while these were seen in 22.7% and 8.7% of the women , respectively . baseline characteristics of study subjects stratified by gender the roc curve analysis was performed to find out optimal cut - off points for bmi , wc , whr , and whtr . the point on the roc , which represented the largest sum of sensitivity and specificity , was chosen to obtain the optimal cut - off point for each of these four measurements in predicting metabolic syndrome . figure 1 shows the roc curves of bmi , wc , whr , and whtr for predicting the presence of two or more non - adipose components of metabolic syndrome as defined for both men and women . the optimal cut - off value of wc in urban and rural males was > 89 cm , which was higher than that in urban and rural females , i.e. 83 cm and 79 cm , respectively , while the optimal cut - off for whtr was > 0.51 in rural females , 0.52 in rural males , and 0.53 in both urban males and females . whr cut - off for the prediction of metabolic syndrome was 0.87 and 0.93 , respectively , in rural females and males and 0.92 and 0.95 , respectively , in urban females and males . kg / m in both urban and rural males as well as urban females , while it was > 21 kg / m in rural females . table 2 shows aurc , optimal cut - off values , and the associated measure of each cut - off value . in both men and women , irrespective of their place of residence , wc was found to be a better predictor of metabolic syndrome than whtr [ table 2 ] . ( a ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban females . ( b ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban males . ( c ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural females . ( d ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural males . ( e ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in the entire study population irrespective of gender and place of living areas under the roc curve , cut offs , sensitivity , specificity of wc , bmi , whr and whtr when we compared these anthropometric variables as a predictor of metabolic syndrome irrespective of the place of living and gender , whtr was found to have the best aurc as a predictor of metabolic syndrome at a cut - off value of 0.52 [ table 2 ] , and it scored over wc as a predictor of metabolic syndrome ( p = 0.001 ) .", "varying degree of prevalence of metabolic syndrome is reported from various regions of the world and the indian subcontinent , but very few data is available regarding the prevalence of metabolic syndrome separately in the urban and rural population of haryana and also regarding which anthropometric parameter of adiposity is best suited for urban and rural population . we found a prevalence of 23.8% ( 168/704 ) , 14.9% ( 122/814 ) , 42.6% ( 275/645 ) , and 36.3% ( 319/879 ) in urban men , rural men , urban women , and rural women , respectively , using the idf diagnostic criteria . moreover , the prevalence is more common in urban population than in rural population , which is likely due to the higher educational status and sedentary lifestyle of urban population . we evaluated and compared the extent to which four different anthropometric variables of adiposity ( bmi , wc , whr , and whtr ) are able to predict two or more non - adipose components of metabolic syndrome using the idf criteria . it was observed that in both urban and rural men and women , wc is a better predictor of metabolic syndrome than the other three anthropometric variables ( bmi , whr , and whtr ) . however , when the entire population was clubbed together and analyzed irrespective of their place of residence and gender , it was found that whtr scored over wc as a predictor of metabolic syndrome . the optimal cut - off of whtr for this prediction was 0.52 , which is in accordance with several reports from other asian countries where a cut - off value of whtr > 0.5 appears to offer a simple and reliable index of identifying individuals who face increased future risk of metabolic complications . traditionally , bmi is a widely popular index of obesity used . wang et al . , in their study of chinese population , found that bmi and wc are more useful than whr for predicting two or more non - adipose components of metabolic syndrome . however , the majority of asian population is obese and at risk of developing metabolic complications even at bmi cut - off level of 25 kg / m , which is not in accordance with the who bmi cut - off level used to define obesity . this has prompted experts across the globe to redefine bmi standards for asian population , and which is now set at 23 kg / m or above for asian population . in the present study , the bmi cut - off of > 23 kg / m was also predictive of metabolic syndrome , except in rural females where optimal cut - off for bmi was > 21 kg / m . despite this change in bmi criteria , its use a large number of studies clearly suggest that the degree of central fat distribution is more clearly related to metabolic risk than bmi . in the present study , the comparison of roc of bmi with that of wc showed that bmi is inferior to wc in predicting metabolic syndrome ( p = 0.0001 ) . wc and whr have been used as measures of central adiposity and evidences suggest a greater association of these anthropometric variables with a future metabolic risk than bmi , which is a measure of general obesity . between wc and whr , several studies have shown that that wc is a better predictor of metabolic syndrome because of variations in the level of hip measurements , differences in cut - off values between men and women and among different ethnic groups , and the possibility of embarrassment to both examiner and examinee when measuring hip circumference . however , the ability of wc to be used as a universal predictor of central adiposity is limited by the use of different methods for the measurement of wc and different cut - offs used for men and women and for different ethnic groups . in the present study , the optimal cut - off value of wc is > 89 cm each in urban and rural males and > 83 cm and > 79 cm in urban and rural females , respectively , and is clearly superior to whr in the prediction of metabolic syndrome ( p = 0.0001 ) . whtr is another anthropometric variable that has been used and found to be a better predictor of metabolic complications in various studies . this is because the height of an individual influences the distribution of body fat , and this factor should be taken into consideration before adopting any anthropometric variable as a measure of adiposity . on average , men are taller than women and have larger waist circumferences . this means that average whtr values are closer for men and women than average wc values because of adjustments for height , and the same value can be used for both genders to indicate increased risk . earlier reported that whtr is a better parameter of central obesity and obviates the need for numerous wc cut - offs ; it may be useful in children where existing parameters are not useful . they also reported that using the average height in various countries and their respective wc cut - offs as defined by the idf consensus definition , the range of whtr varies from 0.51 to 0.58 among males and from 0.47 to 0.54 among females . in the present study , we also observed that a single value of whtr at more than 0.52 is a better predictor of metabolic syndrome in both genders . moreover , for the first time in the present study , we compared both urban and rural population and found that irrespective of the place of living , a single value of whtr can be used as a marker to identify individuals with a high likelihood of contracting central adiposity and metabolic syndrome . a study conducted on adult females from singapore demonstrated that whtr can act as the best screening tool for cardiovascular risk . in japanese men and women , whtr was found to be a better predictor of metabolic risk compared to other anthropometric indices . other studies also reported that the whtr is a simple and effective screening tool for cardiovascular risk factors in both men and women . in contrast , a dutch study revealed that height did not significantly influence the differences in measures of adiposity or intra - abdominal fat volume in women , or intra - abdominal fat areas in both genders . nakamura et al . reported that whtr did not confer an improved discriminatory performance compared to wc . kato et al . concluded that the predictive power of wc was not inferior to whtr , and wc is practically the most convenient measure for predicting metabolic syndrome because of its simplicity . in the present study , the roc and aurc values for wc are better than that for whtr in case of men and women in both urban and rural areas ( p = 0.0054 ) , but if one has to use a single value for the ease and simplicity of prediction of metabolic syndrome , irrespective of gender and place of residence , then at a value of 0.52 , whtr is a good predictor of metabolic syndrome in comparison to wc where one has to use different values for different gender and place of residence . also , for disease with a high prevalence in population , such as metabolic syndrome , there is the need for a test with high specificity to ensure all true negative cases are picked up and the test will have a high positive predictive value . the same is true for whtr , when used irrespective of gender and place of residence , which has high specificity as well as youden 's index .", "to conclude , although the predictive value of different gender - specific wc values is clearly superior to other anthropometric measures for the diagnosis of metabolic syndrome in both urban and rural population of both genders , a single value of whtr > 0.52 irrespective of gender and area of residence can be used as a universal screening tool for the identification of individuals at high risk to the development of metabolic complications ." ]

[ "the constructive , or synthetic , biology approach is a fascinating avenue for exploring the boundary between living and nonliving matter . because the cell is the minimum unit required for life as we currently understand it , various researchers have attempted to construct artificial cells from simple , well - understood chemicals so that phenomena that occur within such artificial cells can be studied , with the ultimate goal of elucidating the origins of life and studying the fundamental functions of living cells123 . in particular , vesicles4 , which are spherical microcompartments made of amphiphilic molecules and can encapsulate biological molecules such as proteins56 and dna78910 , have often been used as models of biological membranes . vesicles can be classified as small ( defined as having a diameter of < 100 nm ) , large ( diameter < 1 m ) , or giant ( diameter > 1 m ) . giant vesicles ( gvs ) have been studied extensively because they are similar to living cells in size , shape , and structure . owing to the size of gvs , morphological changes in gv membranes several methods for preparing gvs have been reported11 , including the hydration method1213 , the freeze - thaw method14 , the electroformation method1516 , and the fluidic device method1718 . however , encapsulating proteins and other macromolecules in gvs at high concentrations by means of these methods is difficult . in particular , it is extremely challenging to encapsulate biological materials in sufficient quantity ( 20 - 30 vol% ) to mimic the crowded environment inside cells1920 . to form gvs instantly , weitz and coworkers established a water - in - oil ( w / o ) emulsion centrifugation method2122 . first , because gvs prepared by this method have low lamellarity2324 , their membranes are so thin that they can be deformed easily . gv membrane deformation induced by ftsz ( a bacterial cell division protein ) , tubulin , and other macromolecules has been studied25262728 , and we observed polyhedron - like deformation of gv membranes induced by encapsulation of microspheres2930 . second , membrane proteins can be inserted into the vesicular membrane by this method , albeit with difficulty31 . for example , the yomo group used this method to study the in vitro synthesis and pore - forming activity of the membrane protein -hemolysin32 . third , it is possible to generate asymmetric gvs in which the lipid components of the inner and outer leaflets are different22 . generated asymmetric gvs with cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides , and with neutral lipids on the outer leaflet to decrease toxicity and nonspecific cellular uptake33 . fourth , the concentration and volume fraction of substances inside the gvs can be relatively high2834 . for example , nishimura et al . encapsulated an in vitro transcription - translation system into gvs and used the system to express green fluorescent protein ( gfp ) within the gvs36 . these five features make w / o emulsion centrifugation an indispensable method for generating cell - mimicking gvs . in previous work , gvs generated by centrifugation were collected by means of a syringe equipped with a long 16 g stainless steel needle containing some of the final aqueous solution22 . in the hands of inexperienced technicians , this collection method could easily result in contamination of the gv with some of the oil . in this study , we used the w / o emulsion centrifugation protocol developed by the yomo group2337 , in which precipitated gvs are collected through a hole opened at the bottom of the centrifuge tube in which they are prepared . we prepared gvs encapsulating 1.0 m microspheres , which are similar in size to intracellular organelles . the use of microspheres allowed us to estimate their concentration by calculating their volume fraction . establishment of a method for preparation of gvs in which materials are densely packed is an important step for creating artificial cells . to confirm the utility of our protocol for various types of inner materials , we also demonstrated that gfp and a small water - soluble fluorescent molecule ( uranine ) could be encapsulated in the gvs .", "prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n form a lipid film on the inside surface of a 5 ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest . the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n form a lipid film on the inside surface of a 5 ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas . incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest . the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n lidded microtube , mix 237.5 l of a dispersion of 1.0 m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0 m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water . the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n in a 1.5 ml lidded microtube , mix 237.5 l of a dispersion of 1.0 m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0 m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres . add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water . the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively . after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5 ml lidded microtube . after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5 ml lidded microtube . vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5 ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5 ml microtube . emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5 ml lidded microtube . gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5 ml lidded microtube . collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min . obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n immediately after chilling the microtube , obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube . dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x objectives ) equipped with a 12v100whal - l halogen lamp . \n record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n fit the units with 470 - 495 nm and 565 - 585 nm excitation filters , respectively , and with emission filters that transmit 510 - 550 nm and 600 - 690 nm light , respectively . \n fit the units with 470 - 495 nm and 565 - 585 nm excitation filters , respectively , and with emission filters that transmit 510 - 550 nm and 600 - 690 nm light , respectively .", "prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n form a lipid film on the inside surface of a 5 ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest . the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n form a lipid film on the inside surface of a 5 ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas . incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest . the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n lidded microtube , mix 237.5 l of a dispersion of 1.0 m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0 m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water . the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n in a 1.5 ml lidded microtube , mix 237.5 l of a dispersion of 1.0 m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0 m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres . add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water . the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively . after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5 ml lidded microtube . after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5 ml lidded microtube . vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5 ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5 ml microtube . emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5 ml lidded microtube . gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5 ml lidded microtube . collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min . obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n immediately after chilling the microtube , obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube . dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult .", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x objectives ) equipped with a 12v100whal - l halogen lamp . \n record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n fit the units with 470 - 495 nm and 565 - 585 nm excitation filters , respectively , and with emission filters that transmit 510 - 550 nm and 600 - 690 nm light , respectively . \n fit the units with 470 - 495 nm and 565 - 585 nm excitation filters , respectively , and with emission filters that transmit 510 - 550 nm and 600 - 690 nm light , respectively .", "the w / o emulsion centrifugation method is illustrated photographically and schematically in figure 1 . the schematic image in figure 1 suggests that the most important determinant of the success of this method is that the specific gravity of the inner aqueous solution must be larger than that of the outer aqueous solution , so that the gvs will precipitate during centrifugation . in addition , the formation of a lipid monolayer at the w / o interface requires that the system be chilled for 10 min after the emulsion is layered on the outer aqueous solution . because the gvs form by transfer of emulsion droplets across the w / o interface , the osmotic pressures in the inner and outer aqueous layers must be the same . as a control experiment , we also prepared gvs containing no microspheres by means of the process shown in figure 1 and step 1.3 , except that the inner aqueous solution was prepared without microspheres . collection of the precipitated gvs after centrifugation is shown in figure 2 . in addition to the semitransparent phase at the very bottom of the microtube , we also observed a white , turbid intermediate phase in the outer aqueous solution ( figure 2a ) . this intermediate phase was rich in aggregations of microspheres and oil , whereas the bottom phase contained the gvs . therefore , after piercing the bottom of the microtube with a pushpin ( figure 2b ) , we collected only the first drop ( figure 2c ) , which contained massive amounts of gvs . it is important to make sure that no more than two drops are collected ; any additional drops may contain aggregations of microspheres and lipids , which will result in a lower density of gvs . the obtained vesicular dispersion often contained encapsulated materials outside of the gvs because the gvs often rupture during centrifugation . to obtain only gvs , a sorting method such as dialysis , gel filtration , or fluorescence - activated cell sorting if necessary for the purpose for which the precipitated gvs are to be used , they can be diluted with the outer aqueous solution . in some cases , the intermediate phase extended to the bottom of the tube , suggesting that any vesicles that formed were held together by the oil . we obtained differential interference microscopy and fluorescence microscopy images of the gvs without microspheres ( figure 3a , 3b ) and with microspheres ( figure 3c-3f ) . lipids conjugated with texas red dhpe , which emits red fluorescence , were used so that vesicle formation could be directly confirmed by visualization of the thin membrane . of the 160 gvs that we obtained , 55 encapsulated microspheres and 105 were empty , giving a ratio of encapsulation of 34% . we determined the volume fraction ( , vol % ) of microspheres in the gvs by means of the following method . because each gv contains dozens to several hundred microspheres , counting all the microspheres under an optical microscope therefore , we mixed the nonfluorescent 1.0 m microspheres with a small amount of fluorescent microspheres , which were manually counted under the fluorescence microscope . the total number ( n ) of encapsulated microspheres was calculated by multiplying the number ( n ) of manually counted fluorescent microspheres by 20 ( based on the original 95:5 [ v / v ] ratio of nonfluorescent to fluorescent microspheres ) . the value of was then estimated as nv100/v , where v is the volume of the microspheres and v is the volume of the individual gv . note that estimation of n from n gives rise to counting errors , and these errors must be taken into account when calculating . the value of was estimated from n , which was directly calculated as 20n , and this in turn resulted in the probability that accurately represents the true value is < 50% . in fact , n fluctuates to some extent around 20n , so we need to consider it as 20(n i ) , where i is the error in n. we estimated i in order that a probability of n i could be more than 50% obtained on the basis of a poisson distribution . we estimated i , which in turn allowed us to calculate 20(n i ) and values of that included counting errors for gvs with diameters of 10 and 15 m ( table 1 ) . according to this procedure , the volume fraction of microspheres in the gv shown in figure 3c was estimated to be 11 3 vol % . our results indicate that we successfully encapsulated 1 m microspheres in gvs at a high volume fraction . we were also able to encapsulate other materials into 100 mol % dopc gvs using the same outer aqueous solution and the same protocol ( figure 4 ) . specifically , gvs containing 0.1 m microspheres were prepared from a tris - buffered solution containing 0.1 m tris - hcl ( ph 7.5 ) , 0.15 m sucrose , and 0.5 vol % fluorescent microspheres by means of the protocol described for gvs containing 1.0-m microspheres ( figure 4a ) . following the protocol described above , dopc gvs containing gfp ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 100 g / ml gfp ; figure 4b ) and gvs containing uranine ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 30 m uranine ; figure 4c ) were also prepared . table 1 : numbers and volume fractions ( , vol% ) of microspheres . errors were determined as described in the text ; n = number of manually counted microspheres ; n = total number of encapsulated microspheres . figure 1 : flow chart and schematic depiction of w / o emulsion centrifugation method . ( a ) oil solution consisting of dopc and texas red dhpe ( 100:0.3 molar ratio ) in liquid paraffin . ( b ) inner aqueous dispersion consisting of sucrose and microspheres in tris - hcl buffer . ( d ) mixture of 1 ml of oil solution and 300 l of inner aqueous dispersion . ( g ) layering of 300 l of the emulsion on 1 ml of the outer aqueous solution . ( i ) schematic depiction of the principle of the w / o emulsion centrifugation method . ( a ) precipitated gvs just after centrifugation ( also depicted in figure 1h ) . the pellet containing the gvs was obtained by piercing the microtube near the bottom with a pushpin and collecting droplets from the hole . ( c ) droplet of the precipitated gvs ( indicated by the yellow arrow ) dispersion diluted with the outer aqueous solution . ( d ) fluorescence microscopy image of 1.0 m microspheres ( yg carboxylate microspheres ) inside a gv . then we estimated total number of inner microspheres ( n = 20 ( n i ) ) was 120 40 . it was calculated that the gv contained 1.0 m microspheres at a volume fraction ( = nv100/v ) of approximately 11 3 vol% , where v is the volume of the microspheres and v is the volume of the gvs . ( f ) merged image of the images in panels d and e. please click here to view a larger version of this figure . differential interference contrast microscopy ( top ) and fluorescence microscopy ( bottom ) images of gvs containing ( a ) 0.1 m microspheres , ( b ) gfp , and ( c ) uranine .", "the specific gravities of the inner aqueous dispersion medium and the outer aqueous solution must be chosen carefully . for the w / o emulsion to precipitate into the outer aqueous solution during centrifugation , the specific gravity of the inner aqueous dispersion medium must be larger than that of the outer aqueous solution . we tried to prepare gvs using inner and outer solutions without sugars , but we obtained no gvs under these conditions , because the inner aqueous solution did not have enough mass to cross the interference between the two phases . if there is a large osmotic pressure difference between the two solutions , gvs that precipitate into the outer aqueous solution may shrink or rupture . therefore , the osmotic pressure inside and outside the gvs must be equal . to accomplish this , we used sucrose as a solute in the inner aqueous dispersion and glucose as a solute in the outer aqueous solution ; both sugars were at the same concentration . both salt22 and sugar233538 have been used for such purposes , but sugar is usually employed because it is less toxic and more soluble than salt . however , if too much sugar is added , the gvs may come into contact with the bottom of the cover glass and collapse . one strategy is to reduce the specific gravity difference between the inner and outer aqueous solutions so that the gvs precipitate under mild conditions ; specifically , the supernatant of the precipitated gv dispersion can be exchanged with a solution that is identical to the inner aqueous solution to reduce the possibility of vesicular rupture by adhesion to the cover glass as a result of buoyancy . because these materials vary in specific gravity , viscosity , and surface tension , different numbers of vesicles form even when the same centrifugation conditions are used33 . to obtain the vesicles with the target properties , it is essential to optimize the specific gravities and viscosities of the inner and outer aqueous solutions as well as the centrifugal acceleration.for preparation of the oil phase , incubation must occur at high temperature and in a dry environment such as an incubator or a dehydrator . in this study , we heated the liquid paraffin to 80 c to completely dissolve the lipid molecules.in addition , the emulsion should be prepared only as needed and should be immediately subjected to centrifugation because it is unstable just after it is prepared and the w / o droplets readily fuse to one another . the emulsion can be prepared in large quantities by sonication , vortexing , or tapping . however , using a homogenizer allows for rapid preparation of large amounts of emulsion and easier emulsification in oil with a high viscosity . it is also important that the emulsion be layered on the outer aqueous solution gently and rapidly and then chilled at 4 c . to shorten the time between emulsification and centrifugation , the oil - outer aqueous solution system can be prechilled before the emulsion is layered on it , and the whole system can then be centrifuged immediately.if the white turbidity appears faster or slower than usual , the mechanical homogenizer must be thoroughly rinsed to remove cleaning detergents . in addition , before emulsification , the oil solution , inner aqueous solution , and emulsifier must be returned to room temperature . , we were able to prepare gvs with a single interior material encapsulated at a high concentration . when the encapsulation of multiple materials is required , it is better to reduce the centrifugal acceleration . for example , an actin assembly system encapsulating seven compounds was achieved with centrifugation at less than 350 x g35 . in cases in which centrifugation is undesirable , gvs can be obtained by adjusting the sugar concentration or by precipitating the emulsion under the influence of its own mass384041 . one is that oil molecules ( paraffin , in this case ) can be solubilized in the gv membrane , as has been pointed out by the weitz group21 . when insertion of a membrane protein into the gv membrane is desired , the effects of co - existing oil molecules on the protein must be considered . , we estimated that the volume fractions of microspheres in the gvs ranged from 4 - 30 vol% ; the volume fractions were not identical to the volume fraction of the inner aqueous solution used for gv preparation . although we were able to encapsulate microspheres in the gvs at a volume fraction high enough for microscopy observation , this method is not suitable for the preparation of gvs with a uniform volume fraction distribution . the w / o emulsion centrifugation method is commonly used for the formation of gvs containing encapsulated materials . recently , molecular robots containing an encapsulated dna device or a molecular device have been constructed4344 . gvs with compartments are the first choice for these kinds of applications ; therefore , techniques , such as ours , that could be used for encapsulating magnetic microspheres and microspheres with diverse surface functionalization can be expected to be useful44 .", "" ]

[ "right sided valvular involvement in infective endocarditis has been well - described , but lesions affecting eustachian valve are distinctly rare . in 1986 , edwards et al . first described the entity of eustachian valve endocarditis ( eve ) in an autopsy study of a patient with overwhelming streptococcal sepsis . in the literature only few cases have been reported so far . herein , we review the literature and describe 2 new cases in which the causative organisms were determined to be vancomycin resistant staphylococcus aureus ( vrsa ) , which is reported for the first time , and staphylococcus hominis .", "a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) . blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) . suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation . transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation . transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava . transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery . she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics . a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) . laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs . tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified . the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative .", "a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) . blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) . suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation . transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation . transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava . transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery . she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics .", "a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) . laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs . tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified . the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative .", "eustachian valve is an embryological remnant of the sinus venosus , directing oxygenated fetal blood from inferior vena cava across foramen ovale , and into the left atrium . in adults , it is non - functional and is considered a benign rudimentary structure . we present 2 new cases of eve with a review of literature [ table 1 ] . in the previously reported cases , the age of patients ranged from 22 years to 76 years with a median age of 44.2 years , with a male to female ratio of 5:3 . there seems to be an increasing trend in elderly population . in the present study , the incidence of eve is not well documented ; however , a retrospective review by san roman et al . reported an incidence of 3.3% in patients with right sided endocarditis . summary of reported cases of eustachian value endocarditis , their location , microbial agents involved , and prognosis a predisposing factor was present in all but three cases,[35 ] with intravenous drug use ( 40% of the cases ) being the most common . other predisposing factors were presence of indwelling catheters , insertion of pacemaker wires , a history of rheumatic heart disease , and immunologic compromise ( chronic alcoholism , human immunodeficiency virus [ hiv ] ) status . the increasing prevalence of indwelling catheters / devices may be the reason for increasing incidence of eve in the elderly population . in the present study , a history of intravenous drug use was obtained in one case , and insertion of implantable venous catheter for repeated blood transfusions in the other case . the blood culture revealed staphylococcus aureus to be the most common pathogenic organism in 53% of the cases , which is consistent with previous reviews sawhney et al . the association of staphylococcus aureus positivity on blood cultures with intravenous drug abuse ( ivda ) and indwelling catheters was found to be in 100% of the cases . other organisms that have been reported so far include staphylococcus hominis , enterococcus cloacae , escherichia coli , proteus vulgaris , streptococcus viridans , klebsiella pneumonia and actinomyces israeli . in our present report , this is the first case of vrsa eve reported in the literature . in the second case , staphylococcus hominis , a harmless commensal on human skin was the causative organism , likely related to the implantable venous access catheter insertion for chemotherapy ( which is reported for the first time also ) . like many other coagulase - negative staphylococci , staphylococcus hominis may occasionally cause infection in patients with compromised immune system . both cases in our series the finding was in concordance with previous studies in which the vegetations were seen more frequently on tee than tte . as the eustachian valve is situated posteriorly , the superiority of tee over tte is seen frequently . only in reports by punzo et al . and in 2 other cases reported by georgeson et al . , tte was frankly misleading , suggesting a ruptured chordae tendinae in one patient , and chiari 's network in the other . tee allows not only better visualization of the eustachian valve and chiari network , but also easily differentiates pathological masses from these normal structures . nevertheless , tte remains the first imaging investigation of choice ; and , tee should be performed if clinical picture strongly suggests endocarditis and no lesions are identified on tte . though eve was first described in a post - mortem autopsy case , eve seems to follow a benign clinical course presenting as right sided endocarditis , resolving with a 4 - 6 week course of culture sensitive antibiotics , as seen in 78% of patients described till date . few patients had a transient worsening of symptoms requiring intubation , open heart surgery with removal of eustachian valve .", "it should be strongly considered when a patient has the clinical syndrome of right - sided endocarditis , but no vegetations are identified on tte . we found the incidence of eve to be highest in young intra - venous drug users , and staphylococcus aureus to be the most common microbial agent . however , over the past 6 years eve has been seen more frequently in the elderly population with culture positivity for diverse microbial agents . although eve is rare , it may be unwise to rule out this diagnosis based solely on tte , especially in the setting of persistent bacteremia or pulmonary emboli ." ]

[ "x - ray micro computed tomography ( mxct ) has many applications in various fields including \n medicine , geology , materials science , dentistry , art and archaeology , among \n others . the methodology combines a non - destructive and \n noninvasive analysis with the ability to quantify the geometrical characteristics of \n irregular specimens such as bones , archaeological remains , teeth , etc . the technique is \n constantly being improved through hardware and software developments and the subsequent increase in \n resolution provides researchers with new information and approaches for the \n investigation of the material properties . in the dental field , mxct has been extensively used to quantify the geometrical changes \n in the root canal anatomy during endodontic therapy , comparing pre and post \n instrumentation analyses . root canals receive a chemo - mechanical treatment with \n successively larger endodontic files ( figure 1 ) in \n order to prepare the anatomy and surface of the root canal to support the dental posts . \n this is a very demanding task as endodontic files must cut the dentin and remove the \n dental debris , preserving the curvature of the root canals without fracture . root \n fracture is the biggest complication during instrumentation , jeopardizing the outcome of \n endodontic therapy . despite the evolution of endodontic files over the last \n four decades and the large number of available endodontic systems , no system is free of \n problems and drawbacks . b : depth marking \n facilitates determination of the exact position of the file on x - ray . c : silicon \n rubber for working length marking . color coding indicated according to iso \n requirements ( yellow : n 20 , red : n 25 , blue : n 30 . ) . e : \n the number of engraved rings on the shaft determines the taper of the instrument . \n one ring represents taper .02 , 2 rings for .04 and 3 rings for taper .06 . f : \n specially designed shaft to be fitted in the handle of the low torque motor . the \n scale on the left is in mm endodontic files are made from different grades of austenitic stainless steel or ni - ti alloys characterized by a variety of geometrical \n features such as taper , cross - section , helix and rake angle , and the distance between \n the successive cutting blades . these \n features are critical factors in the mechanical resistance of endodontic files to load \n forces ( i.e. , bending and torsion ) , their cutting capacity , and the clearance of dental \n chips . the introduction of the ni - ti alloy in file \n manufacturing was followed by the development of a variety of new cross - sectional \n designs with an increased taper compared to the 0.02 employed in k- and h - type files \n made of stainless steel as dictated by the international standard organization ( iso ) \n specification 3630 . nevertheless , the determination of other important \n geometrical features such as the surface area and volume of the endodontic files remain \n unknown , limiting the assessment of other parameters derived from these basic features . \n precise estimation of the basic geometrical features might provide an additional \n approach to explain the differences in the clinical performance among various file \n designs and establish a valuable tool for development of new and more efficient \n geometrical designs . apart from the k - files , where the surface area and volume can be calculated based on the \n untwisted tapered blank values employing mathematical formulas , the calculation of these \n features was impossible before the introduction of mxct . this technique has been \n extensively used to determine the geometrical features , such as shape , surface area and \n the volume of the root canals before and after treatment with endodontic \n instruments but has never been utilized to determine the geometrical features of \n endodontic files . the aim of this study was to determine , for the first time , the surface area , volume and \n specific surface area of successive file sizes of a commercially available ni - ti system . \n additionally , we tested the hypothesis that mxct is capable of discriminating the \n quantitative differences in the aforementioned geometrical properties between the \n successive sizes of endodontic files . these differences exist by definition due to the \n progressively increased size of the successive files used in root canal therapy . the \n hypothesis tested was that there are significant differences in the surface area , volume \n and specific surface area among the different sizes of endodontic files .", "three sets of flex - master ni - ti files ( vdw ; munich , germany ) , 6 files each ( iso sizes \n n 20/lot 0411310306 , n 25/lot 0410310302 and n 30/lot \n 0406310290 ) all of 0.04 taper , were utilized in this study . to avoid any inclination \n from the vertical axis , the files were placed on the stage , employing a custom made \n attachment and then scanned by a mxct scanner ( skyscan model 1072 ; aartselaar , belgium ) \n operated under the following conditions : wo ka source ( 100 kv , 98 a ) , 2.36 \n m pixel size , 180 rotation , 0.9 rotation step , 1.9 sec exposure time \n averaging by two frames , and 1 mm al filter . the surface area and volume of all files \n were determined from the tip up to the 16 mm level , taking 6,782 horizontal slices . the \n files were successively scanned using the automated routine of oversize mode , for 16 h \n each . a fixed threshold was applied to discriminate the crosssection from the air , \n providing high contrast images of the horizontal cross - sections . the surface area ( sa ) and \n volume ( v ) of each file were determined by the embedded software ( ctan , skyscan , \n aartselaar , belgium ) employing the following formulas : with n = number of slices , s = slice thickness , pi = measured periphery in slice i \n and ai = measure area in slice i the specific surface area ( the surface to volume ratio ) was also calculated . the \n percentage differences between the successive file sizes for the aforementioned \n properties were calculated based on the formula : horizontal slices of all scanned files were also inspected for the presence of internal \n defects such as cracks or pores . all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable . linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis .", "all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable . linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis .", "figure 2 demonstrates two representative \n horizontal sections ( perpendicular to the longitudinal file axis ) from the mxct . the \n left image has been taken from a region near the handle and the right from a region near \n the cutting tip of a ni - ti file , demonstrating the difference in the cross - sectional \n surface area . the cross - section was found to be free of internal defects such as pores \n or cracks . left and right images show two horizontal cross sections of the same file . the right image has a smaller cross- \n sectional surface , as it is closer to the cutting tip the quantitative results for the surface area , volume and specific surface area of \n successive file sizes are presented in table 1 . \n the surface area of the n 30 files showed the highest value followed by \n n 25 and n 20 . the increase in total surface area was 8.9% between \n n 20/n 25 and 16.6% between n 25/n 30 files , \n demonstrating close to a 100% increase for the latter . similar results were found for \n the volume of the ni - ti files tested . however , the increase in volume declined towards \n the larger file sizes . the percentage increase was estimated as 21.1% between n \n 20/n 25 and 17.2% between n 25/n 30 ) . the file n 20 \n showed a significantly higher specific surface area followed by n 25 and \n n 30 . the specific surface area showed a 10.1% decrease between n 20/n 25 , but \n only 0.4% between n 25/n 30 . regression analysis revealed a strong \n linearity between the surface area ( figure 3a ) and \n volume ( figure 3b ) versus file sizes . the raw \n data , the fitted line , the 95% confidence intervals , the yielded analytical formula , and \n the regression coefficient ( r ) are all presented in figures 3a and 3b for the \n surface area and volume , respectively . mean values and standard deviations for the surface area ( sa ) , volume ( v ) , and \n specific surface area ( ssa ) of flexmaster ni - ti files . the percentage differences \n ( dif ) between successive file sizes are also presented the same superscripts indicate mean values without statistical significant \n differences ( p>0.05 ) raw data , fitted line with the 95% confidence intervals , mathematical equations \n and liner regression coefficients ( r ) , for the surface area ( a ) and \n volume ( b ) of the flexmaster ni - ti system", "the results of the current study showed significant differences for the surface area and \n volume between the files tested , thus the original hypothesis is accepted . it is \n noteworthy that this analysis provided significant differences for the endodontic files \n with different iso sizes , demonstrating the ability of this analysis to discriminate the \n quantitative differences of the geometrical characteristics among the different \n endodontic file sizes . this discrimination has also been facilitated due to the \n increased resolution of 2.36 m obtained in this study . the transition to scanner \n technology has a beneficial effect on the resolution of mxct from 1000 m for \n conventional tomography , which was insufficient for endodontic applications , to roughly 30 m , providing the ability \n for more extensive research , especially in the root canal anatomy after chemo - mechanical \n treatments . however , beyond the technological advancement , the \n resolution of a specific analysis with a given mxct scanner is limited by the size of \n the object , as it is magnification dependent . this is a disadvantage in addition to the \n restriction that all horizontal projections produced during rotation must be fitted to \n the maximum horizontal width of the ccd camera . the mxct device used in \n this study has a maximum isotropic resolution of 1.8 m but the maximum \n achievable isotropic resolution for endodontic file scanning remained at 2.36 m , \n due to dimensional constraints . the scanning of an alloy with a homogeneous elemental \n atomic ratio and a uniform x - ray \n absorption through its bulk facilitates image contrast and thus the selection of the \n proper threshold level to discriminate the edges of the cross - sections . all tested files \n were found free of internal defects such as pores or cracks , a finding which is in \n agreement with previous cross - sectional and mxct analyses . despite the increased isotropic resolution available through technological advancements , \n the accuracy of the quantitative analysis remains unknown , at least for endodontic \n applications dealing with the quantification of the geometrical features of the root \n canals and endodontic files . it is logical to assume that this accuracy is dependent on \n the isotropic resolution and might increase towards smaller isotropic resolutions . \n however , such an approach requires the development of specific standards made of the \n same material as the unknown sample , in order to preserve the same contrast . this could \n be a milestone for the application of mxct in the quantification of the geometrical \n features and could be an interesting subject for future research . the surface area and volume of the endodontic files , as presented for the first time by \n the present study , was found to increase towards higher file sizes with significant \n differences between the successive file sizes ( table \n 1 ) . linear regression analysis showed a linear correlation of both the surface \n area and volume with the nominal file sizes . the surface area and volume are not \n physically dependent on the nominal iso sizes and the equations presented in figure 3 are simply an engineering relationship \n between two different physical magnitudes . finally , the aforementioned equations are not \n universal and should be used exclusively for the tested ni - ti endodontic system . for the \n other ni - ti files , linear models with different coefficient values are anticipated . although the geometrical configuration and design of the ni - ti files are important \n factors in controlling the clinical performance of these micro - instruments , the \n assessment of their clinical performance with experimental testing remains \n questionable . some studies provide results with great differences , \n not only among the different brands but also within one brand and type . the main \n weakness of the currently available experimental protocols is that the clinical \n performance of the endodontic files has been biased as to the influence of various \n factors and has been determined separately . on the other hand , the \n results of the current study do not have any immediate clinical implications , as they \n must be combined with further experimental results of the clinical properties ( such as \n cutting efficiency , loading of dental chips , etc . ) in order to determine any possible \n correlation . for instance , it is logical to assume that a file with a greater surface \n area has the increased ability of loading dental chips but this requires experimental \n documentation . the surface area and volume between the successive files showed significant differences \n and thus there is no overlap between the successive file numbers , as occurs with the \n first diameter ( d1 ) below the tip . as expected , both the surface area and \n volume show an increase towards the higher file sizes . the percentage difference between \n n 25/n 30 ( 16.6% ) is almost double the value of n 20/n \n 25 ( 8.9% ) files sizes , whereas the percentage increase in volume was similar ( 21.1% and \n 17.2% for n 20/n 25 and n 25/n 30 , respectively ) showing \n a completely different increasing pattern . it should be mentioned that the percentage \n differences in the surface area show an abrupt increase towards the larger file sizes , \n while volumetric changes exhibit a declining trend . this implies that the surface area \n and volume progress towards larger files with a completely different pattern , a finding \n reflected by the values of the specific surface area ( figure 3 ) . the latter is a property that defines the total surface area \n per unit of bulk volume and has significant importance in cases \n where the surface area has a significant effect on the system studied . the n 20 \n files have the highest specific surface area , indicating that more surface area is \n exposed per volume unit than in n 25 and n 30 sizes . \n this value is characteristic of the geometrical design and it is expected to vary \n significantly among different file designs . files with a non - standard taper ( i.e. , \n protaper ) are anticipated to demonstrate increased specific surface area due to the \n significant reduction in file volume . it is noteworthy that beyond the estimation of the surface area and volume of the \n scanned files , the 2d and 3d analysis of the scanned files provides tremendous \n opportunities . firstly , all the geometrical features ( i.e. , cutting angle , depth of \n flutes , crosssectional diameter ) measured in longitudinal and cross - section analyses , \n with optical and electron microscopy can be readily measured with mxct methodology . this \n analysis is advantageous over traditional techniques , as it is non - destructive and thus \n there is no need for specimen preparation ( embedding , cutting , metallographic polishing , \n conductive coating ) . computational analysis of 3d models can be used to quantify any \n selected geometrical feature for any region of interest . for instance , the real surface \n area of a file working in a root canal can be easily estimated , selecting the relative \n region of interest . secondly , 3d modeling of scanned files can provide more accurate \n models for subsequent finite \n element analyses . on the other hand , \n the drawbacks of mxct methodology are the inferior resolution compared to optical and \n electron microscopy and prolonged scanning times for the model reconstruction in high \n isotropic resolution . another limitation is that the image quality might be affected by the beam \n hardening phenomena . normally , an x - ray beam contains photons with a vast range of \n energies . in physical metallurgy , the beam hardening phenomenon refers to the absorption \n of lower energy photons during the passing of a beam through a metal object and once \n this happens , \" beam hardening \" occurs as the mean energy of a beam increases . in this \n study , to minimize the beam hardening effect an al filter was utilized and a beam \n hardening correction routine was applied . the former was used to pre - harden the beam by \n cutting the lower energy photons , while the latter is a correction algorithm which was \n applied during the reconstruction process . the estimation of the surface area and volume \n of endodontic files might provide a new approach for the characterization of \n clinically - related properties such as cutting efficiency , loading of dentin debris etc . \n this study demonstrates that mxct is a powerful tool with interesting applications for \n endodontic research . continuing technological advancements allowing development of more \n powerful mxct scanners should accelerate the applications of this methodology in the \n near future .", "the surface area and volume demonstrated an almost linear increase , while the specific \n surface area demonstrated an abrupt decrease towards the higher file sizes . for the \n first time , we have shown that mxct is capable of discriminating the quantitative \n differences in the geometrical properties of the successive sizes of endodontic files . \n this type of analysis should be used to investigate the unexplored correlations of the \n various clinical properties of the geometrical characteristics of the endodontic files , \n opening new avenues for research in the field of endodontics .", "the authors would like to thank the research group program for funding this research \n project . this study has been funded by a research grant ( # rgp - vpp-206 ) from the \n research group program , deanship of scientific research , king saud university , \n riyadh , saudi arabia ." ]

[ "this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university . we recruited 123 patients , and all patients gave written informed consent to participate in the study . the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) . genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) . genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) . blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test . total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) . a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . , piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp . promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment . patients glucose challenge responses and serum hormone profiles were compared with the test or the student t test with welch correction . ratios of patients with glucose levels exceeding the threshold were analyzed with the test . all data were presented as mean sem , and the statistical significance cutoff value was 0.05 .", "this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university . we recruited 123 patients , and all patients gave written informed consent to participate in the study . the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) .", "genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) . genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) .", "blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test . total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) .", "a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . , piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp . , madison , wi ) . promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment .", "", "patients glucose challenge responses and serum hormone profiles were compared with the test or the student t test with welch correction . ratios of patients with glucose levels exceeding the threshold were analyzed with the test . all data were presented as mean sem , and the statistical significance cutoff value was 0.05 .", "to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) . the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes , 59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s . residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) . the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations . on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies , select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies . because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) . snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . * the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) . fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig . a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig . 1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig . analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig . this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) . differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations . polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa ) the location of snps in the gip gene region is indicated by a green rectangle . the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel . b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset . population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria . the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations . ( a high - quality color representation of this figure is available in the online issue . ) interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig . in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig . because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well . frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn . most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition . population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria . ( a high - quality color representation of this figure is available in the online issue . ) importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig . a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) . the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) . by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore , these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria . * frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) . because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation . we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig . measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig . because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected , coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig . importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) . together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations . the gip promoter reporter activity is haplotype - dependent . a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter . the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked . genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter . b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 . the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . , , equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection , cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp . the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells . ( a high - quality color representation of this figure is available in the online issue . ) given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence . we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 . the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively . in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 . measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively . consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig . by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) . gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests . a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test . the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype . b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype . association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio . significantly different from patients with a gip haplotype ( test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants . we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig . moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis .", "to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) . the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes , 59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s . residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) . the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations . on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies , select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies . because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) . snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . * the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) . fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig . a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig . 1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig . analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig . this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) . differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations . polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa ) the location of snps in the gip gene region is indicated by a green rectangle . the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel . b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset . population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria . the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations . ( a high - quality color representation of this figure is available in the online issue . ) interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig . in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig . because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well . frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn . most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition . population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria . ( a high - quality color representation of this figure is available in the online issue . ) importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig . a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) . the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) . by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore , these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria . * frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) .", "because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation . we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig . measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig . because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected , coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig . importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) . together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations . . a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter . the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked . genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter . b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 . the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . , carlsbad , ca ) . in each well , equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection , cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp . , hercules , ca ) . the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells . ( a high - quality color representation of this figure is available in the online issue . )", "given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence . we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 . the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively . in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 . measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively . consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig . by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) . gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests . a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test . the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype . b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype . association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio . * significantly different from patients with a gip haplotype ( test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants . we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig . moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did . therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis .", "on the basis of studies of signatures of selection , in vitro promoter assays , and glucose challenge tests in humans , we show that it is possible to identify causal variants related to energy - balance regulation by focusing on genic snps that were subject to environmental selection in a subset of candidate genes . specifically , we demonstrated that gip variants at the 5 gene region represent metabolic modifiers that contribute to phenotypic variation in gip response and glucose metabolism . further characterization of these causal variants would open a new venue for understanding the molecular mechanisms that underlie phenotypic variations in energy - balance regulation and improve our ability to stratify and interpret clinical outcomes associated with the gip signaling pathway . for decades , adaptive selection was assumed to be rare ; however , recent studies have demonstrated that adaptive substitution is pervasive in human genomes ( 1,7,37 ) . despite this progress , it is obvious that population differentiation characteristics of human genomes have yet to be fully explored because physiologic consequences of almost all of these past gene environmental interactions remain to be verified experimentally ( 1,2 ) . on the other hand , because many selection pressures could be heterogeneous or reversible in a short time , the signature of selection may have eroded in genes that were responsive to cultural selection pressures ( 12,38 ) compared with those shielded from heterogeneous selection ( e.g. , the adaptation to environmental oxygen levels and altitude ) ( 5,39 ) . we therefore reasoned that important metabolic modifiers could be hidden in the trove of snps that showed limited evidence of positive selection and that this limitation could be particularly pertinent to modifiers associated with adaptations in response to shifts of subsistence cultures . consistent with this hypothesis , a survey of earlier studies of genome - wide or chromosome - wide positive selection using the so - called outlier approaches in which candidate loci are identified in the extreme tails of empiric distributions ( 40)showed that gip variants have not been reported as positively selected ( 1,7 ) . the selection of gip variants was inferred after we focused the analysis on local genomic regions and assessed the significance of integrated haplotype score using coalescent simulations ( 22 ) . therefore , our investigation provided a proof - of - concept study for identifying causal mutations that underlie phenotypic variation of complex disease - related traits . this approach could open new venues for improving the translation of common variant association signals into biologic mechanisms that underlie physiologic variability or disease risk . neel hypothesized that mismatches between prior adaptations and new environments , or a conflict of adaptations , could lead to changes in fitness or health risks ( 11 ) . because ancient variants could have been selected for the organism s reproductive success but not for its health or longevity , the ancient alleles could confer disease risks as selection pressures change . therefore , studies of positively selected variants that are associated with adaptations in energy - balance regulation could point not only to novel genotype phenotype relationships but also to novel molecular mechanisms that mediate the potential phenotypic variation , thereby providing much - needed insight into how and which phenotypic variations in energy - balance regulation can be attributed to the selected variants . in support of the thrifty genotype hypothesis , human capn10 and house - mouse insulin genes have been shown to exhibit characteristics of adaptive evolution after the emergence of agricultural societies ( 41,42 ) . conceptually , the high gip response associated with the ancestral gip haplotype could have been a beneficial energy - conserving mechanism when the food supply was irregular . the ancestral haplotype could become deleterious in the last 10 millenniums as agricultural practice became widespread . one possible deleterious effect of the ancestral gip haplotype in an environment that supplies abundant high - starch food resources is the hypersecretion of insulin and insulin resistance ( 43,44 ) . on the other hand , a reduced gip response associated with the derived gip haplotype could be protective by decreasing the extent of insulin secretion in the face of oversupply of energy inputs ( 45,46 ) . in support of this speculation , it has been well documented that in the absence of modern medicine , diabetes - associated complications and , possibly , obesity posed detrimental effects on survival when human culture shifted ( 47 ) , even though type 2 diabetes is generally considered a chronic disease in modern society . alternatively , an elevated glucose level associated with the derived haplotype may have improved the survival of fetuses if the population faced serious famine an event frequently experienced by agricultural societies ( 48)despite the reality that an impaired glucose - tolerance response represents a risk to both the mother and fetus under normal circumstances . moreover , the derived haplotype could have been beneficial by reducing the obesity - promoting effect of gip ( 16 ) . in vitro and in vivo studies have shown that gip promotes fatty liver and other obesity - associated metabolic disorders , whereas gip antagonists suppress lipid accumulation induced by a high - fat diet ( 23 ) . therefore , the derived gip haplotype could be selected for its effects on the enteroadipocyte or the enteroinsular axis , or both . although we speculated that the derived gip haplotype may have provided protective effects in famine - plagued agricultural societies , the observation that the derived haplotype has not been fixed in any population suggests that the selection of the derived gip haplotype(s ) ( e.g. , cycles of famine ) could be opposed ( balance selection ) as populations experienced temporal changes in selection pressure ( e.g. , resumption of population growth with stable food supply ) . alternatively , the derived gip haplotype could simply be too young to become fixed , or the spread could be limited by the transgenerational effects associated with abnormal gestational glucose metabolism , which raise the risk of macrosomia and diabetes in the offspring ( 11 ) . although an association between gip variants and glucose - metabolism regulation has not been reported , gipr variants were associated with glucose and insulin levels after challenge tests as well as with bmi in gwa studies that evaluated > 29,000 individuals ( 35,36 ) . the finding that patients with a homozygous gip genotype have significantly higher glucose levels compared with those carrying an ancestral gip haplotype within the pool of gipr homozygotes suggested that there is a confounding effect stemming from interactions of gip and gipr variants , and that gip and gipr variants represent novel markers for the stratification of the capability to maintain glucose homeostasis during pregnancy . we also speculate that the significant results observed in pregnant women could be related to the fact that the success of pregnancy has a significant impact on reproductive fitness and that a major fraction of gene environmental selections probably occurred before birth ( 49 ) . recent studies have corroborated this idea by showing that associations between many risk alleles and type 2 diabetes can be replicated with smaller sample sizes in patients with gestational diabetes mellitus ( 50,51 ) . furthermore , given the evolutionary signatures at the gip locus , the plausible molecular mechanism , and the significant results in east asian women , we speculate that the gip variant mediated phenotypic divergence could also exist in most human populations . it is also important to note that the selection of gip variants represents a unique example in which the selection process involves regulatory variants that alter the glucose - induced gip response as well as a nonsynonymous variant that affects peptide bioactivity ( 22 ) . thus , the gip signaling pathway could represent a hotspot for selection in recent human history and play an important role in the manifestation of phenotypic variation in energy - balance regulation among individuals . in addition to gip variants , our study identified several cdkal1 , cyb5r4 , gad2 , and pparg variants as potential metabolic modifiers . recent studies have shown that variants in cdkal1 , pparg , and more than two dozen genes are associated with glycemic traits in diabetic patients ( 10 ) . surprisingly , none of the cdkal1 , cyb5r4 , gad2 , and pparg variants identified here have been implicated in earlier gwa studies , which suggests that these variants could be related to novel energy - balance regulatory mechanisms that operate at certain life stages or under specific physiologic conditions that have not been specifically investigated . future investigations of these variants could reveal additional metabolic modifiers that have arisen recently and their contributions to phenotypic variation in normal human physiology and metabolic syndrome related traits . in conclusion , our data demonstrated a strong association between regulatory gip variants , and gip response and glucose metabolism , reinforcing the indication of an important role of gip signaling in diabetes - related traits from earlier gwa studies of gipr . importantly , our study also provided a novel approach to reveal metabolic modifiers by studying consequences of previous mismatches of physiologic capabilities and environments .", "" ]

[ "the conventional definitions of pediatric adiposity depend on a measured body mass index ( bmi , kg / m ) interpreted relative to a reference distribution ( bmi normative growth charts ) for sex and age [ 13 ] . because it has body weight in its numerator , bmi reflects generalized ( total - body ) enlargement with a simplified correction ( as height ) for skeletal size . pediatric adiposity has been defined alternatively by abdominal size , most commonly a waist circumference , because increased truncal adipose tissue is correlated better than generalized adiposity with cardiometabolic dysfunction [ 4 , 5 ] . since waist circumference is a regional measurement , it reflects specifically only abdominal or central enlargement . thus , waist circumference also requires interpretation relative to its own reference distribution for sex and age . comparisons of risk assessments in youth have generally found little difference between bmi and waist circumference in the ability of these indicators to identify cardiometabolic risk [ 6 , 7 ] . the waist - to - height ratio ( whtr ) is an adiposity indicator with waist circumference in the numerator and a simplified correction ( as height ) for skeletal size . whtr does not depend on sex- or age - specific reference criteria [ 810 ] . in a large sample of us sixth - graders , we examined the performance of bmi z - score ( bmiz , referenced to cdc 2000 growth charts for the united states ) and whtr for the purpose of cardiometabolic risk assessment .", "participants came from the baseline enrollment ( sixth - grade students in 2006 ) of the healthy study , a cluster - randomized , controlled , primary prevention trial designed to improve indicators of adiposity and glycemic dysregulation among us middle - school children [ 12 , 13 ] . seven research centers recruited 42 middle schools with at least 50% of students eligible to participate in the federally subsidized national school lunch program or belonging to an ancestral minority group at increased risk of type 2 diabetes ( hispanic , non - hispanic black , or native american ) . a detailed protocol and background details about the healthy study are available for download at http://www.healthystudy.org/. we restricted a priori our analytic sample to students with integer ages of 11 or 12 years who were able to participate in physical education and did not have known diabetes . from 5950 eligible enrollees , we excluded 13 students due to missing or invalid data for the adiposity indicators ( height , weight , and waist circumference ) . additional 239 students were excluded for lack of outcome cardiometabolic risk variables , and 216 were excluded for lack of information on pubarche ( an adjustment variable associated with adrenarche , growth pattern , and insulin resistance ) [ 1416 ] . participant ancestry ( hispanic , non - hispanic black , non - hispanic white , other ) and sex were self - reported . the study was approved by each research center 's institutional review board , and informed consent from parents and assent from students were obtained prior to data collection . from measured height and weight , we calculated the sex- and age - specific bmiz based on the centers for disease control and prevention 2000 growth charts [ 11 , 17 ] . waist circumference was measured to the nearest 0.1 cm on bare skin just above the iliac crest following procedures of the national health and nutrition examination survey , and the whtr was calculated . blood pressure was recorded three times using an omron automated blood pressure monitor ( with appropriate - size cuff ) after the participant sat quietly for 5 minutes . pubarche was identified dichotomously from the participants ' response to a standardized question on the appearance of underarm and pubic hair . fasting blood samples were processed onsite and shipped to the healthy central blood laboratory ( northwest lipid research laboratories , university of washington ) . insulin was measured by an immunoenzymometric assay using a tosoh 1800 autoanalyzer ; the interassay and intra - assay precision analysis consistently showed a coefficient of variation ( cv ) < 10% . the assay had low cross - reactivity with human c - peptide ( 0% ) and proinsulin ( 2% ) . the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from glucose ( mg / dl ) and insulin ( u / ml ) concentrations using the following formula : \n ( 1)homa - ir=(glucose0.05551)(insulin)22.5 . measurements of total plasma cholesterol , cholesterol in the lipoprotein fractions , and triglycerides were performed enzymatically on the roche modular - p autoanalyzer using well - standardized methods . the interassay cvs were consistently < 1.5% for total cholesterol and triglycerides and < 2% for hdl cholesterol . we calculated the total - to - hdl cholesterol ratio ( tc / hdlc ) , a variable that strongly predicts cardiovascular disease in adults and may be more strongly associated than ldl - cholesterol or hdl - cholesterol concentrations with pediatric adiposity [ 22 , 23 ] . in addition to stratification by sex , we chose a priori to stratify our analyses by high fatness or lower fatness because the ability of adiposity indicators to identify adipose tissue mass , ectopic fat , and cardiometabolic risk variables may be stronger among children in a higher fatness category [ 2326 ] . as defined for this report , the high - fatness level included students who were above the sex - specific median value for both bmiz and whtr ; any student below the median for either adiposity indicator was designated lower fatness . for each fatness level , we prepared sex - specific , linear - regression models adjusted for ancestry ( 4 categories ) and pubarche ( yes / no ) to estimate the associations of continuous adiposity indicators with the continuous cardiometabolic risk factors ( outcomes ) . since blood pressure varies with height in children [ 27 , 28 ] our models for blood pressure outcomes included an additional adjustment for height which was entered as a continuous variable . for all variables except bmiz , we calculated descriptive statistics without using reference - based corrections for sex or age . for indicators or outcomes that departed markedly from a normal distribution ( whtr , homa - ir , tc / hdlc , hdl cholesterol , triglycerides , and ( only for lower - fatness students ) diastolic blood pressure ) , we transformed the variable by loge or inverse square root to approach normality prior to their use in regression models . our adjusted models estimated standardized beta coefficients ( change in the outcome variable ( in standard deviations ) associated with change of one standard deviation in the adiposity indicator ) for each cardiometabolic risk factor . cary , nc ) was used to account for variability both within and between the school clusters . in these mixed models , the proportion of variation explained ( r ) by each adiposity indicator was calculated as the full model r minus r for a model omitting the adiposity indicator . to compare linear slopes among ancestral groups , mixed - regression models estimated nonstandardized , beta - regression coefficients ; interactions were tested between each adiposity indicator and the three ancestries represented prominently in our sample ( hispanic , black , and white ) .", "characteristics of the analytic sample are presented by sex in table 1 . as expected for sixth - grade students , following further stratification by fatness level , the distributions of adiposity indicators and ancestry are summarized in table 2 . compared to the high - fatness groups in our sample , the lower - fatness groups had bmi and whtr distributions that resembled more closely the general population of us youth in the same age range [ 31 , 32 ] . within the high - fatness subpopulations of either sex ( table 3 ) , adiposity indicators explained 19%28% of the variation in homa - ir , 4%9% of the variation in circulating lipids ( tc / hdlc , hdl cholesterol , triglycerides ) , and 5%9% of the variation in diastolic blood pressure . adiposity indicators explained < 3% of the variations in systolic blood pressure and fasting glucose . for each outcome variable in these high - fatness subpopulations , the effect sizes ( standardized beta coefficients ) associated with bmiz were similar to those associated with whtr . within the lower - fatness subpopulations of either sex ( table 4 ) , adiposity indicators explained 8%13% of the variation in homa - ir and 2%7% of the variation in circulating lipids . for tc / hdlc and triglycerides , the standardized beta coefficients tended to be weaker for bmiz ( 0.130.20 ) than for whtr ( 0.170.28 ) . adiposity indicators in these lower - fatness subpopulations explained < 1% of the variations in systolic blood pressure , diastolic blood pressure , and fasting glucose . a comparison between the two levels of fatness ( table 3 versus table 4 ) demonstrates that for either adiposity indicator the associations with homa - ir were stronger among the high - fatness students ( beta coefficients 0.430.52 ) than among the lower - fatness students ( 0.300.37 ) . similarly , both adiposity indicators were associated with diastolic blood pressure more strongly among high - fatness students ( 0.230.32 ; p < 0.001 for each of 4 beta coefficients ) than among lower - fatness students ( 0.030.05 ; p > 0.05 for each of four beta coefficients ) . for identification of lipid outcomes , however , we found steeper beta coefficients only among high - fatness boys ( compared to lower - fatness boys ) whose adiposity was assessed by bmiz . for both sexes assessed by whtr , the associations between adiposity indicators and risk variables were not notably different between the hispanics , non - hispanic blacks , and non - hispanic whites except when related to blood pressure outcomes . although adiposity explained 5%7% of variation in diastolic blood pressure in the complete sample of high - fatness girls ( table 3 ) , this relationship was extremely weak for high - fatness girls who were black , as indicated by slope point estimates close to zero ( figure 1 ) . however , for high - fatness girls who were hispanic or white , bmiz and whtr had significant associations ( p < 0.05 ) with diastolic pressure . among high - fatness boys systolic blood pressure was not significantly associated with bmiz or whtr for high - fatness blacks of either sex , but the association was present for high - fatness students who were hispanic or white . an ancestral contrast ( blacks compared to whites ) related to systolic blood pressure was significant , however , only among the high - fatness girls assessed by bmiz ( figure 1 ; p < 0.01 ) .", "in this large sample of middle - school students at increased risk of obesity and type 2 diabetes , we found that adiposity indicators bmiz ( with reference to cdc 2000 growth charts ) and whtr ( without reference to sex and age ) had similar utility for identifying adverse levels of cardiometabolic variables . our findings are generally consistent with previous published reports , most of which were based on populations that had a wider age range or included less ancestral diversity . earlier cross - sectional studies that compared continuous whtr against bmi either without a normative growth reference [ 3335 ] or with reference - based bmi z - scores / percentile ranks [ 23 , 36 ] generally found that whtr provided stronger associations with lipid outcomes , but bmi was superior for blood pressure outcomes . a recent report on sixth - grade students from switzerland found that bmiz ( referenced to cdc 2000 growth charts ) and whtr provided associations with blood pressures that were weak but nearly identical . in studies of youth from the southern us , bmiz provided a slightly stronger association than whtr with homa - ir and fasting insulin , a relationship that was complicated by nonlinearity . nationally representative , cross - sectional data from the us demonstrated that whtr at ages 417 years was more strongly associated than bmiz with resting heart rate . a longitudinal analysis from the same survey of adolescent and young - adult participants found that categorical whtr predicted all - cause mortality before age 55 better than categorical bmiz ( baseline ages 1218 years ) or bmi ( ages 1939 ) . from the united kingdom , a large study recently reported that whtr and bmiz obtained at ages 79 years had similar associations with cross - sectional and prospective cardiometabolic risk factors in adolescence . given an approximately equal utility of bmiz and whtr for pediatric health assessments , we should consider how these adiposity indicators might perform in different settings or in the future . bmiz values reported in the research literature depend on standardized protocols for measuring height and weight using calibrated , high - quality scales . in nonresearch settings , however , staff training and time pressures might not be so favorable to careful measurements . the dependence of bmiz on normative growth references can be problematic because bmi - for - age reference values can yield discrepant inferences between populations , time periods , and ethnicities within a single country . worldwide bmi growth reference based on large datasets from six countries , but subsequent reviews found that this international growth reference provided no advantage over national bmi growth references for the definition of excessive fat mass in youth or prediction of subsequent cardiovascular risk in adulthood . the world health organization ( who ) more recently developed a bmi - based growth reference , the utilization of which has been described as a cumbersome task in need of simplification . surveys from various clinical settings have found generally that the use of bmi - for - age reference values is suboptimal [ 4750 ] . advocates of the whtr must address problems associated with the available protocols for measuring waist size . while tape measures are inexpensive and generally need little calibration , protocols for circumference measurement are still unfamiliar to many pediatric practitioners or clinic assistants . our study carefully measured the waist circumference just above the iliac crest , an anatomic location endorsed by prominent researchers in the united states [ 18 , 51 ] and canada . the who , however , recommends measuring waist circumference at the approximate midpoint between the lower margin of the last palpable rib and the top of the iliac crest . minimal waist , and other sites [ 54 , 55 ] . in an anthropometric study of diabetic youth , the iliac - crest protocol and who protocol demonstrated comparable reproducibility , but these alternative protocols yielded notable differences in the absolute value of a waist circumference obtained from the same participants . a study of overweight youth found that the who waist - circumference protocol had a stronger association than the iliac - crest protocol with cardiometabolic risk , and studies of adult waist circumference have likewise suggested an advantage for the who protocol [ 5860 ] . it follows that the whtr values calculated from the iliac - crest and who protocols should not be casually substituted for each other . it is possible that if our healthy study had adopted the who instead of the iliac - crest protocol for its baseline anthropometry , the re - calculated whtr indicator might have demonstrated stronger associations with cardiometabolic risk variables than those we report in this paper . standardization of a single waist - circumference protocol would probably advance the widespread adoption of the whtr as a low - cost adiposity indicator [ 55 , 61 ] . as an alternative to the circumference , some pediatric investigators have described waist size in selected participants by measuring the external diameter sagittally ( back - to - front ) in the supine position [ 6264 ] . protocol might further enhance studies that are cross - sectional or involve short - term follow - up of central adiposity , but this anthropometric method needs to be tested in larger datasets that represent general youth populations . the physiological importance of tissues accumulated in the waist may help to explain why whtr was more closely associated than bmiz with variations in the levels of circulating lipid markers among our lower - fatness participants ( table 4 ) . an increase in waist size primarily marks expanding amounts of adipose tissue , including notably the visceral depot which is most strongly associated with an adverse metabolic phenotype [ 5 , 65 ] . variation in the waist circumference can explain more than 64% of the variance in the area or volume of visceral adipose tissue . an increase in bmi , by contrast , may substantially mark also the variations in gain of muscular weight or subcutaneous fat patterning that precede adulthood . along with the changes in fat mass , these variations in lean mass or superficial adipose tissue contribute to the bmi calculation while contributing relatively little to metabolic risk . although high - fatness hispanic and white girls in our study demonstrated the expected associations between adiposity and blood pressure , we found among the high - fatness black girls ( but not black boys ) that neither bmiz nor whtr had a significant association with blood pressure outcomes ( figure 1 ) . in comparison to young hispanics and whites , young blacks tend to have abdominal adipose tissue relatively less in the visceral depot and more in the abdominal subcutaneous regions . another study has also reported that black girls ' waist circumference around the same age was unrelated to their diastolic blood pressure . since both the bmiz and whtr indicators demonstrated similar patterns of nonassociation with black girls ' blood pressure , it may be that this absence of a correlation with blood pressure is due to factors operating primarily outside the abdominal region . perhaps black girls benefit from an increased capacity to expand their lower - body ( gluteofemoral ) , subcutaneous , adipose - tissue stores in a manner that would increase their total body weight yet protect them from cardiometabolic risk [ 7073 ] . if this protective characteristic of black girls extends into their later years , it could help explain why adult black women in the us experience no increased cardiometabolic risk or mortality until their bmi reaches approximately 33 kg / m .", "if a well - standardized waist measurement comes into widespread use for clinical assessments of pediatric adiposity , patients and their families may improve their intuitive understanding of how excess adiposity contributes to adverse health risk . pediatric health care providers , too , may find it more useful to recognize risk associated with a waist increment ( corrected for height ) than with a weight increment ( corrected for height squared ) . adoption of the whtr could optimize both patient education and the tracking of risk . compared to bmi , the whtr allows a simpler calculation without the necessity of squaring the child 's height . of interest to those concerned with child adiposity and cardiometabolic risk observed in different cultures or distinct time periods , the whtr will facilitate comparisons based directly on anthropometric observations without using normative reference tables that may not be suitable to all populations [ 7779 ] ." ]

[ "the bucky shuttle being the combination of nanosize carbon structures fullerene and nanotube , has many possible applications : nanoscale storage cells , devices for directed medicine transfer and containers for effective and safe gas storage [ 7 - 13 ] . nanosize containers and capsules of various shapes that allow reaching a higher safety level and mass content of gas stored have been investigated for a number of years [ 11 - 13 ] . the engineering of nanostructured carbon opens the ways for the production of nanocapsules of complex structural shapes [ 14 - 16 ] . in this work , the processes of methane molecule adsorption , storage and desorption by the nanocapsule are investigated with molecular - dynamic modeling method . the nanocapsule - specific structure defines its adsorption qualities : at the storage stage under normal conditions , the nanocapsule contains the amount of methane that was adsorbed at normal temperature and under 40 mpa . the nanocapsule desorption takes place at the temperature elevation up to 350 k. there is no need to apply electric field during storage and desorption .", "methane adsorption , storage and desorption processes were modeled with the method of molecular dynamics . the values of hydrogen and carbon atom charges in methane molecule were obtained using the combination of hartree fock and becke exchange with lee yang the following atom charge values in methane molecule were obtained : carbon atom 0.628204 mulliken and hydrogen atom + 0.157051 mulliken . 1 . nanocapsule for natural gas storage , consisting of storage chamber , junction and blocking chamber the nanocapsule consists of three parts : storage chamber , junction and blocking chamber . the junction consists of the nanotube ( 10,10 ) and nanotube ( 8,8 ) , moreover , the nanotube ( 8,8 ) is connected with the storage chamber , and nanotube ( 10,10 ) with the blocking chamber . the blocking chamber is opened and closed by the transfer of the k@c60 endohedral complex under electrostatic field action . the charge of + 1|e| of the k@c60 endohedral complex is uniformly distributed over the c60 shell . the nanotube ( 8,8 ) in the junction prevents the k@c60 from entering the storage chamber . the nanotube ( 8,8 ) diameter is rather large for the penetration of methane molecules , but small for the transition of the k@c60 . each hole in the blocking chamber is formed as a result of removing 24 carbon atoms . dangling the holes obtained are large enough for free penetration of methane molecules into the nanotube internal space . the experiment on obtaining similar holes with the application of electron beams is described in . it is shown that the beams can be focused on the area 1 in diameter . the holes in the nanotube can exist at the temperatures up to 400 k ; when the temperature elevates , the hole diameter in the nanotubes considerably decreases due to the motion and fusion of single vacancies [ 8 - 10 ] . during modeling , it is imitated that the nanocapsule is placed on the substrate , i.e. , the nanotube base is fixed the nanocapsule left end the change in the nanotube diameter is also possible with the methods of nanostructural engineering . the charged endohedral complex k@c60 moves in the blocking chamber and junction under the action of external electric field . the electric field direction defines the nanocapsule state in the operation cycle : methane adsorption , its storage and desorption . the value of external electric field intensity , required for the k@c60 to move , equals 3.045 10 v / m . the motion of charged fullerene in the nanotube with the help of electric field is described in detail in .", "the nanocapsule operation can be split into several stages : methane adsorption , storage and desorption . at the adsorption stage ( fig . 2 ) , the k@c60 endohedral complex is retained at the end of the blocking chamber under the action of van der waals forces . the methane molecules from the environment freely penetrate through the three holes into the blocking chamber and adsorb into the external space of the storage chamber . adsorption stage ( t= 300 k , p = 40 mpa ) the calculations are based on the following formula ( 1 ) : where number of methane molecules , nc number of carbon atoms in the nanotube , mass of one methane molecule and mc mass of one carbon atom . figure 3 demonstrates the nanocapsule filling dynamics with methane at 300 k and under 40 mpa . it is clearly seen that 4 ps is enough to complete the filling of the nanocapsule storage area . thermodynamic conditions are p = 40 mpa and t = 300 k to block methane molecules in the storage chamber , it is necessary to move the k@c60 ion into the junction with the help of electric field as shown in fig . 4 . external thermodynamic conditions are t = 300 k and p = 40 mpa . further motion of the k@c60 is blocked by the junction narrow part nanotube ( 8,8 ) . nanocapsule closing stage ( t = 300 k , p = 40 mpa ) figure 5 demonstrates the dependencies of the change in the k@c60 position and its kinetic energy under the electric field action in the blocking chamber upon time . a sharp increase in the k@c60 kinetic energy equaled to 0.254 ev is observed at 0.5 ps , at the same time the velocity reaches 327 m / s . = 1 ps , the k@c60 kinetic energy decreases significantly , reaching 0.000255 ev and further up to 15 ps its value does not considerably increase . this is explained by minor oscillations of the k@c60 near the right end of the blocking chamber . the k@c60 is retained due to van der waals forces . under the constant action of electric field , the k@c60 kinetic energy increases again and reaches 0.61 ev at t = 16.5 ps , i.e. , the k@c60 breaks off the blocking chamber and moves to the storage chamber to block its entrance . results of molecular - dynamic modeling of the k@c60 motion in the blocking chamber under the electric field action at the adsorption stage . the k@c60 ion position with respect to the initial ( time = 0 ps ) ion position as a function of time . the change in the k@c60 ion kinetic energy as a function of time in the time period from t = 16.5 ps to t = 26.5 ps , the considerable attenuating oscillations of the kinetic energy conditioned by the k@c60 motion along the blocking chamber walls adjacent to the junction entrance are observed . in this time period , each peak of the k@c60 kinetic energy corresponds to the time moments after passing the pentagonal rings in the structure of the blocking chamber . under the electric field action , the k@c60 penetrates into the right end of the junction nanotube ( 10,10)and blocks the outlet of methane molecules from the storage chamber . during the penetration , a considerable increase in the kinetic energy is observed , its maximum value reaches 0.63 ev at t = 32 ps . after the k@c60 passes the nanotube ( 10,10 ) , the kinetic energy sharply decreases conditioned by the k@c60 deceleration in the portion of the nanotube ( 8,8 ) . in the interval from t = 41.5 ps to t = 48.5 ps , the insignificant fluctuations of the k@c60 position connected with the compressed gas pressure from one side and electric field action from another the value of the k@c60 kinetic energy does not exceed 0.033 ev . in the process of methane molecules adsorption , the maximum velocity of the k@c60 motion is 515.5 m / s ( t = 32 ps ) . when transferring to the storage stage , the electric field switches off , and external thermodynamic conditions are brought to normal . the k@c60 , which is located in the junction , moves to the blocking chamber under the methane pressure . however , its transfer is insignificant ( d~5 a ) that is conditioned by the necessity to overcome the considerable energy barrier ( e = 90 kcal / mol ) to move to the blocking chamber . the calculations made show that there are no abnormal extensions of bonds between carbon atoms in the nanocapsule . methane storage stage ( t = 300 k , p = 0.1 mpa ) at the desorption stage when the temperature elevates from 300 k up to 350 k and the external pressure is normal , the k@c60 endohedral complex in the junction is pushed into the blocking chamber under the expanding methane pressure , as shown in fig . methane under pressure in the storage chamber freely desorbs into the external space through the holes in the blocking chamber . the availability of three holes in the blocking chamber and their configuration allow forcing out methane molecules and preserving the required rigidity of the construction . the holes are located in such a way that the k@c60 , moving under the electric field action in the blocking chamber , is unable to block all its holes at once . the k@c60 in the blocking chamber does not prevent methane desorption as the gas molecules flowing out do not touch it , and it is retained near the wall of the blocking chamber adjacent to the junction inlet nanotube ( 10,10 ) due to the action of van der waals forces without the external electric field involved . methane desorption stage ( t = 350 k , p = 0.1 mpa ) figure 8 demonstrates the dependencies of the k@c60 location stage and its kinetic energy upon time at the temperature elevation from t = 300 k up to t = 350 k at the stage of the k@c60 desorption into the blocking chamber . at 3 ps , the significant increase in the k@c60 kinetic energy equaled to 2.05 ev ( the k@c60 velocity reaches 930 m / s ) is observed . the k@c60 kinetic energy is consumed for overcoming the considerable energy barrier ( e = 90 kcal / mol ) formed under the action of capillary forces . when the k@c60 passes the energy barrier , the kinetic energy decreases considerably to 0.41 ev ( t = 5 ps ) . after passing the junction at 7 ps the k@c60 , kinetic energy increases again and when the k@c60 gets into the blocking chamber , the oscillations between its walls are observed . each zero value of the k@c60 kinetic energy in the interval from 7 to 23 ps corresponds to the k@c60 impact against the blocking chamber wall . then , the insignificant impacts of the k@c60 against the blocking chamber wall with further state stabilization near one of the pentagonal rings of the blocking chamber are observed ( fig . 7 ) the maximum kinetic energy is 2.05 ev , which is considerably smaller than the known value ( 200 ev ) required for carbon nanotube destruction . the availability of even insignificant number of methane molecules in the blocking chamber considerably decreases the velocity of the k@c60 motion , and , respectively , the kinetic energy of the k@c60 impact against the blocking chamber wall . results of molecular - dynamic modeling of the k@c60 transfer at the temperature elevation up to 350 k. k@c60 ion position with respect to the initial ( time = 0 ps . ) the change in the k@c60 ion kinetic energy as a function of time the methane molecules desorption process is shown in fig . the desorption stops at 25.4 ps , there are 53 methane molecules in the storage chamber , which is 1.38 mass%", "we demonstrated the functioning of the nanocapsule of complex structural shape for methane storage using the method of molecular dynamics . an obvious advantage of the nanocapsule is its operation cycle : methane is adsorbed under the elevated pressure ( 40 mpa ) and at normal temperature with further blocking of methane molecules by the k@c60 endohedral complex in the nanocapsule with the external electric field , the storage is performed in normal external conditions , and methane desorption is performed at temperature elevation up to 350 k , at which methane molecules push out the k@c60 and are desorbed from the nanocapsule . methane content in the nanocapsule at the storage stage is ~11.09 mass% . at the storage and desorption stages , the electric field is not used , this significantly simplifies the use of nanocapsules in automobile applications . the multiple techniques of nanostructural engineering developed are the prerequisites for the creation of similar nanocapsules .", "calculations are made in interdepartmental supercomputer center of the russian academy of science ( moscow ) ." ]

[ "it is an unusual \" atypical \" antipsychotic , as the \" atypical \" profile of the new antipsychotics clozapine , olanzapine , quetiapine , and risperidone has been linked to combined antagonism of serotonin 2 ( 5-ht2 ) and dopamine 2 ( d2 ) receptors , whereas amisulpride has negligible affinity for 5 ht2 receptors and is specific for dopamine d2 and d3 receptors in the limbic rather than striatal structures . there are reports of amisulpride being used for the management of bipolar disorders . however , there are very few reports of mania induced by amisulpride . here we describe a young male who developed mania while on amisulpride , a newer antipsychotic drug introduced in india .", "a 18-year - old unmarried hindu male presented with a history suggestive of schizophrenia for the last 2 years . the illness had an acute onset with complaints of suspiciousness , hearing voices not heard by others , violent abusive behavior , disturbed biological functions , and decreased self care . he was started on risperidone 4 mg along with trihexyphenidyl 2 mg and lorazepam 6 mg per day . gradually , over a period of 1 month , he showed improvement in the symptoms . subsequently , trihexyphenidyl and lorazepam were tapered and he was maintained on risperidone 4 mg per day for the next 1 and a half year . however , he would still be lethargic , would prefer to remain alone and would be inattentive at class . he would not interact with others and not take active part in various household functions . he was prescribed amisulpride for the negative symptoms along with risperidone 4 mg that was continued . it was initiated at 50 mg per day and was increased to 100 mg per day after 4 days of initiation it . after 10 days of 100 mg dose , the patient developed a manic episode , characterized by decreased need for sleep , over talkativeness , hyperactivity , persistent elevated mood , disinhibited behavior , over - grooming , and distractibility . amisulpride was stopped and he was prescribed lorazepam 4 mg on as and when required basis . the patient was followed up after 1 week and during this time there was substantial improvement in his manic symptoms with ymrs score of 17 .", "to the best of our knowledge of mania due to first amisulpride was reported by murphy in 2003 . however , in the case described by murphy , the patient was also on citalopram , an antidepressant , though its discontinuation did not led to improvement in manic symptoms . also , the patient was initiated on olanzapine immediately which would also probably have antimanic effects . in our case , use of the naranjo adverse drug reaction probability scale and edward 's criteria both indicate a probable relationship between the manic episode and short - term exposure to amisulpride therapy . the manic symptoms with amisulpride can be postulated to be due to the fact that amisulpride in low doses ( < 10 mg / kg ) , preferentially blocks presynaptic d2/d3 receptors , resulting in enhanced dopamine transmission . increased dopamine metabolites and increased dopamine transmission this case report highlights the fact that close monitoring of patients on amisulpiride should be done for manic symptoms . this is also important because of the fact that atypical antipsychotic drugs ( including amisulpride ) are used for the treatment for bipolar disorder . open label studies have shown that amisulpride may be useful for patients with bipolar disorder . also , as low doses enhance dopamine transmission , and high doses reduce dopamine transmission , it might be prudent to start amisulpride at the recommended target dose at the initiation of therapy ." ]

[ "aging leads to senile diseases , including alzheimer disease ( ad ) , which has become a serious medical and socioeconomic problem . therefore , progress in early diagnosis and effective treatment of ad is necessary , especially in the preliminary or early stages of the disease . most elderly people experience physiological memory disorders ; these disorders require a careful clinical assessment , as it is extremely difficult to delineate the line between physiology and pathology . thus , when disorders of memory and other cognitive functions go beyond the standards of age and educational level , but have not yet reached the standards of dementia , we diagnose mild cognitive impairment ( mci ) , which includes several states that either match the standard in its wide meaning , for example age - associated memory impairment ( aami ) , age - related cognitive decline ( arcd ) , or we diagnose pathology like mci , which , although clinically similar , has a different prognosis than aami and arcd [ 14 ] . currently , base on criteria established by the mayo clinic group , mci is define as a transitional state between normal aging and alzheimer s disease , in which memory impairment is greater than expected for age , but general cognitive function and daily living activities are preserved . people suffering from mci , especially amnestic form ( amci ) , are at risk for developing dementia , particularly alzheimer disease ( ad ) [ 35 ] . according to various reports , the percentage of conversion from amci into ad ranges from 1% to 25% per year , while the mean time of dementia progression from the mci diagnosis is 4.4 years . obviously , the early identification of patients at risk for alzheimer disease from among those with mci would facilitate proper treatment help to delay the symptoms of dementia . therefore , searching for methods that would help us detect a higher risk of dementia in those patients , as well as looking for objective methods of early diagnosis of patients with mci , seems reasonable . many reports confirm a typical repeatable topographical location of neuropathological lesions at various stages of ad , as described by braak et al and pantel et al . , while emphasizing early involvement of the limbic system , including the hippocampus and entorhinal cortex . earlier pathology in these strategic regions in patients with a higher risk of alzheimer - type dementia , as well as in certain patients with amnestic mci , seems likely . atrophy of the medial temporal lobe in mci patients with a positive correlation of atrophy level and a risk of conversion to ad , inadequate for age and observed in imaging examinations ( ct , mri ) , has been clearly demonstrated [ 813 ] . the level of atrophy is judged by descriptive , volumetric and planimetric methods , or indirectly with the use of linear measurements of particular fluid spaces , such as fissures or certain parts of the ventricular system . despite many years of experience in structural diagnosis of the dementia diseases , the use of functional methods seems to be very helpful . in spect and pet examinations we observe the lowering of perfusion and metabolism of glucose and oxygen in temporal and parietal areas in patients with possible ad , as well as in those with mci . however , these methods have certain limitations such as relatively low linear resolution , which hinders precise determination of the topography of the measuring ( this problem relates to the middle temporal structures and not to the hemispheres cortex ) . a wider clinical application of magnetic resonance spectroscopy ( mrs ) , described by bootomley as a window for metabolism enabled an intravital assessment of regional metabolic disorders in certain structures of the brain . there have been several reports on such dysfunctions in the limbic system in patients with alzheimer disease . a relatively small number of spectroscopic examinations have been carried out on patients with mci . some of these were presented by catani et al . , who located voxels in the white matter at the ventricular triangle level and kantarci et al . who examined the posterior part of the cingulate gyrus in patients with mci and ad . however , these are the locations where anatomopathologic changes , typical for ad , appear later than in the medial temporal lobe ( including hippocampal formation ) . many authors give up on spectroscopic examinations of hippocampal structures due to their technical limitations , such as the size of examined structures and topographical relations of the surrounding area . the problems mentioned most often are : difficulties with field homogeneity , artifacts from cranial base area , a disqualifying weakening of the signal - to - noise ratio , and the need to reduce the volume of interest ( voi ) . there have been reports on the technical capacities of such examinations in various brain diseases [ 1923 ] . according to available the available literature , in the majority of papers h1mrs in mci and ad metabolites were measured in the posterior cingulated gyrus , but parts of papers evaluated the concentration of metabolites in the bilateral hippocampi of mci patients . this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up .", "this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up .", "the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science . all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations . patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded . mci diagnosis was established by a team of specialists after analyzing all available information and test results . mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner . morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence . we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence . morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders . changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study . the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation , it was divided into 2 areas the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi . necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm . additionally , each subject had frontal measurement taken . h mrs examination was carried out with a single - voxel method using press sequence . routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used . the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression , spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 . assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm . the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases , it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx ) were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard . results analysis of clinical and biochemical data age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level was performed including initial data of all subjects enrolled into the study . evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case . a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed . the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package .", "the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science . all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations . patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded . mci diagnosis was established by a team of specialists after analyzing all available information and test results .", "mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner . morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence . we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence . morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders . changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study . the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation , it was divided into 2 areas the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi . necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm . h mrs examination was carried out with a single - voxel method using press sequence . routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used . the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression , spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 . assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm . the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases , it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx ) were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard .", "results analysis of clinical and biochemical data age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level was performed including initial data of all subjects enrolled into the study . evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case . a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed . the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package .", "all patients had annual clinical follow - up at least twice . at the beginning of the study , subjects were divided into 2 groups 9 subjects who had amnestic mci , and the others who had multidomain mci . there were no statistical differences between groups of patients with multidomain mci and amnestic mci , including age , m - m score , level of vitamin b12 , folic acid and homocysteine ( p>0.05 ) . comparison of mean value in some selected parameters including both groups is presented in table 1 . during clinical follow - up ( median 3 years ) 8 subjects had stable disease ( sd ) , 13 had progression of disease ( dp ) and 10 developed ad . there was no significant difference between age , m - m score , vitamin b12 , folic acid and homocysteine level among these 3 groups of subjects ( p>0.05 ) . statistical analyses of baseline metabolic ratios measurement using h mrs between the final 3 groups of patients , ( sd , dp and ad ) found significant difference in frontal lobes in mi / h20 ratio on left , between patients with stable disease ( 0.27 ) and those with progression ( 0.22 ) ( p=0.03 ) ( figure 1 ) . in the groups of patients with dp and those with conversion to ad , there was a significant difference on the left side in ratio naa / cr ( 1.77 vs. 1.43 ) , ( p=0.02 ) ( figure 2 ) . significant difference within temporal external lobes were found between patients with sd and dp in naa / h2o ratio on the left side ( 0.55 vs. 0.51 ) , ( p=0.04 ) ( figure 3 ) . additionally , there were significant differences between patients with sd and ad in chol / cr ratio ( 0.99 vs. 0.88 ) on the right side ( p=0.04 ) and in mi / cr ( 1.09 vs. 0.62 ) on the left side ( p=0.03 ) . a significant difference within the medial temporal lobe was found between patients with dp and ad in glx / h2o ratio ( 0.44 vs. 0.34 ) on the right side ( m - w u - test , p=0.02 ) . there were no other significant differences within h mrs metabolic ratios . an example of h mrs study in patient with initial diagnosis of mci and clinical deterioration during 2 years of follow - up with final diagnosis of ad is presented in ( figure 4 ) ( a initial h mrs , and after final diagnosis , control h mrs study b ) . summary of initial h mrs ratios of some metabolites in frontal , external and medial temporal lobes both sides are presented in table 3 .", "proton magnetic resonance spectroscopy h mrs enables an in vivo noninvasive assessment of the degree of biochemical disorders in a specified voi of the examined tissue , as well as monitoring the progress of those changes in the course of the disease and its treatment [ 5,1725 ] . the method we used , along with the parameters mentioned above , resulted in satisfactory quality of spectra in examined patients . recent studies have demonstrated the value of h1mrs in many types of cognitive disorders , not only in degenerative diseases . in mrs diagnosis , normal tissues present a constant proton spectrum , while ratio changes of the metabolites can be considered as reflections of certain biochemical transformations . several cytological and histochemical investigations have contributed to the study of the relationship between certain chemical substances and precisely defined intracellular structures or biochemical processes physiological as well as pathological . n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells . there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction . it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia . however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group . in our study , we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe . we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe . we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides . this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient . the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects . our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad . others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] . medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur . therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of successive metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al . single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data , numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] . some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) . we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression . others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad . others publications did not report any difference in this metabolite ratio in the group of patients with mci . the differences in results of various groups of researchers could be explained by selection of the study population . the same research group indicated different results , dependent on study group for instance , no difference was found in selected population of patients with amnestic mci compare to a heterogenous population with significant difference in mi signal . some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis . this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear . there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low . recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve . additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction . the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group . the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe . our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad . this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres . this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad . impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously . in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx . this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences . detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease . according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results . some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement . this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy .", "n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells . there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction . it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia . however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group . in our study , we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe . we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe . we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides . this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient . the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects . our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad . others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] . medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur . therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of successive metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al . single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data , numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] .", "some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) . we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression . others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad . others publications did not report any difference in this metabolite ratio in the group of patients with mci . the differences in results of various groups of researchers could be explained by selection of the study population . the same research group indicated different results , dependent on study group for instance , no difference was found in selected population of patients with amnestic mci compare to a heterogenous population with significant difference in mi signal . some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis . this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear . there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low . recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve . additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction . the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group . the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe .", "our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad . this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres . this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad . impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously .", "in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx . this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences . detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease . according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results . some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement . this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy .", "h mrs seems to be a very sensitive method that provides biochemical information using an in vivo approach in patients with initial mci , who in significant numbers developed disease progression and/or converted to ad . a potential advantage could be achieved by using 3 t systems , which can better discriminate quantization of glu + gln / cr and gln / cr ratios ." ]

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

[ "cyclodialysis cleft is a rare clinical finding , but one that can have significant ocular complications , including decreased vision , shallowing of the anterior chamber , corneal edema , hypotony , choroidal effusions , maculopathy , vascular tortuosity , and optic disc edema . while it can be caused by surgical manipulation of the eye , it is more frequently a consequence of trauma.1 traumatic injury to the eye results in separation of the ciliary body from the scleral spur , which creates an aberrant outflow pathway for aqueous humor through the suprachoroidal space and often leads to hypotony . cyclodialysis cleft has been reported to occur in 3.4% of cases of ocular injury or globe rupture.2 additionally , one large review of 291 cases involving cyclodialysis suggested that hypotony occurs 9% of the time.3 medical treatment with atropine ophthalmic drops and laser therapy to promote reattachment of the ciliary body to the scleral spur are options for repair of this entity , but larger and more chronic cyclodialyses often require surgical repair.4,5 because of their rarity , reports on surgical repair techniques in the literature are limited . we share a novel , simple surgical approach to management of a case of chronic traumatic cyclodialysis cleft with a successful outcome .", "a 70-year - old caucasian male followed at the martinsburg veterans affair hospital ophthalmology clinic , martinsburg , west virginia , usa , since 2002 was found consistently to have intraocular pressure ( iop ) asymmetry , with the right eye iop ranging from 615 mmhg and usually having an iop 04 mmhg lower than the left eye . he was monitored for primary open - angle glaucoma and was on latanoprost eye drops in the left eye for field defects in that eye . in april 2008 , the patient s history was significant for trauma to the right eye with a piece of wood over 30 years ago , which was thought to be the cause of the cleft . while the patient had hypotony , he maintained good visual acuity , ranging from 20/25 to 20/40 , and was therefore observed for approximately 3 years until december 2011 . at this visit , his visual acuity dropped to 20/150 and he clinically developed descemet s folds with a shallow anterior chamber and an iop of 02 mmhg . on dilated fundus exam , he had a shallow choroidal hemorrhage and effusion in the far nasal periphery . the patient was initially treated medically with atropine and prednisolone acetate 1% ophthalmic drops for approximately 2 months , without improvement in iop and with persistence of choroidal hemorrhage and effusion . postoperatively , the choroidal effusion resolved , but the patient s iop did not improve . on repeat gonioscopy therefore , the patient was taken for additional surgical repair of the persistent cleft by the same technique , this time with successful closure . on postoperative day 1 following the second surgery , the patient complained of severe eye pain and nausea , and was found to have an lop of 58 mmhg . the patient was treated with topical anti - glaucoma drops and oral acetazolamide , with reduction in the iop to the mid - teens and resolution of his pain . on exam , there was no distortion of the pupil by sutures and the anterior chamber appeared deep ( figure 1 ) . the patient was subsequently tapered off the oral acetazolamide and topical drops , with stabilization of the pressure in the mid - teens and maintenance of 20/70 visual acuity at 1 year postoperatively . final visual acuity was limited by subsequent progression of ocular comorbidities in that eye , including cataract and proliferative diabetic retinopathy . a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft . a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy . interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass , the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull . additionally , as more sutures were placed , the eye appeared to become more firm . we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required .", "a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft . a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy . interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass , the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull . additionally , as more sutures were placed , the eye appeared to become more firm . we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required .", "multiple approaches to cleft closure have been described in the literature , including medical management , laser photocoagulation , cryotherapy , trans - scleral diathermy , gas tamponade , and anterior scleral buckle , along with various surgical repair techniques.6 while different surgical approaches have been described , many are technically challenging , particularly in the hypotonous eye . in this case report , we discuss the repair of a chronic traumatic cyclodialysis with indirect surgical cyclopexy , which we found to be technically easier and less invasive than other reported techniques , but equally successful . compared to the direct cyclopexy described in a review by ioannidis and barton,6 our indirect technique eliminated the need for direct visualization of the ciliary body , thereby reducing technical difficulty along with bleeding and wound dehiscence risk . additionally , compared to cyclopexy described by mccannel,7 our approach eliminated the additional need for clear corneal stab incision with retrieval of suture , which can also be difficult in a soft , hypotonous eye and may prolong surgical time . while our technique was successful , it required two operations to achieve successful cleft closure with improvement in iop . our first attempt failed , likely due to the spacing of our sutures in one particular quadrant , which we determined were too far apart . additionally , we used a needle with less curvature and length initially . we found that spacing the sutures approximately 1 mm apart achieved good apposition of ciliary body to scleral spur and , ultimately , closure of the cleft with restoration of physiologic iop . the length and curvature of the cs175 - 6 ( ethicon , cincinnati , ohio , usa ) needle also assisted with passage of each suture through the targeted tissues . the patient s postoperative iop spike after the second surgery was suggestive of successful closure of the cleft.1 additionally , successful closure was later confirmed by gonioscopy . another challenge to our case was the chronicity of the injury which was sustained 30 years prior to presentation as per the patient s self - reported history . there was concern that , even with successful anatomic closure , iop and visual function may not be restored due to chronic ischemic changes to the ciliary body . however , our repair achieved both anatomic and functional success , suggesting that even chronic cyclodialysis clefts can be effectively repaired . delgado et al reported a similar finding in a case of hypotony maculopathy secondary to cyclodialysis that was resolved with cleft closure 7 years later.8 cyclodialysis is a vision - threatening ocular injury that can often be difficult to treat medically . surgical repair is often necessary but can be technically difficult and challenging , particularly because of how rarely it is encountered in clinical practice . we present a simple , less - invasive approach to surgical cleft closure that was successful anatomically and functionally . additionally , we demonstrate clinical success with cleft closure in an eye with chronic hypotony ." ]

[ "acoh ( 3.00 mmol ) was added to a solution of galactoside 20 or 13 ( 1.57 mmol ) in acetone ( 3.5 ml ) and meoh ( 4.7 ml ) at 0 c . after 8 h , the reaction was quenched by the addition of nahco3 ( 5.95 mmol ) and then filtered . after concentration of the filtrate under reduced pressure , the resulting oil was purified by flash column chromatography ( etoac in hexane ) to afford alcohol 21 ( from 20 ) or 26 ( from 13 ) as a colorless oil . pph3 ( 1.86 mmol ) , diad ( 1.86 mmol ) , and dppa ( 1.86 mmol ) were added sequentially to a cooled solution of alcohol 21 or 26 ( 0.90 mmol ) in thf ( 20 ml ) . concentration under reduced pressure followed by purification of the residue by flash column chromatography ( etoac in hexane ) afforded azide 22 ( from 21 ) or 25 ( from 26 ) as a colorless oil . tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to a solution of azide 22 ( 200 mg , 0.19 mmol ) in ch2cl2 ( 5 ml ) at rt . after 30 min , the reaction mixture was concentrated under reduced pressure to afford pentaol 9 as a colorless oil ( 115 mg , quant . ) : [ ]d + 12.4 ( c 0.5 , cdcl3:cd3od , 2:1 ) ; max(film ) ( cm ) 3282s br ( oh ) , 2114s ( n3 ) , 1696 m ( c = o ) ; h nmr ( 300 mhz , cdcl3:cd3od , 2:1 ) 0.85 ( t , j = 6.0 hz , 3h ) , 1.181.39 ( stack , 22h ) , 1.401.71 ( stack , 4h ) , 1.44 ( s , 9h ) , 3.26 ( a of abx , jab = 12.6 hz , jax = 4.9 hz , 1h ) , 3.513.64 ( stack , 3h ) , 3.653.98 ( stack , 7h ) , 4.89 ( d , j = 3.3 hz , 1h ) ; c nmr ( 100 mhz , cdcl3:cd3od , 2:1 ) 14.3 ( ch3 ) , 23.2 ( ch2 ) , 26.4 ( ch2 ) , 28.6 ( ch3 ) , [ 29.9 , 30.2 , 32.4 , 32.8 ( ch2 , resonance overlap ) ] , 51.7 ( ch ) , 51.8 ( ch2 ) , 68.3 ( ch2 ) , 69.3 ( ch ) , 70.4 ( ch ) , 70.6 ( 2 ch , resonance overlap ) , 72.5 ( ch ) , 75.3 ( ch ) , 80.1 ( c ) , 156.8 ( c ) ; ms ( tof es+ ) m / z 627.3 ( [ m + na ] , 100% ) ; hrms ( tof es+ ) calcd for c29h56n4o9na [ m + na ] 627.3945 , found 627.3956 . tfa ( 1.0 ml , 13.2 mmol ) was added dropwise over 5 min to azide 22 ( 400 mg , 0.38 mmol ) at rt . the resulting colorless oil was used in the next step without further purification ( 192 mg , quant . ) . tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to azide 9 ( 114 mg , 0.19 mmol ) at rt . the resulting colorless oil was used in the next step without further purification ( 96 mg , quant . ) ." ]

[ "ventriculoperitoneal and cystoperitoneal ( cp ) shunts are commonly used for the treatment of hydrocephalus and symptomatic intracranial cysts . abdominal complications , including intestinal volvulus , pseudocyst formation and migration into the gastrointestinal tract have been reported , accounting for 25% of shunt - related complications . bowel perforation by the shunt catheter is uncommon and accounts for 0.010.07% of all abdominal complications [ 13 ] . breast - related complications represent a class of thoracic shunt complication and are characterized by breast cerebrospinal fluid ( csf ) pseudocyst formation , csf galactorrhoea and shunt obstruction . we report a rare case of acute mastitis caused by enteric organisms passing through a cp shunt catheter , which had penetrated the transverse colon , and was related to a previous failed attempt to remove the shunt resulting in a divided catheter .", "the patient is a 56-year - old woman who underwent a surgical treatment of a lateral ventricle meningioma at the age of 29 . the postoperative course was uneventful and she was well without shunt - related problems for 26 years . she developed a brain abscess related to the shunt catheter at the age of 55 . a surgical removal of the catheter was attempted , but was not technically possible , and only the segment from the clavicle to the breast was removed with ligation of the two remaining ends . a year later , she was taken to a local hospital with continuous right - breast pain . the diagnosis of acute mastitis was made and she was referred to our institution for further examination . on presentation , her temperature was 36.7c , and laboratory tests revealed a decreased platelet count of 103 000/l , elevated ast of 97 u / l , alt of 72 u / l and c - reactive protein level of 1.4 mg / dl . cultures taken from the abscess showed enteric organisms including enterococcus avium , klebsiella oxytoca and bacillus . abdominal ct showed that the shunt catheter was in the splenic flexure of the transverse colon with no other abnormalities ( fig . 2 ) . colonoscopy was performed to survey the colon and the shunt catheter was found inside the lumen at the splenic flexure ( fig . acute mastitis caused by enteric organisms which had migrated through the shunt catheter after penetration into the transverse colon was the presumptive diagnosis , and the catheter removed under general anesthesia . the breast to the abdomen segment of the catheter was exteriorized through the right - anterior chest wall without laparotomy . the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) . figure 3:colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) . an inflammatory polyp was present at the penetration site in the colon wall ( b ) . the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) . colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) . an inflammatory polyp was present at the penetration site in the colon wall ( b ) .", "the penetration of shunt catheters into the intestine at the distal end is rare with a reported incidence between 0.01 and 0.07% . the mortality rate may be high , up to 15% , due to intracranial or intra - abdominal infections . several factors have been found to be responsible for this rare complication , including chronic irritation of the gastrointestinal tract , prior abdominal surgery or silicone allergy [ 2 , 3 ] . csf leakage and retrograde flow from the peritoneal cavity to the breast may cause breast - related complications . this is the first report of case with acute mastitis caused by enteric organisms migrating through a cp shunt catheter that had penetrated the transverse colon . the diagnosis of bowel perforation by a shunt may not be easy to make , unless the shunt protrudes from the anus [ 5 , 6 ] . prolonged diarrhea of unknown etiology , as well as abdominal symptoms , serves as warning signs of possible bowel perforation . ct can play a crucial role in the diagnosis of bowel perforation . in the present case , these findings suggested that the infection was due to the retrograde flow from the colon . the development of mastitis was facilitated by previous division of the catheter , leaving a ligated end . presumably , the ligature became dislodged , leaving a direct connection from the breast tissue to the distal end of the catheter . previous study reports that the shunt catheter can be directly removed percutaneously in 69% ( 31/45 ) , whereas 17% ( 8/45 ) of patients require a laparotomy and repair of the bowel . if there is evidence of significant abdominal infection , such as an abscess or life - threatening peritonitis , the fistulous opening may not close spontaneously , warranting laparotomy . in the present case , previous insufficient surgical treatment such as a partial removal of the catheter contributed to this shunt - related complication . however , it is common surgical practice to permit the distal end of a shunt catheter to remain in situ in order to simplify the surgical procedure . although this practice is most commonly uncomplicated , it is not unreasonable to suspect that the tube may irritate the serosal surface of the colon leading to chronic inflammation . it is speculated that local inflammation owing to repeated irritation of the bowel wall by the catheter tip might have contributed to the subsequent perforation . in conclusion , a patient who presents with acute mastitis and has previously undergone shunt placement should undergo assessment of the abdominal segment of the catheter ." ]

[ "we explore , in mice , the possibility of diagnosing an early - melanoma tumor and following its growth by analyzing the changes that occur throughout the pathological process in vocs excreted in urine and feces . we report here total of 29 potential biomarker candidates of melanoma ; 25 of these are novel , and 4 have been previously reported by other research groups in relation to vocs detected from cultures of human melanoma cells . the chemical identity of only 24 of the 29 possible biomarker candidates was revealed with high confidence . this study will contribute to the development of a noninvasive and reliable diagnostic procedure to detect melanoma in the urine of patients . melanoma accounts for less than 2% of all skin cancers , but for the vast majority of deaths from skin cancer . in the united states alone , it is estimated that 76,100 individuals will be diagnosed with melanoma during 2014 and that 9,710 patients will die from it . early detection of malignant melanoma is the key factor in reducing mortality from this cancer . although histopathological examination is the gold standard for diagnosis of melanoma , additional , noninvasive approaches should be developed . here , we explore the possibility of exploiting volatile organic compounds ( vocs ) to develop such an approach ; hundreds of vocs are emitted from the human body and usually reflect the metabolic condition of the individual . therefore , pathological processes , such as cancer , are expected to influence the voc fingerprint of patients either by changing the ratio between different vocs or by producing new vocs . it has been reported that dog scan identifies , by using their olfaction sense , melanoma on the skin of patients or melanoma samples hidden on healthy subjects . used gas sensor array ( also known as an electronic nose ) to differentiate between melanomas and nevus lesions . those authors compared the lesion with the adjacent skin region to reduce the skin headspace variability . collected biopsy samples and compared the vocs released from melanoma tissue to those released from nevus and normal tissue , using matching skin as a control . those authors found 32 potential biomarker candidates ; the concentration of 9 vocs increased in the presence of melanoma , and 23 vocs were detected only when melanoma cells existed and were not detected in normal cells . the authors used the nist 2.0 mass spectral database , with a 60% quality factor for chemical identification of the possible biomarker candidates ; however , they did not report the quality factor obtained for the different compounds by mass spectrometry ( ms ) . in a recent study , kwak et al . compared human melanoma cells to normal melanocytes cultured in vitro . by using gas chromatography- ( gc- ) ms , those authors found increased levels of both isoamyl alcohol and isovaleric acid in the headspace over melanoma cells . the authors also found that the melanoma cells produced some unique compounds , such as dimethyldisulfide and dimethyltrisulfide . in the present study , we analyzed the vocs emitted from the urine and feces of mice before ( healthy group ) and after ( cancer group ) subcutaneous injection of b16 melanoma cells . the present work will serve as a basis to analyze possible vocs in human patients . we expect some voc to be shared between the species and new and different vocs to be detected .", "the sample classification used in the present study is summarized in table 1 . the analysis of the urine samples of healthy and tumor - bearing mice revealed some differences in the vocs composition . there was no significant difference between the first two samples ( samples a and b ) , which were taken before the injection of the b16 melanoma cells , and the first sample taken after injection ( sample c ) . in the second sample taken after the injection ( sample d ) , a significant difference was found in the observed vocs , although tumors were not yet palpable . in the last two samples ( samples e and f ) , tumors were palpable in all mice , and the chromatographic spectra were significantly different from those obtained in earlier samples . therefore , the samples were classified into three groups : healthy , early - melanoma , and late melanoma . typical urine chromatograms obtained from one mouse before ( sample b ) and after injection ( sample e ) are shown in figure 1 . some peaks in the chromatograms were present only in the tumor - bearing mice , and the area under some peaks was markedly higher or lower in the tumor - bearing mice compared to the healthy mice . by using the nist'08 and wiley mass spectral libraries , total of 120 vocs were identified in urine and 139 vocs in feces were identified in all mice samples , with an 80% quality factor ( qf ) . after subtracting the vocs that were found in the background samples , a statistical analysis reduced the numbers of these peaks to 16 vocs in urine and 13 vocs in feces , which could serve as possible biomarker candidates . in addition , three vocs in urine and two vocs in feces , with a qf in the range of 6079% , were also included as possible biomarker candidates . of the 19 possible biomarker candidates in urine , three compounds were detected only in the tumor - bearing mice ( table 2 ) , three vocs were detected only in the healthy and early - melanoma - bearing mice ( table 3 ) , and 13 vocs were detected in markedly higher concentrations in the tumor - bearing mice as compared with the healthy group ( table 4 ) . similarly , from the 15 possible biomarker candidates found in the headspace of feces , two vocs were detected only in the tumor - bearing mice ( table 5 ) and 13 vocs were found with a markedly higher ( 12 vocs ) or lower ( 1 voc ) concentration in the tumor - bearing mice than in the healthy group ( table 6 ) . to examine the significance of the changes in the concentrations of compounds during the different melanoma stages ( see tables 4 and 6 ) , a two - tailed paired t - test was carried out . as can be seen in table s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/841245 , some of the vocs that were identified as possible biomarker candidates in the current study have already been reported previously by other research groups as being predictive for melanoma and other cancers . the four potential melanoma - related biomarkers that have already been reported are isopropyl palmitate ( rt = 18.41 , qf = 72% ) , 1-hexadecanol ( rt = 14.54 , qf = 93% ) , benzaldehyde ( rt = 6.94 , qf = 97% ) , and dimethyl sulfone ( rt = 6.21 , qf = 94% ) . 1-hexadecanol and isopropyl palmitate , a derivate of palmitic acid , were detected only in the urine of tumor - bearing mice . these findings are consistent with a previous study , which reported a 35-fold increase in the level of 1-hexadecanol in the case of melanoma ( as compared with a matching skin sample ) and that isopropyl palmitate was found only in the melanoma sample . the authors suggested that these compounds may reflect an increased de novo synthesis of fatty acids , which is a crucial metabolic alteration that cancer cells require for synthesis of a new plasma membrane . de novo synthesis of fatty acids could be caused by the hyperactivity of the oncogenic fatty acid synthase ( fasn ) , a common phenotype in cancer pathogenesis . benzaldehyde levels in feces were 132% higher in the tumor - bearing mice than in the healthy group ( p = 0.004 ) , but it is important to notice that the increases were from the early - melanoma stage to the late melanoma stage ( p = 0.002 ) , and not from the healthy group to the early - melanoma stage . in other words , benzaldehyde could be a useful possible biomarker candidate for predicting melanoma , but not in its early stage . the levels of dimethyl sulfone in urine showed a 199% increase from the healthy to the tumor - bearing mice groups ( p = 0.026 ) . as shown earlier , significantly higher amounts of this compound were found in metastatic melanoma cells , as compared with normal cells . this finding suggests that the metabolism of sulfur - containing amino acids by melanoma cells differs from that in normal cells . the gc - ms analysis of headspace in b16 melanoma cell cultures yields 12 vocs that were not detected in the headspace of the materials in the phosphate buffered saline ( pbs ) and fresh growth medium without cells ( table 7 ) . the compounds that were identified were isopropyl myristate ( rt = 16.76 , qf = 93% ) , decane ( rt = 7.43 , qf = 93% ) , 2,4-dimethyl-1-heptene ( rt = 4.67 , qf = 93% ) , and hexadecane ( rt = 14.7 , qf = 95% ) . as described above , the concentrations of isopropyl myristate were higher in both urine and feces of tumor - bearing mice . decane and two chemically similar compounds , 1-hexadecene and 2,4-dimethyl - heptane , were previously reported to be present only in melanoma samples . the difference between 1-hexadecene and 2,4-dimethyl - heptane to hexadecane and 2,4-dimethyl-1-heptene ( resp . ) is in the presence or absence of a single double bond . hakim et al . studied two hydrocarbons , decane and 2,4-dimethyl-1-heptene , that were reported as potential biomarkers of lung cancer and suggested that these compounds are probably the outcome of oxidative stress . as mentioned above , the tumor was palpable only in the last two samples ( late melanoma ) . an average lesion volume of 1664 mm ( n = 2 ) was measured three days after the last sample was taken ( see figure s1 ) . among all possible biomarker candidates that were found in the urine and feces , three compounds in urine ( figures 2(a)2(c ) ) and two compounds in feces ( figures 2(d)-2(e ) ) three of these compounds were all ketones , that is , 5-methyl-2-heptanone ( rt = 6.87 , qf = 91% ) , 6-methyl-2-heptanone ( rt = 6.71 , qf = 94% ) , and 6-methyl-3-heptanone ( rt = 6.67 , qf = 97% ) . the quality factor of the two compounds found in the feces headspace was smaller than 80% ; hence they are referred to only by their retention time : rt = 8.9 and rt = 18.41 . 6-methyl-2-heptanone has been found in the blood of patients with liver cancer and 6-methyl-3-heptanone was found in the urine of mice with lung cancer . in addition , other chemically similar ketones , such as 2-heptanone and 6-hydroxy-6-methyl-3-heptanone , were also detected in urine samples from mice with lung cancer tumors . partial least square discriminate analysis ( pls - da ) models of urine and feces were built with the simca p+ software . each model included three latent variables that were calculated by the software and are , in fact , weighted linear combinations of the 12 possible biomarker candidates that most contributed to the separation between the three groups ( all the compounds in tables 26 , except compound 2 in table 2 , compound 3 in table 3 , compounds 3 and 912 in table 4 , compound 1 in table 5 , and compounds 5 and 6 in table 6 ) . the models are shown in figures 3 and 4 for the urine and feces models , respectively . the urine model exhibited good predictability of the mouse condition ( healthy or sick ) with a 92.3% sensitivity , 100% specificity , a 93.1% negative predictive value ( npv ) , and a 100% positive predictive value ( ppv ) . the model for feces gave less significant results , with an 80.8% sensitivity , a 96.3% specificity , an 83.9% npv , and a 95.5% ppv . it is important to note that eight out of nine mice were correctly predicted with an early - melanoma by using the urine model , whereas the feces model did not clearly discriminate between healthy and early - melanoma mice .", "the present study constitutes a novel proof - of - concept for the detection and monitoring of melanoma in urine and feces samples of mice at an early stage . our urine statistical model correctly predicted eight out of the 9 mice bearing an early - melanoma , indicating that mice at that stage can be detected and distinguished from healthy mice and from mice bearing a late melanoma . while other research groups [ 6 , 7 ] focused on comparing human melanoma to normal and nevus skin biopsies , we attempted to distinguish between mice with an early - melanoma and healthy mice . our model was able to distinguish between melanoma bearing mice and healthy mice with sensitivity of 92% and specificity of 100% , as compared to studies of other groups on human samples ( e.g. , 70% sensitivity and 90% specificity reported by and 89% sensitivity and 90% specificity reported by ) . these differences might be related to the low variability in the vocs of mice all having similar genetics and environment , as compared to those of humans . the results of this study also indicate a marked difference between healthy mice and mice bearing a late melanoma , whereas the difference between healthy mice and mice bearing an early - melanoma is less clear . one possible explanation for this finding is that the concentration of the new compounds , which are products of the altered metabolism of the melanoma cells or of the normal cells that it affects , is below the detection threshold of our gc - ms system . in addition , the angiogenesis in mice bearing an early - melanoma is limited and , therefore , metabolites induced by melanoma cell do not disperse efficiently and are thus hard to detect . to overcome these concentration - dependent limitations , one should concentrate on detecting the compounds ( potential biomarker candidates ) that are responsible for the differences between healthy and late melanoma one should also develop methodologies to detect vocs directly from the regions of skin showing suspicious lesions ; a step in this direction has been taken by detecting differences in human melanoma cell line signatures by ftir spectroscopy [ 15 , 16 ] . some of the potential biomarker candidates that were identified in the present study were previously reported to be predictive for melanoma and other malignancies ( table s1 ) . those reports strengthen the validity of our findings and imply the existence of cancer type-specific biomarkers and of common cancer biomarkers . some of the potential biomarker candidates are cross species compounds ; namely , they are found in both mice and human melanoma cells ; therefore , similar experiments with human melanoma cell lines as xenografts should be conducted to confirm these data . two additional possible biomarker candidates that may be good candidates to identify melanoma are dehydroabietic acid ( rt = 21.7 ) and 2-hexanone ( rt = 3.78 ) . it could be a pollutant from the sawdust , although it was not detected in the background samples . another possibility could be wrong identification of this compound by the code used ms database search ( qf = 81% ) . importantly , isopropyl myristate , the concentration of which increased in the urine and feces of tumor - bearing mice , was also found in the vapor phase of the b16 melanoma cells culture . this finding suggests that isopropyl myristate is a valid direct product of melanoma cell metabolism for further study . other compounds that were found in the headspace of the melanoma cell cultures were not detected in the urine or feces samples , and vice versa . this is attributed to the differences between the tumor cells microenvironment in a culture as compared to a tumor in the animal . we assume that there may be also contribution from the tumor microenvironment since tumor cells affect other cells in the tissue . at this point , the origin of the specific molecules we detected can not be determined . the observed increase in the concentration of ketones as a function of tumor growth suggests that ketogenesis pathways , incorporating various ketone formations , may be involved in the model of melanoma cancer . therefore , monitoring these ketones could help in tumor follow - up in melanoma patients . finally , to substantiate the findings in this study , we plan to perform an extended study with larger number of mice focusing on the urine vocs . in addition , to correlate more precisely between tumor size and voc concentration one should collect samples more often ( every 24 days ) and correlate the biomarkers to tumor volume as measured with a caliper . a different way of measuring is by expressing gfp or luciferase in the b16 cells and following their volume with an imaging device such as mri . despite the differences between mouse and human metabolism , possible biomarker candidates found in mice urine and feces if the possible biomarker candidates were formed in biochemical processes related to the activity of cancer cells , we can expect to find some overlap between biomarkers found in human and mice .", "urine and feces samples from nine mice were collected , twice before ( healthy group , n = 18 ) and four times after ( cancer group , n = 35 , one mouse died before the last sample was obtained ) subcutaneous injection of b16 melanoma cells . compounds from these samples were separated and identified by using headspace solid phase microextraction ( hs - spme ) and gc / ms . in addition , vocs above a culture of b16 melanoma cells were also collected and analyzed . comparison between vocs content of the two first samples of each mouse , collected before b16 melanoma cell injection , showed high repetition of peaks in the chromatograms of each mouse . comparison of peak repetition in the chromatograms of different mouse in the two preinjection samples shows a markedly reduced degree of repetition ( see figure s2 in the supplementary material ) . these findings clearly indicate that the metabolism characteristics of each mouse have large influence on the observed chromatograms . consequently , we did not use a control group but compared the chromatograms of each mouse after b16 melanoma cell injection to those obtained before injection . female c57bl/6j inbred mice , 12 weeks old and weighing 1820 g , were obtained from harlan laboratories ( jerusalem , israel ) . all mice were maintained at the animal resource center of ben - gurion university of the negev , in a controlled environment ( illumination , temperature , and humidity ) free of specific pathogens . all animal experiments described in this work were approved by the ben - gurion university committee for the ethical care and use of animals in experiments . each individual mouse was kept in a separate cage ( 9 cages total ) under the same housekeeping conditions . the first samples were taken after a week of acclimation ( at the age of 13 weeks ) . b16f10 cells were cultured in an rpmi 1640 medium supplemented with 10% heat - inactivated fetal calf serum ( fcs ) , 100 u / ml penicillin , 100 mg / ml streptomycin , and 2 mm l - glutamine ( biological industries , israel ) . b16f10 cells ( 10 cells ) were seeded in a 10 cm diameter tissue culture dish . after 48 h , the medium was collected and centrifuged to eliminate cells and cell debris , and the cells were harvested by 0.23% trypsin and washed once in medium and two more times in phosphate buffered saline ( pbs ) . b16f10 cells were harvested by 0.23% trypsin and washed once in medium and two more times in pbs . one hundred microliters , containing a total of 2 10 b16f10 cells in pbs , was injected subcutaneously to the left flank of each of nine c57bl/6 mice . mice with tumors that reached the size of 1.5 cm diameter were sacrificed by co2 . urine and feces samples were collected into vials with a pipette from the new disposable plastic table cover . at the end of the sample collection we could not find in the literature a component in the urine or feces that allow the prediction of the hydration state of mouse ( similar to creatinine in human urine ) . we found 8 vocs that showed up in all the chromatograms ; however , their peak areas did not show any correlation to the total chromatogram area and they could not be used to monitor hydration state of the mice . thus , it was assumed that there should not be significant variations in mice hydration state since they were kept in identical conditions ( temperature and humidity ) with unlimited access to food and water . moreover , urine and feces samples were collected from all mice at the same time . it should be noted that the amount of urine and feces that were collected from the different mice varied , not enabling a direct comparison between amounts of different vocs of different mouse . static headspace sample extraction was achieved by exposing a 65-m polydimethylsiloxane / divinylbenzene ( pdms / dvb ) spme fiber ( supelco , bellefonte , pa , usa ) to the headspace for 10 min at 60c . following the extraction , the fiber assembly was transferred to the gc injection port for desorption at 250c for 5 min , with the split valve closed for 2 min . gc - ms analyses were performed by using an agilent 6890 series gc system ( agilent , usa ) connected to an agilent 5973 network mass selective detector ( agilent , usa ) . the bench top system was fitted with an spme injection sleeve 0.75 mm i d quartz liner ( supelco , bellefonte , pa , usa ) . the analytical column was a zebron zb-5msi fused - silica capillary column , 30 m 0.25 mm i d 0.25 m film thickness ( phenomenex , torrance , ca , usa ) . the carrier gas was 99.9995% pure helium ( maxima , ashdod , israel ) passed through a moisture trap ( model mt-200 - 2s ) and an agilent hydrocarbon / moisture trap ( model hmt200 - 2 ) ( agilent , china ) at flow rate of 1.0 ml min . the gc was operated under the following temperature program : 50c for 3 min , ramp of 12c min to 240c , held at 240c for 5 min , ramp of 50c min to 260c , then held at 260c for 1 min , giving a total run of 25.23 min . for gc - ms , following electron ionization , ions were scanned as the total ion current ( range : 10500 m / z at 2.97 scans s ) . at both the beginning and end of each gc / ms analysis session the headspace over a calibration mixture ( containing equal amounts of chloroform , toluene , and dodecanethiol ) the test is based on examination of retention time and peak shape of the solvents used in the calibration mixture . compounds were assigned a chemical identification by means of spectral library matching , using the nist'08 and wiley libraries . the databases search was performed using the chemstation software ( by agilent ) that was used to control and operate the gc / ms system . the assignment of chemical identity by the software is accompanied by a quality factor ( qf ) . the qf is also termed in some cases as matching factor . a library search procedure that identifies structural features of an unknown compound this procedure first retrieves library compounds whose spectra are most similar to the spectrum of the unknown compound . the algorithm then deduces structural features of the unknown compound from the chemical structures of the retrievals . the significance and reliability of each retrieved spectrum are weighted according to its similarity to the spectrum of the unknown compound . there are different procedures to estimate the reliability of the chemical identification of a gc peak ( see , e.g. , stein ) . the exact algorithm used to obtain the qf in the chemstation software is not described in the documentation ( it is considered as a commercial secret ) ; however , the manual states that qf larger than 80% is considered to be reliable . below this value of qf the reliability of the assignment is too low and the peaks are designated by their retention time only . a gc - ms analysis produces a complex chromatogram , where each peak represents a different volatile compound and the area below the peak is proportional to the amount of the compound in the sample . this threshold was used to ensure that all volatiles with low concentration are detected but noise is eliminated . to obtain estimates of the concentration of each compound , the peak area this normalization is performed by the chemstation software ( by agilent ) used to operate the gc / ms . the total chromatogram area used in the normalization is available in the output file ; hence , the actual peak area of any individual feature can be calculated . examination of the variations in total chromatogram area shows that the standard deviation is 17% of the mean value , a quite narrow distribution . the data analysis described in the present study we repeated some of the analysis using the actual area of the peaks in the chromatograms and found almost identical results to those obtained by the normalized data . some of the volatiles in the chromatogram were eluted at multiple rts and , therefore , we used only the rt with the best match . in addition , to overcome peak shifting across different chromatograms , we first grouped peaks in the same time window ( 0.3 s ) and then grouped adjacent peaks in adjacent time windows that hit the same match . each identified volatile was compared to all the background volatiles ( room air , plastic table cover , vial , vial septum , spme fiber , sawdust , and mouse food ) to ensure that differences were not due to a background alternation . a partial least squares discriminant analysis ( pls - da ) was conducted with the simca p+ code ( version 12.0.1.0 , by umetrics ab , ume , sweden ) to test whether mice with melanoma could be diagnosed by using the potential urine or feces biomarkers . each chromatogram vector was defined as a member in one of three classes , healthy , early - melanoma , and late melanoma in urine samples , and in one of two classes , healthy and melanoma in the feces samples . two mice chosen arbitrarily were scanned three days after the last sample was taken to confirm the existence of the tumor and to estimate its volume . t1-weighted scanning images were acquired at 1 t by means of an aspect m2 high performance mri system ( aspect imaging technologies ltd . , israel ) , by using a gadolinium - dtpa contrast agent ( dotarem , 1 ml / kg body weight plus 0.05 ml for catheter ) ." ]

[ "genome - wide association studies ( gwass ) have emerged as a powerful non - biased technique for identifying pathways related to human diseases with complex genetic architecture . understanding such complex diseases requires a broad understanding of the relevant functional nodes regulating disease - relevant pathways . genes do not function in isolation , and it is clear that both gene - gene and gene - environment interactions contribute to disease susceptibility ( barreiro et al . , 2012 ; cadwell et al . , 2010 ; silver et al . , 2013 ) . more than 140 genetic risk loci have been identified for crohn s disease , a chronic inflammatory condition affecting the gastrointestinal tract ( franke et al . , 2010 ; these studies have highlighted the contribution of multiple immune and cellular pathways to crohn s disease pathogenesis , particularly those involved in microbial defense ( jostins et al . , 2012 ; lassen et al . , 2014 ; murthy et al . , 2014 ; sadaghian sadabad et al . , 2014 ) . atg16l1 ( autophagy - related protein 16-like 1 ) is a component of the core autophagy machinery , and the atg16l1 t300a polymorphism confers a modestly increased susceptibility to crohn s disease despite its relatively high prevalence in the population ( franke et al . , 2010 ) . autophagy is a pro - survival intracellular degradation pathway that functions as a key mediator of a number of processes including central metabolism , cell signaling , cell death , and carcinogenesis , as well as both innate and adaptive immunity ( deretic et al . , 2013 ; yang and klionsky , 2010 ) . the atg16l1 t300a polymorphism has been associated with both impaired antibacterial autophagy and altered production of cytokines by peripheral blood mononuclear cells ( pbmcs ) in response to immune stimuli ( lassen et al . , 2014 ; recent studies have demonstrated that the mechanism underlying these alterations is linked to increased susceptibility of atg16l1 t300a to caspase - mediated cleavage and thus lower levels of functional atg16l1 ( lassen et al . the autophagy pathway is important for defense against a number of intracellular pathogens and pathobionts including salmonella enterica serovar typhimurium , shigella flexneri , listeria monocytogenes , enterococcus faecalis , and mycobacterium tuberculosis ( benjamin et al . , 2013 ; castillo et al . , 2012 ; gutierrez et al . , 2004 ; huett et al . , 2012 ; , 2013 ; ogawa et al . , 2005 ; tattoli et al . , 2012 ; , cytoplasmic bacteria are first marked for degradation primarily by ubiquitination ( deretic et al . , 2013 ) . recent studies have identified key roles for the e3 ligases lrsam1 and parkin in recognition and ubiquitination of cytoplasmic bacteria ; however , there are likely additional cellular e3 ligases that provide specificity to this pathway ( huett et al . , 2012 ; ubiquitinated bacteria are then recognized by the adaptor proteins p62 , optineurin , and ndp52 , leading to the recruitment of lc3 and the autophagy machinery ( cemma et al . , 2011 ; thurston et al . , 2009 ; wild et al . , the autophagy pathway targets only a subpopulation of bacteria within a cell , and little is known about the molecules involved in the selective recognition and targeting of bacteria for autophagosomal degradation , despite the important pathological consequences of this process . in this study , we approached the question of the impact of the atg16l1 t300a risk variant by searching for associated responsiveness quantitative trait loci ( reqtls ) , which are defined as the effects of genetic variation on transcriptional responses of cells ( barreiro et al . , 2012 ) . given that functionally similar genes are often transcriptionally coregulated , using multiple immune stimuli in genetically defined cell populations along with reqtls has enabled the identification of differential effects on molecular circuits ( gat - viks et al . , 2013 ) . in the current study , we perform stimulus - specific perturbational profiling in pbmcs , quantitative mass - spectrometry - based ( ms ) proteomics , and genome - wide rna expression analysis to identify antibacterial genes that are functionally related to atg16l1 . using these approaches , we demonstrate a role for clec12a in antibacterial autophagy and pinpoint a functionally relevant interaction node of clec12a with an e3 ligase complex that is important for this function .", "to identify molecules involved in the atg16l1-t300a - dependent antibacterial autophagy pathway , we used perturbational profiling to measure differential gene regulation in the setting of stimulation with pathogen - associated molecular patterns and bacterial challenge . given that mounting evidence indicates an important role for the involvement of host - microbiota interactions and bacterial defense pathways in crohn s disease ( knights et al . , 2013 ; we selected a panel of three stimuli to perturb pbmcs from healthy individuals homozygous for the ancestral atg16l1300 t allele or the 300a risk allele ( rs2241880 ) . the nod2 ligand muramyl dipeptide ( mdp ) was selected based on the previous association of this stimulus with crohns - disease - specific pathways ( hugot et al . , 2001 ; mdp can also modulate signals mediated through tlr2 activation , a pathway that has been implicated in crohn s disease and experimental colitis ( netea et al . , 2004 ; we therefore also used the tlr2 ligand pam3cys , as well as the bacterium borrelia burgdorferi , from which these lipopeptides were initially isolated , to elicit both nod2-dependent and -independent responses . using microarrays to assess genome - wide rna expression profiles , we found that baseline gene expression was similar between individuals harboring the non - risk and risk alleles . furthermore , upon exposure to innate immune ligands , we observed strong genotype - independent induction of proinflammatory cytokines and chemokines after hierarchical clustering ( figures 1a and s1a ) . 2012 ) , we next used factorial design analysis to examine whether transcriptional responses to pam3cys , borrelia , or mdp differed between groups stratified by the 300 t non - risk and 300a risk alleles . using multiple immune stimuli in genetically defined pbmcs along with reqtls has enabled differential effects on molecular circuits to be determined ( gat - viks et al . , 2013 ) . our analysis identified 20 atg16l1300a - dependent reqtls under at least one stimulation condition ( figures 1b and s1b ) . thus , perturbational profiling of human immune cells revealed genotype - specific responses to stimulation . we focused additional analysis on the autophagy pathway , the cellular degradation system that has been implicated in risk of crohn s disease by gwass , which identified predisposing alleles in atg16l1 and irgm ( immunity - related gtpase family m ) ( rioux et al . , we and others have reported that the atg16l1 t300a polymorphism is associated with impaired antibacterial autophagy as well as altered production of cytokines by pbmcs in response to immune stimuli ( lassen et al . , 2014 ; murthy et al . , 2014 ) . additionally , tlr simulation , particularly through tlr2 , has been shown to induce antibacterial autophagy ( anand et al . , 2011 ; 2010 ) . to determine whether any of the identified genes might be associated with autophagy , we generated protein interaction networks anchored on core autophagy proteins as well as autophagy - associated components previously identified from a high - confidence interaction network derived from a systems - wide autophagy proteomics study ( behrends et al . , 2010 ) . of the genes identified , rab24 was previously reported to have a direct effect on autophagy ( behrends et al . , 2010 ; munaf and colombo , 2002 ) , and three additional gene products ( hck , fez1 , and fyn ) interacted with the autophagy network via known protein - protein interactions ( figure s1c ) . these observations demonstrate that the atg16l1 t300a polymorphism is associated with transcriptional modulation reflecting known protein - protein interactions with the autophagy network . given the previously described role for atg16l1 and irgm in antibacterial autophagy ( singh et al . , 2006 ) , we next used sirna to evaluate whether any of the identified atg16l1300a - dependent reqtls affected antibacterial autophagy . of the 20 genes identified , 13 had detectable expression in hela cells and were suppressed by single sirnas ( figure s1d ) . antibacterial autophagy was assessed in sirna - treated cells using infection with s. typhimurium , a model pathogen that is degraded by autophagy ( birmingham et al . , 2006 ) . knockdown of three of these genes ( clec12a , rab24 , and evi2b ) resulted in significantly decreased salmonella - autophagosome colocalization ( figures 1c , 1d , and s1e ) . we next tested whether rab24 , evi2b , or clec12a play a role in classical autophagy using an lc3 flux assay , in which levels of lipidated lc3-ii are compared to lc3-i by western blot . in this assay , an increase in the ratio of lc3-ii to lc3-i corresponds to an increase in autophagic flux . knockdown of rab24 and evi2 resulted in decreased lc3-ii accumulation when cells were treated with torin 1 , an mtor inhibitor and inducer of bulk autophagy , suggesting that these genes are involved in classical autophagy ; knockdown of atg16l1 served as a control in these experiments . knockdown of clec12a did not impair lc3-ii accumulation under these conditions , suggesting that clec12a functions specifically in antibacterial autophagy ( figure 1e ) ( thoreen et al . , 2009 ) . additionally , to confirm that the antibacterial autophagy phenotype seen with clec12a knockdown was not due to off - target effects , we employed a knockdown rescue approach ( huett et al . , 2012 ) by generating two sirna - resistant constructs that express full - length clec12a during knockdown of endogenous gene expression ( figure 1f ) . the autophagy defect caused by sirna against clec12a was rescued by overexpression of the appropriate clec12a construct ( figures 1 g and 1h ) , demonstrating that the decrease in antibacterial autophagy was specific to a reduction in clec12a expression and not due to off - target effects of sirna . additionally , sirna targeting clec12a did not alter bacterial entry , suggesting the observed effect was specific to the autophagy pathway ( figure s1f ) . given our findings that immune cells harboring the atg16l1300a allele exhibited reduced induction of clec12a under immune stimulation conditions and that clec12a is required for efficient lc3-salmonella colocalization ( figure 1 ) , we next investigated whether a genetic interaction exists between the atg16l1 t300a snp and clec12a with respect to antibacterial autophagy . to investigate this hypothesis , we generated an atg16l1 knockout ( ko ) hela cell line using the crispr - cas9 system ( ran et al . loss of atg16l1 expression was confirmed in single hela clones ( figure s2a ) . as expected , atg16l1 ko cells were unable to convert lc3-i to lc3-ii under basal or stimulatory conditions ( figure s2b ) and had negligible lc3-bacteria colocalization ( figure 2 ) . to determine the effect of the t300a coding polymorphism , we stably transduced atg16l1 ko cells with 300 t ( atg16l1 ko + atg16l1300 t ) or 300a ( atg16l1 ko + atg16l1300a ) alleles , achieving equivalent expression levels of atg16l1 in these lines ( figure s2a ) . as expected , we found that basal autophagy was restored in both atg16l1 ko + atg16l1300 t and atg16l1 ko + atg16l1300a cell lines , with modest impairment of antibacterial autophagy in the atg16l1 ko + atg16l1300a cells , consistent with published findings ( figure 2 ) ( conway et al . , 2013 ; lassen et al . , 2014 ; murthy et al . , 2014 ) . targeting clec12a with sirna resulted in impaired antibacterial autophagy in the atg16l1 ko + atg16l1300 t cell line similar to wt cells . we observed a more - pronounced impairment in atg16l1 ko + atg16l1300a cells , suggesting functional interaction of the t300a polymorphism and reduced clec12a expression . clec12a is a c - type lectin receptor ( clr ) belonging to a family of transmembrane proteins that recognize pathogen - associated molecular patterns and engage downstream immune signal transduction pathways ( dambuza and brown , 2015 ; osorio and reis e sousa , 2011 ) . clec12a is highly expressed in human myeloid cells ( figure s3a ) , and targeting antigen to clec12a results in enhanced antigen presentation on dendritic cells in mice ( lahoud et al . , 2009 ; recent studies have demonstrated that clec12a binds to uric acid crystals and helps to dampen inflammation through its itim motifs ; however , it remains unclear whether this is the only ligand for clec12a ( neumann et al . to determine whether clec12a plays a role in pathogen defense in vivo , we used clec12a mice . bone - marrow - derived macrophages ( bmdms ) from wt or clec12a mice were infected with a strain of listeria ( egde ) known to be susceptible to autophagy in primary macrophages ( anand et al . , 2011 ; birmingham et al . , 2007 ; huett et al . , consistent with the salmonella infection results in sirna - treated cells , clec12a bmdms displayed lower levels of bacterial colocalization with lc3 compared with wt bmdms ( figures 3a and 3b ) . additionally , clec12a bmdms did not show differences in torin-1-induced autophagy , suggesting that bulk autophagy is normal in these cells ( figure s3b ) . taken together , these data suggest that clec12a functions selectively in the antibacterial autophagy pathway for multiple pathogens in both mouse and human cells . we next investigated whether clec12a plays a role during infection with intracellular pathogens in vivo . wt or clec12a mice were pre - treated with streptomycin and then infected with salmonella ( conway et al . four days post - infection , stool was collected from infected mice and colony - forming units ( cfus ) were measured . clec12a mice had significantly higher cfu levels compared to wt mice ( figure 3c ) . clec12a mice also exhibited increased systemic bacterial dissemination as assessed by measuring cfus in spleens ( figure 3d ) . this increase in cfu was associated with a more - severe clinical disease score as assessed by overall appearance , piloerection , mobility , and posture ( figure 3e ) , as well as decreased survival ( figure 3f ) . taken together , these data suggest that clec12a plays a role in intracellular microbial defense both in vitro and in vivo . to further demonstrate the precise role for clec12a in antibacterial autophagy , we next tested whether knockdown of clec12a altered intracellular bacterial replication . using knockdown of atg16l1 as a positive control , we found that knockdown of clec12a in hela cells resulted in significantly increased intracellular replication of salmonella ( figure 4a ) , demonstrating that clec12a is required for restriction of intracellular bacterial replication . s4a ) were predominantly membrane associated and could be found surrounding intracellular dsred - labeled salmonella at both early and late time points after infection . association of clec12a with salmonella peaked at 30 min post - infection and declined gradually , whereas lc3-salmonella colocalization peaked 60 min after infection , consistent with previous data ( figure 4b ) ( huett et al . , 2012 ) . live cell imaging confirmed that gfp - clec12a surrounds bacteria and then dissociates at approximately 1 hr post - infection ( figure s4b ) , consistent with the time course of peak lc3 colocalization . given the early association of clec12a with bacteria , we next determined whether clec12a was present at sites of bacterial entry into the host cell . we found that gfp - clec12a colocalized with bacteria and ruffled actin , an indicator of recent bacterial entry , suggesting that clec12a associates with bacteria concomitant with bacterial cellular entry ( figure s4c ) ( lhocine et al . we confirmed that clec12a colocalizes with salmonella and rab5 , a marker of early endosomes ( figure s5a ) . however , clec12a recruitment was also readily detected around salmonella without colocalization of rab5 , suggesting that clec12a is not exclusively associated with endosomes ( figure s5a ) . next , we evaluated the colocalization of clec12a with galectin 3 and galectin 8 , which are both known to bind to sites of bacterial - induced intracellular membrane damage as well as sites of sterile membrane damage ( thurston et al . , 2012 ) . clec12a colocalized with both galectin 3- and galectin 8-salmonella complexes ( figure 5a ) . these data confirm that clec12a associates with bacteria early after infection concomitant with membrane damage . clec12a also colocalized with the antibacterial autophagy adaptor ndp52 ( figure 5b ) as well as the autophagosome proteins gfp - lc3 , the atg8 homolog gabarapl2 , and atg16l1 ( figures 5c and 5d ) . in all cases , these proteins surrounded intracellular bacteria in conjunction with either endogenous clec12a or gfp - clec12a ( figures 5b5d ) . of note , expression of gfp - clec12a gave a more - intense fluorescent signal and demonstrated increased levels of clec12a puncta within the cells , regardless of stimulation or bacterial infection . physical interactions between clec12a and lc3 , gabarapl2 , and atg16l1 were also confirmed by co - immunoprecipitation ( figure s5b ) . we first determined whether knockdown of clec12a altered recruitment of ubiquitin to bacteria . at 1 hr post - infection , sirna knockdown of clec12a significantly reduced salmonella - ubiquitin colocalization ( figures 5e and 5f ) . consistent with this observation , recruitment of ndp52 to salmonella was also decreased upon sirna knockdown of clec12a ( figures 5 g and 5h ) . colocalization of salmonella with galectin 3 , galectin , 8 , and p62 were unchanged in cells treated with clec12a sirna ( figures s5c s5e ) . these data reveal clec12a as an early bacteria - associated factor that binds to the autophagy machinery and helps initiate antibacterial autophagy through the ubiquitin - ndp52 pathway . recently , clec12a has been shown to be a receptor for dead cells ( neumann et al . , 2014 ) . we hypothesized that clec12a could also recognize sites of membrane damage triggered by vacuolar disruption after bacterial entry ( tattoli et al . galectin 3 served as a marker of damaged endosomes ( thurston et al . , 2012 ) . gfp - clec12a colocalized with galectin 3 puncta after hypertonic shock and treatment with polyethylene glycol ( peg ) ( figure 5i ) . these results demonstrate that clec12a is recruited to sites of membrane damage and could explain the high proportion of clec12a recruitment early after bacterial entry . to develop a more - integrated model of clec12a function in pathogen response , we utilized time course rna - seq data and targeted quantitative proteomics to pinpoint clec12a - dependent protein interactions that may be important for pathogen response . first , we employed 3 rna - seq to quantify the transcriptome of listeria - infected wt or clec12a bmdms at various times post - infection . using this approach , we identified genes with statistically significant differences ( fdr - adjusted q value 0.05 ) in fold - change response to listeria infection between wt and clec12a bmdms at any time point post - infection ( figure 6a ) . of note , expression of lamp1 , a lysosomal membrane protein and marker of salmonella - containing vacuoles , was significantly downregulated in clec12a bmdms , suggesting endomembrane dynamics might be perturbed in these cells . we next performed clustering and pathway analysis using an expanded list of 674 nominally significant genes that were differentially regulated in clec12a bmdms at baseline as well as in response to listeria . genes were grouped into ten clusters using short time - series expression miner ( stem ) , where each cluster represents one of the predefined model profiles in stem that capture potential distinct patterns of infection response ( figure 6b ; table s1 ) ( ernst and bar - joseph , 2006 ) . differences in these model profiles represent distinct regulation patterns of gene expression . gene ontology enrichment analysis was applied to assess the functional significance of each cluster of genes . the top scoring pathway that was differentially regulated in clec12a bmdms was the negative regulation of the intracellular signal transduction pathway that includes key signaling components such as ndufs3 , tmem161a , and mapkapk5 , among others ( cluster 2 ; table s1 ) . additionally , antifungal response genes ( s100a9 , myd88 , and ptx3 ; cluster 6 ) were also differentially regulated in the clec12a bmdms . selective upregulation of glucose transport ( ppbp and sorbs1 ; cluster 7 ) but downregulation of lipid transport ( scarb1 , slmo1 , and soat2 ; cluster 5 ) in clec12a cells suggested preferential regulation of metabolism by clec12a in response to listeria infection . these results highlight the central role of clec12a in pathogen - induced defense pathways and suggest how deficiencies in clec12a could result in impaired bacterial defense . next , we sought to identify important transcriptional nodes that control these responses by integrating known transcription factor - dna interaction data with our temporal gene expression profiles of infected wt and clec12a bmdms into a unified model . to identify important regulation points in the clec12a pathway , we used dynamic regulatory events miner ( drem ) , which searches for clec12a - dependent bifurcation events , defined by a transition in which a set of genes that were previously coregulated instead show divergent response / regulation profiles ( figure s6a ) ( schulz et al . , 2012 ) . based on an extension of a hidden markov model , the analysis identified egr1 , a transcription factor involved in transcriptional response to pathogens ( de grado et al . , 2001 ; mcdermott et al . , 2011 ) , as the earliest stage regulator that induces clec12a - dependent downregulation of specific genes ( figure s6a ) . interestingly , egr1 is also known to be induced by either amino acid deprivation or er stress , which are both stimuli known to trigger autophagy ( shan et al . , 2014 ) . these data suggest that reduced expression of egr1 target genes could contribute to the reduced pathogen response and autophagic targeting of bacteria in clec12a bmdms . other notable transcriptional regulators identified in our analysis that are known to be important for pathogen response include rela , nfe2 , hif1a , and arnt2 . taken together , this analysis suggests that deletion of clec12a results in an alteration of the response to pathogens at the transcriptional level and identifies key regulators involved in this response . to further understand the role of clec12a in antibacterial autophagy , we next applied an integrated systems approach using quantitative ms - based proteomics and coupled these results with our transcriptomic profiling analysis to uncover functional clec12a interactions relevant to bacterial defense ( figure 6c ) . proteomic interactors were defined as those that demonstrated significantly increased binding to flag - clec12a relative to flag vector alone after immunoprecipitation ( table s2 ) . interactome analysis identified highly connected nodes within the clec12a interactors ( figure s6b ) . a specific interaction between clec12a and lc3 or atg16l1 was not identified by ms - based proteomics ; however , this may reflect the fact that a meaningful interaction between these proteins occurs only under stimulation and represents a fraction of the total cellular pool of these proteins . to identify clec12a protein interactions that may lead to the measured transcriptomic changes in response to bacterial infection , we used a network optimization approach to identify high - probability sub - networks , composed of signaling and transcriptional factor interaction cascades ( figure 6c ) ( basha et al . , 2013 ) . we then used a hypergeometric test to rank order clec12a - interacting proteins with the most significantly enriched set of target genes within the network to identify relevant functional nodes ( table s3 ) . some of the key transcriptional regulators identified in these sub - networks overlapped with those identified by drem analysis , including rela , tp53 , hif1a , and egr1 . this analysis pinpointed the ubiquitin - like protein , nedd8 ( neural precursor cell expressed , developmentally downregulated 8) , as both a clec12a interactor and a regulator of egr1 . these results suggest that the interaction between clec12a and nedd8 is a functional interaction and contributes to an early clec12a - dependent transcriptional response . this integrated approach also linked an e3 ubiquitin ligase complex that includes cul3 ( cullin 3 ) and the substrate adapters klhl9 ( kelch - like family member 9 ) and klhl13 ( kelch - like family member 13 ) to clec12a - dependent transcriptional responses controlled by the transcription factors cebpb and cebpd ( table s3 ) ( chen et al . , 2014 ) . importantly , proteomic interactome analysis demonstrated a physical interaction between nedd8 , klhl13 , cul3 , and klhl9 , indicating that this is a functional e3 ligase complex that interacts with clec12a ( figure 6d ) . nedd8 is a target of some cycle - inhibiting factors ( cifs ) , which are virulence factors produced by bacterial pathogens that alter the host cell cycle ( crow et al . , 2012 ; jubelin et al . , cif can associate directly with nedd8-modified cullin - ring complexes and inhibit their activity ( crow et al . we therefore investigated whether cul3 , nedd8 , klhl9 , and klhl13 had roles in antibacterial autophagy similar to clec12a ( huett et al . 2013 ) . upon sirna knockdown in hela cells , klhl9 , klhl13 , and nedd8 had a dramatic effect on both lc3-salmonella colocalization and bacterial replication , consistent with a role for these proteins in antibacterial autophagy ( figures 6e and 6f ) .", "human genetics provides a powerful tool to identify and understand physiologically relevant host responses to pathogens given that host - pathogen interactions are known to have a role in many complex diseases . gene - gene interactions and environment - gene interactions add to the complexity of identifying causal genes and variants from gwas data , as well as determining how these variants affect pathogenesis . pathway - specific perturbational profiling and integrated systems approaches have previously been used in genetically defined organisms to identify genetic nodes of regulation underlying disease ( gagneur et al . , 2013 ; gat - viks et al . , 2013 ; smith and kruglyak , 2008 ; zhang et al . , 2013 ) . using this strategy , we uncovered bacteria- and bacterial - ligand - specific programs affected in healthy individuals homozygous for the t300a polymorphism in the autophagy gene atg16l1 . interestingly , 15% of the t300a - dependent response genes were found to play a role in autophagy pathways , highlighting important snp - pathway interactions . our network analysis identified the clr family member clec12a as a gene that is less responsive upon tlr2 or bacterial stimulation in individuals carrying the atg16l1 \n 300a snp . pursuing the functional implication of this snp - environment interaction , we identified a role for clec12a in antibacterial autophagy and demonstrated that clec12a controls microbial replication in vitro and in vivo . using cells engineered to express the crohn s disease non - risk or risk allele of atg16l1 , we interrogated the snp - gene interaction with clec12a to demonstrate an exacerbated effect on antibacterial autophagy in the setting of the disease - associated snp . these findings are consistent with clec12a functioning at an early step in the antibacterial autophagy pathway at the level of pathogen recognition caused by membrane damage , with atg16l1 functioning downstream at the level of autophagosome formation . these data suggest a model in which individuals who carry the 300a risk allele have reduced clec12a responsiveness that serves to compound defects in antibacterial autophagy . these results highlight how non - redundant genes in the same pathway can be identified through ligand - specific transcriptional regulation . clrs are a large family of pattern recognition receptors , many of which are important signaling mediators in antimicrobial defense ( drummond and brown , 2013 ) . clec12a was recently shown to be a receptor for uric acid crystals and dead cells ( neumann et al . , 2014 ) , and we extend this finding to demonstrate that clec12a also recognizes sites of intracellular membrane damage triggered by bacterial entry ( tattoli et al . , 2012 ; additionally , we show that clec12a likely serves as an adaptor to help recruit ubiquitin and ndp52 to sites of vacuolar damage caused by bacteria and thus induce antibacterial autophagy . these data suggest parallels between the functions of galectins and clrs as early sensors of damage in selective autophagy pathways . additionally , a dendritic cell clr , dngr-1 , regulates endocytic handling of necrotic cell antigens to modulate cross - priming , suggesting that clrs could function broadly in the selection of endocytic cargo ( zelenay et al . , 2012 ) . recent data also indicate a role for another clr , clec16a , in mitophagy , indicating that these lectins can function broadly to control target selectivity in autophagy ( soleimanpour et al . , 2014 ) . here , we integrated time course rna - seq data and quantitative proteomics to identify relevant clec12a - dependent protein interactions that are involved in antibacterial autophagy . we identified an e3 ligase complex including klhl9 , nedd8 , and klhl13 that interacts with clec12a and is required for antibacterial autophagy . transcriptomic data suggested that the association of clec12a with this e3 ligase complex is important for the clec12a - dependent response to bacterial infection . virulence factors produced by some bacteria are known to directly target neddylated cullin - associated ubiquitin ligase activity , suggesting that these proteins might be broadly involved in inhibiting bacterial pathogenesis ( jubelin et al . , 2010 ) . additionally , klhl9 has been previously associated with early onset autosomal dominant distal myopathy , a disease in which alterations to the autophagy pathway are thought to contribute to pathophysiology ( cirak et al . , 2010 ) . taken together , these data identify a role for the recently evolved substrate adapters klhl9 and klhl13 as well as nedd8 in the restriction of bacterial infection . previous studies have suggested roles for the e3 ligases smurf1 , lrsam1 , and parkin in pathogen - specific autophagy ( huett et al . , 2012 ; manzanillo et al . , 2013 ; orvedahl et al . , 2011 ) . recent studies have also highlighted roles for other e3 ubiquitin ligases in the control of bulk autophagy . these include a role for rnf5 in regulating the levels of atg4b ( kuang et al . , 2012 ) , a role for cullin-5 and cullin-4 in autophagy regulation through ambra1 ( antonioli et al . , 2014 ) , and a role for rnf216 in autophagy regulation through beclin1 ( xu et al . , 2014 ) additionally , several members of the trim family of proteins can act as a platform for assembly of the autophagy machinery ( mandell et al . , expression of rnf5 and rnf216 were also shown to increase pathogen susceptibility through autophagy ( kuang et al . , 2012 ; xu et al . , 2014 ) . taken together with our data , these findings demonstrate that e3 ubiquitin ligases play an important role in regulating autophagy at different stages of the pathway . specifically , e3 ubiquitin ligases create selectivity in autophagy regulation by recognizing and integrating specific signals from pathogens and cellular states . utilizing e3 ligases to tightly control the response to pathogens is likely a highly conserved mechanism of innate defense ( pollier et al . , 2013 ) . here , we use integrated genomics to demonstrate a role for clec12a in antibacterial autophagy and identify an e3 ligase complex that provides insight into the selectivity of pathogen degradation . our in vivo results highlight a strong role for clec12a in the restriction of microbial replication . it is possible given this dramatic effect that clec12a functions not only in antibacterial autophagy but in other pathogen defense pathways as well . this study highlights how perturbational profiling can be used to study pathways underlying immunity and pathogen defense and illustrates the potential of combining whole - genome experimental data sets to understand functional gene interactions within a relevant pathway .", "for perturbational profiling , blood was collected after written informed consent ( or waiver as approved by the institutional review board ) from healthy volunteers at radboud university nijmegen medical centre ( runmc ) . the study was approved by the institutional review boards and was performed in accordance with the declaration of helsinki . separation and stimulation of pbmcs from healthy individuals was performed as described previously ( netea et al . , 2004 ) . microarray hybridization and genotyping of volunteers was performed as previously described ( smeekens et al . , 2013 ) . factorial design analysis was performed as described previously ( cadwell et al . , 2010 ) . s. typhimurium infections of hela cells and gentamicin protection assays were performed as previously described ( huett et al . , 2012 ) . for entry assays , cells were infected for 20 min , washed , fixed in 4% paraformaldehyde for 15 min at room temperature , and stained as described below . for antibacterial autophagy assays in sirna - treated cells , rnai knockdown for 48 hr was performed as described above on hela cells plated on glass coverslips and infected as above . autophagy was induced in hela cells by treatment for 4 hr with 100 nm torin-1 , 10 g ml of e64d - pepstatin a ( sigma - aldrich ) , or mock treatment with dmso . cells were treated with sirna for 48 hr as described above and then autophagy was induced followed by cell lysis ( 25 mm tris [ ph 7.5 ] , 0.5% np-40 , 150 mm nacl , and protease inhibitors [ roche ] ) . western blotting to demonstrate lc3 lipidation was performed after equalization of protein amounts and sds - page on an anykd polyacrylamide gel ( bio - rad ) . following transfer to immobilon - p membranes ( millipore ) , detection was performed using rabbit anti - lc3 primary ( sigma - aldrich ) , mouse anti - actin ( sigma - aldrich ) , and appropriate fluorescent secondary antibodies ( li - cor biosciences ) as previously described . the second exon of atg16l1 was targeted in hela cells using the px330 plasmid crispr system as described ( ran et al . , 2013 ) . all animal studies were conducted under protocols approved by the subcommittee on research animal care ( srac ) at massachusetts general hospital . clec12a mice were obtained from the laboratory of g.d.b . at the university of aberdeen . clec12a mice were produced by conventional gene targeting of exons 14 on a c57bl/6 background ( p. redelinghuys , a. augello , r.a . protocols for bacterial growth and infection were performed as previously described , with slight modification ( barthel et al . , 2003 ; conway et al . , 2013 ) . quantitative proteomics using itraq labeling of peptides was performed as described previously ( lassen et al . , 2014 ; mertins et al . , 2014 ) . hela cells were reverse - transfected with plasmid expressing gfp - clec12a on glass coverslips in 12-well plates the night before the assay . osmotic lysis of endosomes was accomplished by exposing cells to hypertonic medium ( 0.5 m sucrose in pbs with 10% peg100 ) for 10 min . cells were washed twice with 1 pbs and then incubated in 60% pbs for 3 min as described previously ( thurston et al . , 2012 ) . cells were returned to complete medium for 20 min and then fixed with ice - cold methanol .", "" ]

[ "randomized controlled studies have shown that noninvasive ventilation ( niv ) reduces short - term mortality , risk of intubation and complication rate when given as an additional treatment to selected patients with an acute exacerbation in chronic obstructive pulmonary disease ( copd ) complicated by hypercapnic acute respiratory failure.1,2 plant et al2 showed a decrease of the in - hospital mortality rate from 20% to 10% when niv was given to patients with mild acidosis and in a stationary or intermediary ward in a multicenter setting . as with most other treatment modalities , there is likely to be a less noticeable effect when comparing real - life and clinical trial efficacy . this was documented in an audit performed in the uk,3 where the aim was to explore factors related to reported high mortality rates . niv was implemented as an add - on modality in an acute medical ward and a respiratory ward at odense university hospital , odense , denmark , in 2004,4 based on local instructions and national recommendations,5 and the modality has since been used as a standard additional treatment primarily for patients with hypercapnic acute respiratory failure and verified or presumed copd . specifically , patients with arterial measures of ph < 7.35 and paco2 > 6.0 kpa and mono - organic symptoms of dyspnea , respiratory rate > 25 and pao2 < 7.0 kpa without oxygen therapy are eligible for niv . exclusion criteria for niv treatment in the ward are untreated pneumothorax , unstable hemodynamics , and affected alertness , indicating need of intensified monitoring and intubation capability when required . there are few reports on long - term survival after receiving niv in a clinical setting,6 and the issue of whether there is an advantage by implementing niv in patients with severe prognosis and expected high mortality rates has not been addressed . the aim of this study was to analyze long - term all - cause mortality following the first years of implementing niv in a real - life acute medical ward at a university hospital in denmark , with the null hypothesis that niv has no impact on long - term survival .", "all niv treatments were registered prospectively at the acute medical and respiratory ward of the odense university hospital , with patients receiving niv for the first time in the period january 1 , 2005 to december 31 , 2007 included for this analysis . baseline data registered were age , sex , when registered in the records , arterial blood gas analyses initiating the indication for niv ( ph and pco2 are presented ) , diagnoses at discharge , and forced expiratory volume in 1 second ( fev1 ) . patients were included at the time of their first niv episode , and were followed until death or january 31 , 2012 using the danish central person register to ensure complete follow - up . patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital . when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics . patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward . criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 . where possible , patients initiated on niv were referred to the respiratory ward the next day . since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study . initiation was done by internal medical staff and a respiratory physician was available for consultation if required . patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a do - not - intubate order , based on the severity of the underlying illness(es ) . on a few occasions , niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately .", "patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital . when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics . patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward . criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 . where possible , patients initiated on niv were referred to the respiratory ward the next day . since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study . initiation was done by internal medical staff and a respiratory physician was available for consultation if required . patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a do - not - intubate order , based on the severity of the underlying illness(es ) . on a few occasions , niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately .", "in the study period 20052007 , niv was initiated on 390 occasions : 107 times in 2005 , 143 times in 2006 , and 140 times in 2007 . of the 302 patients registered , 253 patients received niv for the first time and were included in this study . copd was listed as the primary diagnosis in 216 patients ( 85.4% ) ; the diagnosis was confirmed by documented spirometry in 182 patients ( 8% ) and was judged as highly probable in 34 patients ( 16% ) . other diagnoses registered at discharge in patients presenting with acute hypercapnic respiratory failure were congestive heart failure , thoracic malignancy , and hypoventilation by other causes ( see table 1 ) . arterial gas analyses resulting in initiation of niv was registered in 51.4% of cases ; in these patients , the median ph was 7.24 ( 7.077.43 ) , and median pco2 ( kpa ) was 10.1 ( 6.0 , 20.3 ) . after 5 years , 23.7% of the patients were still alive as shown in figure 1 . for patients with documented copd , the corresponding 30-day mortality was 24.3% and 5-year survival was 23.1% ( see figure 2 ) . there was no statistically significant difference in survival for men and women , although we did see a trend towards better long - term 5-year survival in females compared to males ( 25.7% vs 19.2% , p = 0.25 [ logrank ] ) . presence of copd did not affect prognosis , but the trend of better long - term 5-year survival in females compared to males was maintained ( 27.2% vs 17.9% , p = 0.083 [ logrank ] ) . the only statistically significant differences found between the long - term survivors and patients who died in the observation time were age and issue of a not - to - intubate order . survivors had a median age of 66 years compared to 74 years in nonsurvivors , and the not - to - intubate order was associated with 100% mortality . there was no difference in registered ph or pco2 between survivors and nonsurvivors at initiation of niv . descriptive data of copd patients versus non - copd patients receiving niv for the first time are shown in table 2 , and supplementary information in figure 1 .", "data on long - term survival of patients receiving niv for the first time revealed an expected high 30-day mortality rate , considerably higher than that seen in pivotal trials of niv in copd,1,2 but in line with other observational audits and studies.3,6,7 however , the 5-year survival rate was higher than earlier anticipated , which could be due to more stringent selection criteria for initiation of niv , and the fact that the study was only conducted in one center , giving patients more homogenous treatment . one significant difference between survivors and nonsurvivors was age , survivors being , as a group , younger than the group of patients who died , but the oldest patient ( 91 years ) registered in this study was alive at the end of 5 years of observation . we observed a trend of better long - term survival in females compared to males , in the whole niv group and especially in patients with copd , but further studies are needed to evaluate confounding variables . unfortunately , the registration of comorbidities was not done routinely or stringently , but , in the last couple of years , more focus has been made on screening and registration of comorbidities in copd , a benefit for future surveys . historically , males have had a higher exposure of cigarette smoke and occupational lung irritants8 and these factors could be contributing to the trend of worse long - term survival . it is well known that patients entered into clinical trials often differ significantly from patients in the usual clinical setting . in contrast to randomized controlled studies , this patient - based study included a much less selected group of patients having more comorbidities presenting with acute hypercapnic respiratory failure . patients were not limited to patients with respiratory failure due to exacerbation in copd , and , a priori , one could assume that this would affect prognosis . patients with other diagnoses included patients with clearly reversible disorders , such as overdoses of benzodiazepines , but several survivors were patients with other less morbid diagnoses than copd . despite the disadvantages of retrospective and therefore less complete data retrieval from records ( especially concerning arterial blood analyses ) , survivors compared to nonsurvivors were similar with regards to copd and , when registered , fev1 , ph , and pco2 . in the non - copd patients especially severe diseases such as congestive heart failure , thoracic malignancy , and end - stage fibrosis , were present among nonsurvivors but not at all present in survivors . in several of these cases , niv was given as a last treatment option in patients with a not - to - intubate order . it seems apparent that patients labeled not - to - intubate have a particularly poor prognosis and , based , on our findings probably did not benefit from the treatment . the study included all patients initiated in an acute medical ward setting or a respiratory ward . unlike two previous prospective studies by berkius et al,6 including 93 copd patients in a multicenter icu setting , and harris et al,7 reporting use of noninvasive ventilation ( continuous positive airway pressure and bilevel positive airway pressure ) in clinical practice over 5 years , we have not included patients receiving the modality of niv initiated only in the icu setting . berkius et al6 reported survival data ( 5-year ) for copd patients receiving niv in the icu ( 17% ) , which was lower for patients needing intubation ( 6% ) . a national british copd audit from 2008 on acidosis , niv , and mortality in hospitalized patients with copd exacerbations , including 232 hospital units and 9716 patients , showed that 20% of the patients had acidotic artery blood analyses at admission.3 niv treatment in clinical practice was given to severely ill patients , many with mixed metabolic acidosis . further , patients fulfilling clinical criteria for niv treatment failed to receive niv , and others received it inappropriately . niv was also seen used as a ceiling of treatment in which efficacy was uncertain . the primary focus of this present study has not been evaluation of the indication for niv treatment ; since 2008 , an annual national surveillance of patients with copd ( dansk register for kol [ drkol ] ) has been launched , registration practice ( international classification of diseases [ icd]-10 ) has been reviewed , and audits performed . in denmark , treatment with niv was provided to 9% of all copd patients admitted with an exacerbation in 2009 and 10% in 2010 and 2011.9", "our study confirms that patients with hypercapnic respiratory failure are a high - risk group , often with a poor prognosis . it casts some doubt as to what is achievable from niv in a non - icu setting and suggests that regular audits are required to ensure that the right patients are offered the right treatment when admitted with mild hypercapnic respiratory failure . however , the 5-year survival rate was higher than earlier anticipated , justifying the broad approach to niv treatment in this setting . the high 30-day mortality rate for all niv - initiated patients was higher ( 29.3% ) than for the group of patients with copd ( 24.3% ) , and this supports the notion that niv should be dedicated primarily to patients with copd in exacerbation . the 30-day mortality rate for copd patients receiving niv was similar in an audit from 2010 , and patients with ph < 7.25 had a worse outcome than patients presenting with higher ph at niv initiation ( unpublished data ) . monitoring of patient outcomes in a real - life setting is necessary to ensure optimal selection of patients for niv in a non - icu setting ." ]

[ "takayasu arteritis ( ta ) is a chronic inflammatory arteritis affecting the aorta and its main branches . the disease has a world - wide distribution , but has a high prevalence in japan . although there are no population based studies from india , reports have shown than the prevalence may be as high as in japan . most of the patients diagnosed to have this condition are women in their second and third decades of life . the occurrence of the disease in young children and infants is extremely rare with only a few cases reported all over the world .", "the present case report is about a 2-year - old female patient who was brought to the hospital with a history of incidentally detected hypertension of 2 months duration . she was born out of a non - consanguineous marriage and had an uneventful antenatal and postnatal course with normal developmental milestones and she had received vaccinations according the national immunization program , which includes bacillus calmette - guerin ( bcg ) . on examination , she was afebrile , active and well - nourished ; her weight was 16 kg and height was 80 cm . blood pressure was 140/90 mmhg , which was more than the 99 percentile for her height . her renal functions were normal ( blood urea : 8.4 mg / dl ; serum creatinine : 0.39 mg / dl ) . serum sodium was 133.5 mmol / l , s. potassium 3.3 mmol / l , s. calcium 9.9 mg / dl and s. phosphurus 3.94 mg / dl . erythrocyte sedimentation rate ( esr ) was 20 mm/1 h and c - reactive protein was 6 mg / dl . the size of the right kidney was 7.1 cm 3.6 cm and that of the left kidney was 6.1 cm 2.9 cm . computed tomography aortogram showed critical stenosis of the left renal artery , at a length of 8 mm from the origin , a short segment stenosis of the right renal artery at its origin , a tight focal stenosis at the origin of the celiac artery with post - stenotic dilatation , mild stenosis at the origin of the superior mesenteric artery and narrowing of the abdominal aorta at the level of renal arteries ; there was no para - aortic lymphadenopathy [ figure 1 ] . the diameter of aorta at the level of diaphragm , renal arteries and just above bifurcation into iliac arteries were 6.2 mm , 5.2 mm and 6.6 mm respectively , proving that there was a narrowing at the level of renal arteries followed by post - stenotic dilatation . with these angiographic findings , in this infant with hypertension , a diagnosis of ta was made according to the european league against rheumatism / pediatric rheumatology international trials organization / pediatric rheumatology european society ( eular / printo / pres ) criteria - 2008 . the child is being treated with nifedipine 5 g thrice daily and clonidine 50 mg thrice daily and her blood pressure is 90/55 mmhg . computed tomography angiogram showing bilateral renal artery stenosis and narrowing of abdominal aorta ( arrows : a : short segment stenosis of right renal artery , b : stenosis of left renal artery with small left kidney , c : narrowing of the abdominal aorta at the level of renal arteries )", "ta , also known as pulseless disease , occlusive thromboaortopathy and martorell syndrome , was first systematically described by a japanese ophthalmologist mikito takayasu . the disease remains an enigma as the exact cause of the disease is still not elucidated . a human leucocyte antigen ( hla ) association and viral etiology have also been postulated . the clinical presentation of ta includes three phases : \n the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade feverthe pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertensionthe occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . \n the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade fever the pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertension the occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . the diagnosis of ta in children is based on the eular / printo / pres criteria [ table 1 ] . diagnostic criteria for takayasu arteritis in children ta is certainly underdiagnosed and underreported from india , due to the fact that the disease may not present with classical features and the imaging modalities are available only in tertiary care centers . a study that compared the clinical manifestations of ta in india and japan reported that there was a significant variation in the manifestations of the disease in the two countries . in contrast to japanese patients in whom proximal aorta involvement ( takayasu conference classification - type i and ii ) was common , in indian patients descending and abdominal aorta involvement ( type iv ) was common . moreover , hypertension was the most dominant sign in indians compared with pulse lessness in the japanese . it was also noted that the age at diagnosis of the disease was almost a decade earlier in indians compared to japanese ( 28 10 vs. 37 14 years , p < 0.01 ) . in an indian study , it was found that aortoarteritis was the most common cause of renovasular hypertension and accounted for 59.4% of all cases . however , the mean age of the subjects was 27 years and the youngest child was 5 years of age . to the best of our knowledge , there are no reports from india of a case of ta in an infant . our patient had type iv disease , with the involvement of abdominal aorta , coeliac , superior mesenteric and both renal arteries . although it is suggested that glucocorticoids are the mainstay of treatment in the early or prepulseless phase , other immunosuppressive agents like methotrexate , azathioprine and anti - tumor necrosis factor agents have also been used , especially in steroid resistant cases . however , no evidence is available for benefit of therapy in the pediatric age group . however percutaneous intervention is less likely to succeed when stenosis or occlusions affect lengthy portions of an artery or the artery is heavily scarred . an angioplasty to the left renal artery was attempted in this case , but was unsuccessful . hence it was decided to continue medical management with anti - hypertensives as blood pressure could be controlled with medications and she was not given steroids as there were no clinical or laboratory indicators of an active inflammatory disease in this infant .", "although the world - wide prevalence of ta is very low , it is more prevalent in asian countries like japan and india . the clinical manifestations of the disease in india are different from that seen in japan . type iv disease involving abdominal aorta and renal arteries is more common in indians and hypertension is the most common sign . if the disease is active , steroids can be tried and the lesions amenable to angioplasty may be corrected by a percutaneous transluminal angioplasty ." ]

[ "currently , liver transplantation is the only definitive treatment for the end stage liver diseases . however , its widely used was limited by the expensive costs , shortage of donor organs and invasive procedure ( 1 ) . hepatocyte transplantation has emerged as a feasible alternative to liver transplantation in some liver diseases ( 2 ) , but it is limited by organ donors and the low cell quality of available liver tissues ( 3 , 4 ) . although human embryonic stem cells ( hescs ) and umbilical cord blood stem cells ( ucbsc ) could be induced into hepatocyte - like cells in vitro ( 5 ) , their widely used were limited by the low differentiation quality , ethics , and teratoma formation ( 6 ) . recently , the adult stem cells ( ascs ) derived from various tissues , including bone marrow derived mesenchymal stem cells ( bmscs ) , adipose tissue derived mesenchymal stem cells ( adscs ) , and peripheral blood mononuclear cells ( pbmcs ) have been developed as new cell sources contributing to liver regeneration for their high efficiency of hepatogenic differentiation using simple procedures and no ethnic issues ( 710 ) . in the present study , we investigated whether ascs are the ideal seed cells for the liver regeneration from the followings : ascs biology , including isolation , culture , differentiation to hepatocytes , and the further role of ascs in cell therapy and tissue engineering", "in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee . most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) . side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) . adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time , g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) . then pbmcs were collected by means of aphaeresis . conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) . ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 . more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 . however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues . more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii . cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs . besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) . adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) . some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types . however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently . the hepatocyte - like cells from ascs were confirmed from the gene and protein expression . gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) . protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs . however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) .", "in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee .", "most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) . side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) . adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time , pbmcs were mobilized with recombinant g - csf at 510 g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) .", "conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) . ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 . more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 . however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues . more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii . cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs . besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) . adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) .", "some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types . however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently .", "the hepatocyte - like cells from ascs were confirmed from the gene and protein expression . gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) . protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs . however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) .", "in the present study , we described different ascs could be induced into hepatocyte lineage cells in different culture systems in vitro . it is very safe and easy to acquire the enough ascs , and then induce them into functional hepatocytes in vitro . based on this progress , ascs transplantation might be a novel therapy for the severe liver diseases , and also will be ideal seed cells for liver tissue engineering . however , which ascs are better still needs us to investigate from their preparation , molecular characterization , and functional assay . this paper firstly provides such a concise review focused on ascs biology and differentiates potential , indicating ascs might be an ideal seed cells in cell transplant therapy and tissue engineering . we also believe in the future , some studies will show us the most appropriate ascs for cell transplant or tissue engineering ." ]

[ "waldenstrom 's macroglobulinemia ( wm ) is the result of a clonal proliferation of lymphocytes that produce monoclonal immunoglobulin m ( igm ) . it is now considered to correspond to lymphoplasmacytoid lymphoma as defined by the world health organization classification system ( 1 , 2 ) . many central nervous system ( cns ) complications have been described in wm patients ; the majority have been associated with blood hyperviscosity caused by igm . the hyperviscosity syndrome is characterized by headache , tinnitus , vertigo , blurred vision , and chronic bleeding from the nose and gums ( 3 ) . however , cns infiltration by plasmacytoid lymphocytes ( bing - neel syndrome ) has only rarely been reported ( 4 ) .", "a 51-yr - old woman was diagnosed with wm three years prior to this presentation . she received six courses of chlorambucil ( 0.3 mg / kg per day on day one to four orally ) and prednisone ( 45 mg / m per day on day one to four ) . next , she received fludarabine chemotherapy ( 25 mg / m per day on day one to five intravenously ) with a limited response . high - dose cyclophosphamide and granulocyte colony - stimulating factor were administered to induce peripheral blood progenitor cell ( pbpcs ) mobilization for autologous stem - cell transplantation ; however , the number of pbpcs collected was not sufficient . we then decided on a conservative treatment approach , because the patient did not have specific symptoms and the serum igm level was stable ( 3,000 - 3,500 mg / dl ) . the laboratory values on admission were as follows : white cell count 8,050/l with normal differential counts , hemoglobin 9.6 g / dl , platelets 296,000/l , erythrocyte sedimentation rate 144 mm / hr , total protein 9.44 g / dl , albumin 2.85 g / dl , igg 3144.4 mg / dl , iga 25.4 mg / dl , igm a brain computed tomography scan revealed multifocal extra - axial tumorous lesions along the dura matter . a brain magetic resonance imaging showed an extra - axial soft tissue tumor along the left cavernous sinus and tentorium , right frontal convexity and tentorium and falx ; the brain parenchyma appeared to be unremarkable ( fig . cerebrospinal fluid analysis showed the following : white cell count 43/l with neutrophils 1% , lymphocytes 52% and monocytes 47% , total protein 1.81 g / dl , glucose 104 mg / dl , igm 64.5 mg / dl and a few plasmacytoid lymphocytes on cytology . stereotactic biopsy of dural tissue at the falx showed a diffuse infiltration with atypical cells , which were identified immunophenotypically as plasmacytoid lymphocyte with expression of lca ( + ) , cd3 ( - ) , cd20 ( + ) , and vs38a ( + ) ( fig . 2 ) . the patient was confirmed to have cns infiltration by atypical plasmacytoid lymphocyte infiltration ( bing - neel syndrome ) . the paitent received a total dose of 1,980 cgy with irradiation therapy of the whole brain that was administered in 11 fractions , and then her headache subsided , but the igm level was elevated persistently to 3,379.8 mg / dl . following radiation therapy , fludarabine chemotherapy was performed ( 25 mg / m per day on 1 to 5 intravenously , two courses ) . a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ( fig . the patient had no evidence of cns recurrence during the follow - up period of one year . however , the igm level has been increasing slowly , so further chemotherapy , including rituximab may be considered .", "in 1936 bing and neel reported the association of hyperglobulinemia , cns symptoms ( paresthesias , headache , and paralysis ) , and brain infiltration composed of plasma cells and lymphocytes in two patients ( 5 ) . in 1944 , waldenstrom described the syndrome that bears their name ( bing - neel syndrome ) ( 6 ) . the bing - neel syndrome , originally placed in a \" toxic - infectious \" category , appears to be the result of involvement of the cns by diffuse neoplasm infiltration . although they are most often peripheral , they can involve the cns . in wm , the cns may be involved by a variety of mechanisms , including hyperviscosity and direct infiltration by neoplastic cells ( 7 , 8) . patients with bing - neel syndrome have sometimes presented with a mass containing neoplastic cells , but the masses have been intraparenchymal rather than meningeal ( 8 , 9 ) . this syndrome can be subdivided into diffuse and tumoral forms . in the diffuse infiltrative form such as this case , malignant cells are localized mainly in leptomeningeal spaces , periventricular white - matter , pons , and medulla ( 7 , 10 - 12 ) . radiological findings in this syndrome have been reported , but a typical pattern has not emerged ( 13 - 15 ) . an imaging technique would be the preferable diagnostic test , although histological confirmation is necessary to establish the definitive diagnosis . intraventricular chemotherapy for bing - neel syndrome was reported to be effective in 1984 ( 15 ) . however , a review of the literature reveals that the outcome for most patients who underwent chemotherapy was poor , and the patients died within several months ( 10 , 13 , 14 ) . therefore , patients with bing - neel syndrome may benefit from cranial radiation therapy prior to chemotherapy ( 9 , 12 , 16 ) . recently , a great deal of interest has been noted by treatments with purine nucleoside analogs ( fludarabine , cladribine , and pentostatin ) because of their remarkable activity in lymphoproliferative disorders . it has been reported that a patient with bing - neel syndrome ( in its diffuse form ) was successfully treated with cladribine administration ( 17 ) , or radiation therapy and combination of cladribine , cyclophosphamide , and prednisone ( 18 ) . in the present case further treatment was not possible because of persistant bone marrow suppression . a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ; the patient had no evidence of cns recurrence during the follow - up period of one year , but the igm level has been increasing slowly . we think that the major effect of deceased mass size was due to the radiation therapy in the present case . several retrospective and prospective studies have indicated that rituximab may induce an objective response in approximately 30 - 40% of previous treated patients with wm ( 19 ) . however , the effect of rituximab treatment on the cerebrospinal fluid b - cell compartment is limited in comparison with the effect on the b cells in the periphery ( 20 ) , and it has not been tried in bing - neel syndrome as yet . therefore , the effect of rituximab on cns involovement of wm need to be validated by of future studies ." ]

[ " the increasing prevalence of childhood obesity , especially in preschool aged children [ 1 , 2 ] , has stimulated research targeting critical periods of growth . according to freinkel 's hypothesis of fuel - mediated teratogenesis , the intrauterine environment can influence changes in gene expression and affect the development and maturation of fetal organs and tissues [ 3 , 4 ] . the postnatal period up to 2 years of age is also a critical period of growth . indeed , rapid infant weight gain is associated with an increased risk of obesity and metabolic consequences later in life [ 58 ] . these include metabolic parameters such as in utero glucose exposure [ 911 ] , maternal prepregnancy body mass index ( bmi ) [ 1214 ] , and maternal insulin sensitivity during pregnancy . in a cohort of low - income children , maternal obesity during pregnancy more than doubled the risk of obesity in children . maternal diet and physical activity during pregnancy [ 1619 ] , as well as smoking may impact offspring risk of obesity and metabolic risk [ 2023 ] . however , there is a paucity of literature examining these risk factors in very young children . in particular , breastfeeding has a protective role against the development of obesity [ 2427 ] . harder et al . reported a dose - dependent association between breastfeeding and risk of obesity , where each month of prolonged breastfeeding decreased obesity risk in the child . beyond the first few months of life , breast - fed infants gained less weight than formula - fed infants . another study showed weight gain from 6 to 12 months was less in infants exclusively breastfed for 5 months compared to 2 months . earlier introduction to complementary foods may also contribute to increased risk of childhood overweight [ 14 , 31 ] , however the evidence is inconsistent . additionally , reduced physical activity and increased screen time ( television and video ) are associated with obesity risk in older children , although limited data is available in infants [ 33 , 34 ] . there is little known about the impact of maternal and infant lifestyle on weight gain and adiposity at 1 year of life . the effect of screen time in infants on weight gain and adiposity has not been included as a potential factor in prior statistical models . there is also little information on the effect of maternal physical activity on weight outcomes of offspring . the objective of our study was to determine how maternal physical activity , maternal insulin sensitivity ( isogtt ) , prepregnancy bmi , infant feeding practices ( breastfeeding duration , age of introduction of formula , and complementary foods ) , and screen time contribute to infant weight gain and adiposity at 1 year of age .", "the study protocol was approved by the research ethics board at mount sinai hospital and the hospital for sick children . participants were recruited for this prospective cohort study at the time of antepartum gestational diabetes mellitus ( gdm ) screening and were consented to be followed into the postpartum . all consenting women then completed a 3-h oral glucose tolerance test ( ogtt ) in late 2nd or early 3rd trimester . venous blood samples were drawn at baseline , 60 , 120 , and 180 min after ingestion of a standard 100 g glucose load . the pregnant women were then classified into 3 glucose tolerance groups : ( 1 ) gdm , as defined by the national diabetes data group ( nddg ) criteria ( requires at least two of the following : fasting glucose 5.8 mmol / l , 1 h postload glucose 10.6 mmol / l , 2 h postload glucose 9.2 mmol / l , or 3 h postload glucose 8.1 mmol / l ) ; ( 2 ) gestational impaired glucose tolerance ( gigt ) , defined as meeting only one of the above criteria ; ( 3 ) normal glucose tolerance ( ngt ) , defined as not meeting any of the nddg criteria . parental demographics , medical history and anthropometrics were collected during pregnancy at the time of the ogtt . data collected included maternal age , maternal prepregnancy weight , maternal and paternal ethnicity , family history of diabetes , socioeconomic status , and maternal physical activity indices . maternal isogtt was calculated from the ogtt using the matsuda index , which is well correlated with insulin sensitivity derived from the euglycemic - hyperglycemic clamp method . physical activity was assessed using the baecke questionnaire , which has been validated in several populations including women of child - bearing age [ 37 , 38 ] . the questionnaire was completed during the ogtt , with participants reporting on their physical activity in the year preceding the pregnancy . the baecke questionnaire measures three domains of physical activity : ( i ) occupation - associated activity ( work index ) ; ( ii ) sport - related physical activity ( vigorous / sport index ) ; ( iii ) leisure - time physical activity not including sports ( leisure index ) . the work index quantifies the exertion related to occupational activities , including sitting , standing , lifting , and walking , as well as effects on the individual ( e.g. , fatigue and perspiration ) . the sport index characterizes vigorous / sport activity with respect to intensity ( using the updated compendium of physical activities ) . these indices were calculated on a scale from 15 , where 5 represented the highest level of physical activity for each category . for example , a score of 3 out of 5 for leisure index might indicate sometimes walking or cycling for 1530 min and seldom watching television . mean parental education score and occupation score was calculated on a scale of 17 and 19 , based on the hollingshead index , an established surrogate index of socioeconomic status . a maximum averaged parental score of 62 indicated the highest level of education and profession . infant information collected at birth included length of gestation , gender ( male or female ) , and birth weight . at the 1 year visit , infant weight and length were measured , and weight - for - length z - score was calculated according to the world health organization ( who ) 2006 child growth standards to provide a surrogate measure of adiposity . the infant lifestyle questionnaire was completed by the mothers when the infants were 3 and 12 months of age and provided information on exclusive breastfeeding duration , age of introduction to formula and cereal , and screen time based on predictors found to be important for the development of obesity ( average daily exposure to tv or video viewing , meals eaten while tv is on , tv in bedroom ) [ 34 , 42 ] . infants born < 37 or > 42 weeks , twins , or those with medical illnesses that required prolonged hospitalization were excluded from the analysis . bivariate analysis of continuous variables with infant weight gain and weight - for - length z - score were assessed by spearman 's correlation analysis . independent samples t - test or one - way analysis of variance ( anova ) and post hoc analysis ( fisher 's least significant difference ) were used to test differences in the outcome variables with maternal glucose tolerance group ( gdm , gigt , or ngt ) , gdm status ( yes / no ) , family history of diabetes ( yes / no ) , infant gender ( male / female ) , ethnicity ( caucasian or non - caucasian ) , age of introduction to formula ( at birth , 15 months , 6 months or never ) , and average daily screen time divided into 3 categories ( no screen time , < 60 minutes , and 60 minutes ) . we converted age of introduction to formula into a categorical variable because although exclusive breastfeeding duration and age of introduction to formula are similar variables , they are not synonymous because some infants were never formula - fed ( n = 80 ) . variables determined to be significant from bivariate analyses , concurrently with variables known to influence infant weight gain and adiposity , were entered into multiple linear regression models . two separate models were created for weight gain from birth to 1 year and weight - for - length z - score at 1 year . nonmodifiable risk factors ( infant age , sex , ethnicity , birth weight , and family history of diabetes ) were forced into the model and modifiable risk factors ( maternal prepregnancy bmi , gdm status , maternal physical activity indices prior to pregnancy , socioeconomic status , maternal log isogtt , infant exclusive breastfeeding duration , age of introduction to formula , age of introduction to cereal , and daily screen time ) were entered according to the forward stepwise method .", "descriptive characteristics and lifestyle measures are presented in table 1 for mothers and infants at 1-year postpartum ( n = 246 ) . from the time of consent postnatally to the 1-year followup visit , 62 ( 20% ) of the original 311 infants were lost to followup . there were no significant differences in gdm status , prepregnancy bmi , maternal education , or maternal physical activity between mothers retained in the study versus those lost to followup . no significant differences were found for infant weight gain and weight - for - length z - score based on the mother 's gdm status ( yes / no ) or glucose tolerance group during pregnancy ( gdm , gigt , or ngt ) . independent samples t - tests also showed no significant differences in the infant outcomes for family history of diabetes ( yes / no ) and infant ethnicity ( caucasian or non - caucasian ) . males had greater weight gain than females ( p < 0.001 ) , however no differences between males and females for weight - for - length z - score were found ( p = 0.636 ) . one - way anova showed significant differences in infant weight gain for age of formula introduction ( p = 0.001 ) and screen time ( p = 0.032 ) ( figure 1 ) . similar trends for age of formula introduction were found for infant weight - for - length z - score at 1 year ( p = 0.012 ) . however no differences were found between 0 , < 60 min , or 60 minutes of daily screen time ( p = 0.19 ) . spearman 's correlation analysis ( table 2 ) showed a negative association for infant weight gain with maternal pregravid vigorous / sport index ( p = 0.031 ) , exclusive breastfeeding duration ( p < 0.001 ) , and an earlier age of introduction to cereal ( p = 0.02 ) . weight - for - length z - score was positively associated with maternal prepregnancy bmi ( p = 0.003 ) and birth weight ( p < 0.001 ) , and negatively associated with maternal log isogtt during pregnancy ( p = 0.031 ) and maternal pregravid vigorous / sport index ( p = 0.006 ) . maternal pregravid vigorous / sport index was also positively correlated with isogtt during pregnancy ( p < 0.001 ) , as expected from previous data . each month of prolonged exclusive breastfeeding reduced weight gain by 116.4 g , after adjustment for infant age , sex , infant ethnicity , family history of diabetes , and maternal pregravid vigorous / sport index ( p < 0.001 ) . after adjustment , each unit increase in maternal pregravid vigorous / sport index decreased weight gain by 218.6 g ( p = 0.016 ) . in total , 33% of the variance in the model for weight gain was explained by the sample . infant birth weight , infant ethnicity , and family history of diabetes did not significantly predict infant weight gain at 1 year . additionally , socioeconomic status , maternal prepregnancy bmi , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time did not emerge in the model when entered . for weight - for - length z - score , an increase in one unit of maternal prepregnancy bmi ( kg / m ) was associated with an increase in weight - for - length z - score of 0.03 ( p = 0.016 ) , or one standard deviation change on the who child growth standards , after adjustment . thus , mothers with a greater prepregnancy bmi were more likely to have heavier infants normalized for length . in addition , each month of prolonged exclusive breastfeeding and unit increase in maternal pregravid vigorous / sport index decreased weight - for - length z - score by 0.08 ( p = 0.010 ) and 0.20 ( p = 0.031 ) , respectively . infant sex , infant ethnicity , and family history of diabetes were not significantly associated with infant weight - for - length z - score . variables that did not emerge in the model included : socioeconomic status , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time . approximately , 19% of the variance in the model for weight - for - length z - score was explained by the sample .", " these findings demonstrate that maternal pregravid physical activity , prepregnancy bmi , and infant feeding practices have a significant influence on infant weight gain and adiposity . weight gain from birth to 1 year was negatively predicted by infant female sex , maternal pregravid vigorous / sport activity , and exclusive breastfeeding duration and positively predicted by infant age at time of 1 year visit . weight - for - length z - score at 1 year was negatively predicted by maternal pregravid vigorous / sport activity and exclusive breastfeeding duration and positively predicted by birth weight , infant age at time of 1 year visit , and maternal prepregnancy bmi . factors that have been previously shown to influence infant weight gain and weight - for - length z - score , such as socioeconomic status or gdm status , were not significant in our model . this may be due to the fact that women in this study tended to have excellent glycemic control , which was evidenced by normal birth weight in the gdm pregnancies . the effect of maternal prepregnancy bmi on infant adiposity is consistent with existing literature [ 1214 , 31 ] . in mothers with ngt or gdm , maternal prepregnancy bmi was significantly associated with childhood overweight in both groups , even after adjustment for maternal glucose status and infant birth weight . furthermore , knight and colleagues reported that the impact of maternal fasting plasma glucose in nondiabetic mothers on infant growth was transient , while maternal prepregnancy bmi and paternal bmi remained correlated with offspring bmi at 2 years of age . thus , the authors suggested that parental obesity , shared environment , or genetic factors had a greater influence on childhood bmi than maternal fasting plasma glucose during pregnancy . after controlling for confounding factors , maternal pregravid vigorous / sport activity significantly predicted infant weight gain . the mean pregravid vigorous / sport index in our study was 2.4 , which suggests that 1530 min of walking or cycling per day in mothers prior to pregnancy may be beneficial for decreasing obesity risk of offspring . maternal log isogtt did not predict infant growth at 1 year , after adjusting for maternal physical activity indices . prior analyses by our group showed pregravid vigorous / sport activity was an independent predictor of maternal isogtt and reduced glucose intolerance in pregnancy . taken together , pregravid physical activity may affect infant weight gain through a mechanism that partly involves maternal isogtt , however , these results suggest that maternal lifestyle may have a greater impact on infant weight gain than maternal glucose tolerance group or isogtt . although daily screen time was not a significant predictor of infant weight gain or adiposity in our multivariate analyses , it was unsettling to find that more than half of the infants in our study were already exposed to television at 1 year of age and 11% of infants watched more than 2 hours of television per day . infants with 60 minutes of screen time each day were heavier at 1 year of age than infants with 0 or < 60 minutes of daily screen time . one possible explanation may include decreased overall activity levels in infants exposed to television for longer periods of time . preschool children , more than 2 hours of television per day was associated with a higher risk of overweight and at risk for overweight and greater adiposity . despite the known effect of screen time on bmi in childhood [ 4547 ] , there is currently limited information on the effect of screen time under 2 years of age . the mechanisms to explain the effects of breastfeeding compared to formula feeding may include different physiological and behavioural factors . several studies [ 2427 ] , however not all [ 32 , 48 , 49 ] , suggest that breastfeeding has a protective role on later obesity risk . after adjusting for confounding variables , infants exclusively breast fed for a longer duration had reduced weight gain and adiposity at 1 year . this may be influenced by the unique metabolic factors in breast milk compared to formula , such as breast milk leptin [ 50 , 51 ] and adiponectin that may induce earlier satiety . the higher protein content of formula may also contribute to increased adiposity in formula fed infants . koletzko and colleagues demonstrated that infants given formula with high protein content compared to infants given formula with low protein content resulted in increased weight at 2 years of age with no effect on infant length . it is thought that the higher protein consumption results in higher insulin secretion and stimulates the expression of insulin - like growth factor i ( igf - i ) , leading to more adipogenic activity and adipocyte differentiation [ 54 , 55 ] . additionally , behavioural differences between breast - fed and formula - fed infants may affect infant energy intake . studies have shown that there is less maternal control and a greater response to the infant 's hunger and satiety cues with breastfeeding compared to formula feeding [ 56 , 57 ] . there is also a lower frequency of meals and a higher uniformity of feeding volumes in formula - fed infants compared to breast - fed infants . although some studies have reported the timing of cereal introduction as a positive predictor of infant weight gain , it did not emerge in our multivariate analyses . in a recent study , huh and colleagues noted that the timing of the introduction of solid foods on the odds of obesity at 3 years of age varied by breastfeeding status . infants that were introduced to solid foods before 4 months and were never breast fed or stopped breastfeeding before 4 months had a sixfold increase in the odds of obesity compared to infants breast fed for more than 4 months . since our study population was introduced to cereal later than 4 months ( 5.5 1.1 months ) altogether , our findings on infant feeding suggest that exclusive breastfeeding duration may be more predictive of infant weight gain and adiposity than age of formula or cereal introduction . this study should be viewed in context of certain strengths and limitations . due to the assessment of glucose tolerance in the late 2nd or 3rd trimester of pregnancy , we can not completely exclude the possibility that some individuals may have received treatments following ogtt that could affect in utero glucose exposure and isogtt on infant outcomes . although data was available on the type of intervention ( diet therapy or other ) , glycemic control during pregnancy could not be determined . other limitations included the lack of data on maternal diet and maternal physical activity during pregnancy in our study . proposed that maternal energy intake influenced energy intake in children more than intrauterine exposure to diabetes , which suggests maternal energy intake should be included in future studies . additionally , our study population had a large proportion of caucasian infants and highly educated parents , and it is unclear whether results may be extended to other ethnic backgrounds or families with lower socioeconomic status . loss to followup was 20% , and although there were no differences in maternal factors between those who left and remained in the study , feeding practices of the infants may have differed . we were also unable to report on infant physical activity due to challenges in capturing physical activity accurately and objectively in infants . nevertheless , we examined maternal and infant lifestyle variables that have not been previously studied together , providing further insight into the effects of the maternal and infant environment on weight gain and adiposity while controlling for important confounding variables . in summary , increased maternal pregravid physical activity and longer exclusive breastfeeding duration may have a critical influence on reducing infant weight gain and adiposity as early as 1 year of age . results from this study support the recommendation for exclusive breastfeeding in infancy , and suggest maternal physical activity may also influence postnatal outcomes . future directions include examining the additive effects of maternal diet during and after pregnancy , infant physical activity , and sedentary time on infant weight gain and adiposity . the evaluation of paternal physical activity may also provide insight into the impact of family lifestyle on infant growth . longitudinal followup of these children will examine the effects of maternal and infant factors on bmi and adiposity in early childhood beyond 1 year of age ." ]

[ "phylogenetic networks represent the evolutionary relationships of taxa , including sequences , genes , chromosomes , genomes , or species . there are different types of phylogenetic networks , but here , we are interested in reticulate networks , which provide an explicit representation of evolutionary history , meaning that internal nodes represent ancestral species , and nodes with more than two parents correspond to reticulate events such as recombination , hybridization or lateral gene transfer ( huson and bryant 2006 ) . reticulate networks have been extensively used in evolutionary studies , especially at the population level , where reticulate events are , in general , quite common ( posada and crandall 2001 ) . very few studies have tried to assess the performance of the algorithms used to reconstruct phylogenetic networks ( cassens et al . 2007 ) , and only recently , a comprehensive computer simulation study has been completed ( woolley et al . one of the problems that arose during these studies was the comparison of reticulate networks . although several comparison metrics have been already introduced in the literature ( baroni et al . 2008a , 2008b ; cardona , rossell , and valiente 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , metrics for phylogenetic networks ii : nodal and triplets metrics , unpublished data ; cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) , all of them have specific requirements regarding the nature of the networks to be compared in order to be perfect , that is , to have the metric properties of nonnegativity , separation , symmetry , and triangle inequality . before continuing a network contains nodes ( vertices ) and branches ( edges ) that connect them . in general , we will refer to rooted networks , with a direction from the past to the present ( i.e. , directed graphs ) that allows for the identification of parent and child the root node is the oldest node and has no parents . tree nodes when two nodes share the same parent they are siblings . depending on the relationships among the nodes that occur in a network , these can be classified as tree sibling ( cardona , llabrs , et al . 2008a ; cardona , rossell , and valiente 2008a ) , where every hybrid node has at least one sibling that is a tree node ; tree child 2008b ; cardona , rossell , and valiente 2008a , 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , unpublished data ) , in which every internal node has at least one child that is a tree node ; regular ( baroni et al . 2004 ) , where the set of descendant leaves ( clusters ) of the nodes are all distinct ; galled trees ( gusfield et al . 2004a , 2004b ) , where the paths from the most recent common ancestor ( mrca ) of the parents of a hybrid node down to the hybrid node form disjoint cycles ; and ( binary ) trees , which only contain tree nodes . these network classes are nested in this order : tree sibling tree child galled trees trees , meaning a tree is also a galled tree , a tree child network , and a tree sibling network , and so on . regular networks , however , are not related to tree sibling , tree child , or galled tree networks ( cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) . the point we want to make is that the networks resulting from reticulating evolutionary processes do not necessarily correspond with any of the idealized classes of networks described above and for which perfect metrics exist . were this true , it would imply that many phylogenetic networks could not be properly compared with the existing metrics . obviously , it is very important to characterize the size of this perceived gap between the algorithms and the biology , and this is precisely our goal . in order to provide a formal statistical description of this disagreement , independent of particular organisms or genomic regions , we will use coalescent theory ( kingman 1982 ) to generate the type of reticulate networks that result from the evolutionary process . the coalescent describes the probabilities of the different genealogies for a sample of genes generally but not necessarily from the same population , and it was further extended by hudson ( 1983 ) to include recombination events . specifically , we will quantify the different classes of networks ( regular , tree sibling , tree child , and galled trees ) produced by the coalescent as a function of the population recombination rate .", "the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination , the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one . therefore , for a sample of n genes , there will be n 1 coalescent events until the mrca is reached . if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) . therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for ancestral recombination graph ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1 , we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination . the genealogy goes from the present ( bottom ) to the past ( up ) . coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) . two sample sizes ( n = 10 and 50 ) and seven population recombination rates ( = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored . we used a continuous - time approximation where the times of the events are exponentially distributed . for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies . we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) . the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively . the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees . first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig . superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events . although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 .", "the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination , the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one . therefore , for a sample of n genes , there will be n 1 coalescent events until the mrca is reached . if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) . therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for ancestral recombination graph ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1 , we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination . the genealogy goes from the present ( bottom ) to the past ( up ) . coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) . two sample sizes ( n = 10 and 50 ) and seven population recombination rates ( = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored . we used a continuous - time approximation where the times of the events are exponentially distributed . for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies . we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) . the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively .", "the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees . first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig . superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events . although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 .", "in the absence of recombination , all the simulated genealogies were binary trees , as expected . with recombination , different classes of reticulate networks were produced whose complexity was a function of the population recombination rate ( tables 1 and 2 ) . for n = 10 , at small recombination rates ( = 1 ) many networks were tree sibling , but half were already non - tree child . with twice as much recombination , half the networks were more complex than tree sibling networks , and only a few were tree child networks or galled trees . with moderate recombination rates ( = 4 ) , only 15% of the networks were tree sibling , whereas with larger recombination rates . almost no network , or even none at all , could be classified into any of the standard classes . increasing the sample size to n = 50 just increased a little more the complexity of the networks but preserved the same trend regarding the effect of recombination . on the other hand , even at small recombination rates ( = 1 ) rather few networks are regular because hybrid nodes in the genealogies simulated by the coalescent with recombination have two parents and a single child , and a network containing a hybrid node with a single child is not regular unless the child is also a hybrid node . number of simulated networks falling in each class as a function of the recombination rate = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 10 number of simulated networks falling in each class as a function of the recombination rate = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 50 we will argue that the network topologies we do not consider branch lengths produced by the coalescent with recombination will be indistinguishable from those produced by other reticulating processes like gene conversion , hybridization , and lateral gene transfer . note that the coalescent with recombination results , going backward in time , in nonreciprocal exchanges of genetic material as only one of the two recombinants inherits ancestral material and will be therefore represented in the network . conveniently , these types of events are also typical of gene conversion , hybridization , and lateral gene transfer . in addition , the nodes in the arg produced by the coalescent do not have to belong to the same species ; they just represent gene copies . therefore , we believe that these results are likely to be very similar for models of lateral gene transfer and/or hybridization the assumptions underlying the standard classes of networks should eventually fail given sufficient reticulation . we can conclude that the reticulate networks produced by the evolutionary process , at least as modeled by the coalescent with recombination , are much more convoluted than regular , tree sibling , tree child , or galled tree networks . these network classes the only ones for which perfect metrics exist are clearly insufficient to describe reticulating evolutionary processes . indeed , new network metrics need to be developed if we really want to compare reticulate phylogenetic networks estimated from real data ." ]

[ "we recently demonstrated that diabetes directly induced dental caries in rats and mice , although a direct association between the 2 was less evident and conflicting in humans [ 14 ] . we demonstrated that dental caries were produced using noncariogenic diets with a low concentration of sugar in diabetic rodent models [ 57 ] , although many studies usually add large amounts of sugar to the diet to induce dental caries [ 8 , 9 ] . in addition , early studies on the production of experimental dental caries have reported the effects of physical properties of cariogenic diets with high concentrations of sugar on dental caries . these studies mainly used finely ground cariogenic diet [ 1013 ] because it is more cariogenic compared to a coarsely ground diet in rodents . moreover , hard and coarse foods have the ability to prevent caries [ 1417 ] . thus , modifying the dietary formulation may possibly enhance or reduce caries development in diabetic animals . however , no reports have investigated the effects of the physical properties of non - cariogenic standard diets on dental caries in diabetic animals . currently , 2 types of non - cariogenic standard diet formulations ( i.e. , powdered and pelletized diets ) are widely used in experimental rodents . in the present study , we compared the effects of these diets on the development of dental caries in a diabetic rodent model focusing on the difference in hardness between the powdered and pelletized diets .", "six - week - old female f344 rats were supplied by japan slc , inc . the animals were housed in stainless - steel cages at a temperature of 2026c and a relative humidity of 4070% under a 12/12 h light / dark cycle , that were ventilated with filtered fresh air . to prevent infection , the animals were allowed free access to tap water and fed with a widely used standard pelletized or powdered diet ( charles river formula 1 ( crf-1 ) ; oriental yeast co. , ltd . , the animals were handled according to the principles for all experimental procedures outlined in the guide for the care and use of laboratory animals prepared by our institution ( setsunan university ) and the japanese association for laboratory animal science . the glucose levels in the fresh urine were measured semiquantitatively using urine test paper ( wako pure chemical industries , osaka , japan ) daily from day 1 to day 3 after dosing , once every week for 1 month after the first week , and once every month thereafter . the blood glucose levels in the tail vein samples were also measured semi - quantitatively using the glucose oxidase method ( glutest e ; sanwakagaku , aichi , japan ) once every month from the fourth week after alloxan injection . blood samples obtained from the tail vein and fresh urine were collected from 1:00 to 4:00 p.m. the severity of hyperglycemia was defined as follows : normal , < 200 mg / dl ; mild , > 200 mg / dl ; moderate , > 300 mg / dl ; or severe , > 400 mg / dl . the severity of glycosuria was defined as follows : normal , < 100 mg / dl ; mild , > 100 mg / dl ; moderate , > 250 mg / dl ; or severe , > 500 mg / dl . the experimental design is shown in figure 1 . ten rats , aged 7 weeks , were given a single dose ( 35 mg / kg body weight ) of alloxan ( sigma - aldrich japan , tokyo , japan ) via tail vein injection . the dose of alloxan was determined as the given dose at which a rat survives for a long period after the onset of diabetic symptoms . after the confirmation of hyperglycemia and glucosuria following the dosing of alloxan , five rats were given a pelletized crf-1 diet ( diabetes - pelletized diet group ) and the remaining 5 rats were given a powdered crf-1 diet ( diabetes - powdered diet group ) . as a control , each of the 5 intact rats was fed a pelletized or powdered crf-1 diet ( control - pelletized diet group and control - powdered diet group ) . the animals were euthanized by exsanguination under deep anesthesia at the end of the observation period . subsequently , the mandible was removed and fixed in 10% neutral - buffered formalin ( ph 7.4 ) . after a 24 h fixation , the occlusal , buccolingual , and proximal surfaces of all of the molar teeth were intensively observed under a binocular stereoscope . soft x - ray images of the mesiodistal plane were taken under conditions of 35 kv and 2 ma for 4 min . the molar teeth were classified into 5 groups according to caries characteristics by observing and analyzing the radiographs : no radiolucent change ( grade 0 ) , radiolucent area only on the occlusal surface of the crown ( grade 1 ) , radiolucent areas on occlusal surface and either of the mesiodistal surfaces of the crown ( grade 2 ) , radiolucent areas over the entire surface of the crown ( grade 3 ) , and radiolucent areas over most of the surface of the dental root ( grade 4 ) . the mean score of the caries was used as indicator for comparing the severity of the carious lesions between the groups . after soft x - ray examination , a histopathological examination was performed on the mandible in all of the rats . after fixation with 10% neutral - buffered formalin , the sample was decalcified in a 5% solution of ethylenediaminetetraacetic acid 4 na ( edta 4 na ) for 2 weeks at 4c . after decalcification , the specimens were trimmed , dehydrated in a sequential ethanol series using an automated processor , and embedded in paraffin wax . serial 7 m thick sections on the mesiodistal plane were made through the centers of all of the molars and then stained with hematoxylin and eosin for examination using light microscopy . the severity of the caries lesion was graded as follows : slight , dentin caries localized in occlusal surface of dentin ; mild , dentin caries extended into the dental pulp with pulpitis and/or pulp necrosis ; moderate , dental crowns were partially decayed ; and severe , dental crowns were completely decayed ( only the molar roots remained ) . the wilcoxon rank - sum test was employed to compare the differences in the mean scores of the caries lesions using soft x - ray examination between the groups . the chi - square test was used to determine the caries incidence using soft x - ray examination and the incidence of histopathological lesions in each group of rats .", "severe hyperglycemia ( > 400 mg / dl ) and glucosuria ( > 500 mg / dl ) continued from the day after alloxan injection to the last monitoring day in all of the rats in the alloxan - treated groups . in addition , the blood glucose levels ranged from 78 to 120 mg / dl ( normoglycemia ) , and the urine glucose levels were less than 100 mg / dl in the control groups . typical macroscopic appearances of carious molars in diabetic rats are shown in figures 3(a ) , 3(b ) , and 3(c ) . macroscopically , the dental caries developed mainly in occlusal fissures and were identified as partial coronal defects of the molars in alloxan - treated diabetic rats ( figure 3(b ) ) . the carious lesions expanded horizontally , until the crown of the carious molar was completely invisible ( figures 3(b ) and 3(c ) ) . in contrast , control nondiabetic rats showed no changes in any of the molars ( figure 3(a ) ) . soft x - ray images of the carious lesion in diabetic rats are shown in figures 3(d ) , 3(e ) , and 3(f ) . in all of the alloxan - treated diabetic rats , radiographically , the dental caries progressed both horizontally and vertically . in diabetic rats fed with a pelletized diet , the carious lesion was mainly characterized as grade 2 ( figure 3(e ) ) or 4 type ( table 1 ) . in addition , the diabetic rats fed a powdered diet demonstrated carious lesions that were nearly of grade 4 type ( figure 3(f ) ) . in the control non - diabetic rats , there was no change in the molar teeth ( table 1 , figure 3(d ) ) . the histopathological characteristics of the carious lesion in diabetic rats are shown in figures 3(g ) , 3(h ) , and 3(i ) . histopathological carious lesions were detected in the crown as eroded dentin with bacterial colonization in alloxan - treated diabetic rats . in diabetic rats fed with the pelletized diet , many of the molars were moderately affected and the dentin caries spread over a wide area of the dental crown ( figure 3(h ) ) . in the diabetic rats fed with the powdered diet , the dental caries were markedly worsened and the crowns were nearly completely decayed ( figure 3(i ) ) . the incidence of caries teeth on the basis of each scoring in the soft x - ray examination is shown in table 1 . the incidence of dental caries in the diabetic rats fed on both the pelletized and powdered diets was apparently higher ( p < 0.01 ) compared to the non - diabetic control rats ( table 1 ) . in the diabetic rats fed with the pelletized diet , 96.7% of their molar teeth were affected with dental caries , and the mean caries score was 2.7 . in addition , in the diabetic rats fed with the powdered diet , all of their molars ( 100% ) were affected with caries , and the mean caries score was 3.7 , which was significantly higher ( p < 0.01 ) than the diabetic rats fed with the pelletized diet ( table 1 , figure 4 ) . no radiolucent lesions were observed in any of the molars in control non - diabetic rats . the incidence of histopathological carious lesions on the basis of each grade is summarized in table 2 . histopathologically , the severity of the carious lesions in the diabetic rats fed with a powdered diet was significantly enhanced compared to the diabetic rats fed with the pelletized diet ( table 2 ) . in control non - diabetic rats , slight carious lesions , which were macroscopically and radiographically normal , were detected in a few animals ; however , there were no differences observed between the non - diabetic rats on the pelletized and powdered diets ( table 2 ) .", "the physical properties of foods such as hardness , adhesiveness , and cohesiveness are closely related with the caries - producing potential [ 19 , 20 ] . moreover , the development of caries is known to be profoundly affected by food hardness , and hard and coarse foods can exert a detergent activity during mastication , which is effective in the prevention of caries [ 17 , 19 ] . furthermore , a lower degree of caries was reported in animals fed with the hardest food diet . in the present study , the severity of caries in diabetic rats was enhanced in the powdered diet group compared to the pelletized diet group . it was clear that food hardness affected the development of dental caries in diabetic rats fed on a non - cariogenic diet . hard foods are known to help flush away or neutralize undesirable material within the dental plaque and exert a cleaning effect on smooth surfaces by direct mechanical friction . therefore , cariogenicity will be higher in diabetic rats fed with a powdered diet than in those fed with a pelletized diet . in this study , a high incidence and severity of dental caries was confirmed in the alloxan - treated diabetic rats fed with 2 types of standard diets with a low concentration of sugar . furthermore , the mean caries score of the diabetic rats fed on the pelletized diet at 52 weeks after alloxan dosing in this study was almost 2-fold compared to animals at 26 weeks after alloxan dosing described in a previous report , strongly suggesting that the duration of hyperglycemia may affect caries development in diabetic animals . in this study , dental caries developed and extended to all of the molars in diabetic rats fed on both the pelletized and powdered diets . thus , the effect of food hardness on the different diets in caries development in diabetic rats may become clearer when the dental caries mildly develop during the more early stages . in conclusion , food hardness could have an effect on the development of dental caries in diabetic rats , and appropriate consideration of the food formulation should be made in experimental caries studies using diabetic rodent models ." ]

[ "encapsulation of biological macromolecules within lipid vesicles is important for biophysical research and for a variety of applications such as pharmaceuticals , foods , and cosmetics . although attractive interactions between solutes and the lipids used to encapsulate them can lead to high encapsulation efficiencies , such methods are only effective for certain solute / lipid combinations . in contrast , passive encapsulation ( i.e. , without attractive interactions ) is more general but leads to internalization of solutes at or below their concentration in the vesicle formation solution . solute size is a key determinant in the efficiency of passive encapsulation , with larger solutes excluded from the vesicle interior such that the internal solute concentration is lower than the external concentration during vesicle formation . knowledge of solute concentrations in the vesicle interior is important for understanding processes as varied as enzymatic turnover , polymerization , crystallization , or phase separation occurring in this volume . it is also of interest for applications in which vesicles serve as carriers for solute delivery , such as in food formulations or therapeutics . quantification of encapsulated solute is accomplished by different methods depending on the size of the vesicles . encapsulation in large unilamellar vesicles ( luvs ) and other submicrometer vesicles ( svs , which include both luvs and small unilamellar vesicles , suvs ) is quantified in terms of the bulk encapsulation efficiency ( bee ) . bee is the percentage of solute trapped within a batch of submicrometer vesicles as compared to the total amount of solute added . it is typically determined by removing all of the unencapsulated solute ( e.g. , via centrifugation or dialysis ) and then lysing the vesicles to quantify the remaining ( encapsulated ) solute . giant vesicles ( gvs , 1100 m),(12 ) on the other hand , are not generally studied via bulk methods due to their limited stability , but are large enough to be analyzed individually . for example , the concentration of a fluorescently tagged solute encapsulated within an individual giant vesicle can be quantified by comparing the internal solute fluorescence to a calibration curve.(13 ) the ability to determine encapsulation efficiency for individual gvs ( eeind ) also makes it possible to compare encapsulation between vesicles within a batch , which can vary widely . recently , approaches to determination of eeind for submicrometer vesicles have been reported ; these powerful new tools have not yet become routine . the most common gv formation techniques , electroformation(18 ) and gentle hydration,(19 ) passively encapsulate solutes , such that the expected concentration inside ( cin ) is equal to the external concentration during vesicle formation ( cout ) . for solutes with rh < 10 nm , mean cin does tend to be equal to cout ; however , we and others have measured considerable variability in cin for individual gvs in the same batch . for example , mean cin was found to be much less than cout for dextran polymers with hydrodynamic radius , rh > 10 nm.(14 ) solute size is also an important determinant of entrapment in submicrometer vesicles . for example , adrian and huang compared the encapsulation of proteins of increasing molecular weight to that of a small marker protein ( lysozyme , 14.1 kda ) in ca . the 97 kda -amylase was encapsulated only 50% as well as the lysozyme.(7 ) the same authors reported that proteins up to 120 kda could be encapsulated in ca . 200 nm diameter vesicles with the same trapping efficiency as sucrose.(7 ) bee is often quite low for passive uptake because it is defined in terms of the total encapsulated volume ( capture volume , cv ) , which can be < 1% of the total volume , depending on the amount of lipid present and the morphology of the vesicles . although cv can be increased up to ca . 50% by using more concentrated lipid,(20 ) this is not always possible for a given system . indeed , most papers report bees much less than 50% . to increase bee beyond what is possible by passive encapsulation , for example , cationic lipids are routinely used to package nucleic acid drugs , despite concerns about their toxicity . solute encapsulation based on electrostatic attractions can be essentially complete , with nearly 100% of all solute molecules added to the solution being associated with liposomes . despite excellent loading , attractive interactions with encapsulating lipids can lead to more complex liposome morphologies , prevent activity of the encapsulated solute until released , and inhibit effective release of solutes upon vesicle rupture . while these factors are often considered acceptable in drug delivery applications , it is preferable to have free encapsulated solute of known concentration for biophysical studies , e.g. , to evaluate the effects of confinement.(30 ) encapsulation of rh > 10 nm fluorescently labeled dextran polymers in gv has been reported to improve markedly in the presence of several weight percent additional polymeric solutes.(15 ) the additional polymers acted as volume excluders , resulting in condensation of the labeled solutes and allowing them to be more easily encapsulated during vesicle formation.(15 ) macromolecular crowding is an attractive means for improving macromolecule encapsulation because of its generality.(31 ) this approach for improving encapsulation has not previously been tested for biomacromolecules such as proteins , which in many cases already exist in globular conformations and hence may not undergo as large a rh change in response to volume excluders , nor has it been tested for encapsulation in submicrometer vesicles . herein , we report the use of peg as a macromolecular crowding agent to increase the encapsulation efficiency of fluorescently labeled biomacromolecules within gvs and 200 nm diameter luvs . we find in general that the addition of peg increases the encapsulation efficiency of high molecular weight ( mw ) biomolecules compared to those encapsulated in di water alone . we conclude that volume exclusion in the presence of a crowding agent such as peg or dextran can be used as a general route to increase encapsulation efficiency of biomacromolecules in both giant and submicrometer lipid vesicles , up to levels expected for passive encapsulation of lower mw solutes ( i.e. , cin = cout ) .", "volume exclusion induced condensation is expected to have the greatest impact on large , extended molecular conformations . therefore , the macromolecules used in this work were chosen to give a range of molecular sizes and shapes . we first describe the effect of polymeric volume excluders on encapsulation in gvs formed by gentle hydration and then discuss encapsulation in luvs prepared by a freezethaw / extrusion protocol . we began by encapsulating fibrinogen , a 340 kda protein with a hydrodynamic radius ( rh ) of 11 nm , which is important in coagulation and thrombosis and is often administered to patients who have suffered severe blood loss or have lost the ability to clot blood on their own . fibrinogen has been reported to condense to approximately one - half of its original length during polymerization . fibrinogen labeled with alexa fluor 488 ( af488 ) was added during gv formation at a concentration of 33 nm , either in di water or in a 3 wt % peg 8 kda solution . gvs were imaged without removing them from the hydration solution , such that the external fibrinogen concentration remained 33 nm . figure 1 shows representative confocal fluorescence images and histograms of encapsulation efficiency for individual gvs formed in the presence of fibrinogen alone and coencapsulated with 3 wt % peg . here , encapsulation efficiency for the individual vesicles ( eeind ) is defined as the concentration ratio between the vesicle interior ( ci ) and the external solution ( co ) used during vesicle formation . the concentration ratio ( ci / co ) for each vesicle a vertical line is included at ci / co = 1 to facilitate comparison to the external fibrinogen - af488 concentration . in the absence of added peg , encapsulation efficiency is quite poor ; the gv interiors appear dark , and fibrinogen - af488 concentration varied considerably from vesicle to vesicle , ranging from 1 to 54 nm ( 0.041.64 co ) with a mean of 21 nm ( 0.64 co ) . in contrast , vesicles formed in the presence of 3 wt % peg contained fibrinogen - af488 concentrations similar to that of the external solution , with 65% of the gvs encapsulating internal concentrations between 28 and 38 nm and a mean of 34 nm . ( left ) confocal fluorescence images of vesicles prepared in the presence of af488-labeled fibrinogen without ( top ) and with ( bottom ) 3 wt % peg 8 kda . membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine . ( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled fibrinogen in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 33 nm ) . we repeated this experiment with two other proteins having relatively large rh to test the generality of the method of increasing eeind via macromolecular crowding . thyroglobulin is a 660 kda protein with a pi of 4.6 that is important in the synthesis , storage , and secretion of thyroid hormones . the native structure has two slightly elongated subunits , with an rh of 10 nm.(39 ) in vivo , thyroglobulin structure is known to condense in order to increase the storage capacity of the thyroid.(40 ) encapsulation of thyroglobulin - af488 ( 18 nm ) in gvs in the absence and presence of 3 wt % peg 8 kda produced results similar to those for fibrinogen - af488 ( supporting information figure 1 ) , which had similar rh and ability to compact . we then repeated the experiment with catalase - af488 , a 250 kda enzyme with a pi of 5.4 and an rh of 10.4 nm . this protein catalyzes the decomposition of hydrogen peroxide to water and oxygen,(43 ) and has been encapsulated in submicrometer liposomes along with superoxide dismutase and injected to protect against oxygen toxicity in rats.(44 ) images and a histogram for cin are shown in supporting information figure 2 . eeind for was significantly increased vs control by addition of peg 8 kda during encapsulation of all three of these relatively large proteins ( student s t test , p < 0.001 ) . these results are consistent with the role of peg 8 kda as a volume excluder , driving the condensation of macromolecular structures . proteins , nucleic acids , and enzymes are all known to adopt condensed structures in macromolecularly crowded solutions . thus , we interpret the improved encapsulation of these high molecular weight proteins in 3 wt % peg 8 kda as the result of their condensation to give reduced hydrodynamic radii , which in turn caused them to be encapsulated as effectively as lower mw biomolecules . we next evaluated the effect of peg 8 kda on encapsulation of two smaller proteins and a dna oligomer , all of which had hydrodynamic radii less than 10 nm . -1-antitrypsin ( aat ) is a 52 kda protein with an rh of 3.8 nm and a pi between 4.9 and 5.1 . it is responsible for inhibiting elastase in vivo , which destroys elastin on connective tissues.(57 ) aat deficiency often leads to lung diseases , and clinical trials have been reported where patients are given aat infusions , which showed beneficial effects.(58 ) it has also been delivered to mice via encapsulation in submicrometer liposomes.(59 ) we began by encapsulating 1.9 m aat - af488 in gvs formed in with the presence and absence of 3 wt % peg 8 kda . aat - af488 was encapsulated at essentially cin = cout with or without 3% peg ( figure 2 ) . ( left ) confocal fluorescence images of vesicles prepared in the presence of 1.9 m labeled -1-antitrypsin without ( top ) and with ( bottom ) 3 wt % peg 8 kda . membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine . ( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled -1-antitrypsin in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 1.9 m ) . similar results were observed for encapsulation of labeled human serum albumin ( hsa ) in peg solution as compared to di water ( supporting information figure 3 ) . hsa has similar characteristics as aat ( i.e. , hsa has a mw of 67 kda , pi of 4.95.1 , and rh of 3.51 ) . we also studied the encapsulation of a single - stranded 173 base pair oligomer ( 53 kda ) labeled with fluorescein ( fam ) . nucleic acids are often encapsulated in submicrometer vesicles for gene delivery and in gvs to study biochemical reactions . dna is known to undergo condensation in the presence of divalent cations , polycations , or even neutral polymers such as peg . this oligonucleotide was sufficiently small to be effectively encapsulated in the gv even without addition of a crowding agent ( supporting information figure 4 ) . table 1 compares encapsulation for each of the biomacromolecular solutes with and without added peg . in addition to the increase in cin for the larger proteins when encapsulated in the presence of 3 wt % peg , decreased heterogeneity in cin across a population of vesicles in a batch is suggested by the standard deviations in cin ( table 1 ) and the histograms for individual solutes ( figures 1 and 2 and supporting information figures 14 ) . an f - test(61 ) was performed to evaluate whether differences in the variance between the control and + peg populations were statistically significant . we found statistically significant changes in variance for the fibrinogen ( p < 0.001 ) , catalase ( p < 0.001 ) , and aat ( p < 0.05 ) , but not for the other solutes . the increase in encapsulation homogeneity when fibrinogen and catalase were coencapsulated with a crowding agent arises largely from the shift to higher encapsulated concentrations . decreased variance for aat may also be due in part to condensation , which has been reported for aat and other small polymers , but is more likely due to peg increasing the viscosity and osmotic pressure of the solutions , as has been discussed previously . for example , increased viscosity could improve encapsulation heterogeneity by facilitating pore formation in the membrane , allowing more time for solutes to equilibrate between internal and external solutions.(65 ) we note that the proteins were not found to be associated with the lipid membrane of the gvs , but rather to be uniformly dispersed in the exterior and/or interior of the vesicles ( see , for example , figures 1 and 2 , left panels ) . polymerization or aggregation on microscopically visible length scales preparation of the vesicles in 3% peg did not inhibit their formation or change their size or morphology in any obvious way as compared to preparation in peg - free solution , consistent with our previous findings of decreased average vesicle diameter at 10 and 20 wt % peg but no noticeable difference between 0 and 3 wt % peg in terms of gv formation , size , or morphology.(15 ) the improvements in eeind observed in the presence of peg should be applicable to other crowding agents.(15 ) we therefore coencapsulated fluorescently labeled fibrinogen and 1-antitrypsin with 3 wt % dextran 500 kda for comparison . the 3 wt % dextran 500 kda had the same effect on encapsulation as peg 8 kda : the higher mw protein , fibrinogen , showed an increase in eeind magnitude and homogeneity with the introduction of either peg or dextran ; and the lower mw protein , aat , showed essentially no change in mean eeind , but a decreased variance ( f - test , p < 0.05 ) when dextran was added ( table 1 ) . theoretical eeind defined as the solute concentration in the external solution during vesicle preparation , which is given in parentheses for each solute in the first column . fluorescein labeled ; hydrodynamic radius of 7.5 nm for 280 nt ssdna was reported in ref ( 66 ) under conditions where excluded volume effects can be neglected ; extrapolation of length vs rh data in ref ( 66 ) suggests 5 nm hydrodynamic radius for our 173 nt sequence . internal solute concentrations in sv can not be determined via simple confocal imaging as for the gv described above . therefore , we estimated cin and the effectiveness of solute encapsulation by comparing the percentage of total solute that has been encapsulated within a batch of vesicles ( bulk encapsulation efficiency , bee ) to the capture volume . capture volume was estimated based on the moles of lipid present and the size of the vesicles , assuming unilamellarity . bee was determined by removing the unencapsulated solute from a batch of vesicles , then lysing the vesicles and quantifying the remaining ( encapsulated ) solute . for effective passive encapsulation , one can expect to achieve the same concentration inside the vesicles as is present outside ( cin = cout ) . however , for high mw solutes , bee is often less than the capture volume ( i.e. , interior concentrations < exterior concentrations ) . table 2 shows the average bee for three encapsulated solutes ( carboxyfluorescein ( cf ) , 500 kda dextran - fitc , and thyroglobulin - af488 ) with and without added peg in 200 nm diameter vesicles . the theoretical bee for passive encapsulation in this system is 0.38% , calculated by estimating the number of vesicles formed based on the amount of lipid added to the system ( 500 m ) and then calculating the volume encapsulated by those vesicles as compared to the initial volume . we estimated that there were 3.4 10 lipid molecules per 200 nm diameter vesicle , for 4.5 10 vesicles that collectively encapsulated 19 l , or 0.38% of the total solution volume ( 5 ml ) , based on calculations for similar systems . dynamic light scattering verified that vesicle diameter remained constant whether in dilute or crowded solution , and the addition of a fluorescent lipid in the membrane verified that a negligible amount of lipid was lost to the filter during extrusion . errors represent the standard deviation of bee measurements taken for three separate batches of vesicles on three different days . encapsulation of the small fluorescent molecule , carboxyfluorescein ( cf ) , was performed as a control . it was expected that the effect of the coencapsulation of peg would be negligible , since the addition of peg did not significantly impact the encapsulation efficiency of cf in gvs.(15 ) the change in experimental cf bee observed between the solutions with and without peg was not statistically significant based on a student s t test ( p = 0.63 ) ( table 2 ) . encapsulation of fitc - dextran 500 kda and thyroglobulin - af488 did show improvement for luvs prepared in solutions containing peg 8 kda as compared to solutions without peg ( student s t test , p < 0.05 ) . experimentally determined bee for fitc - dextran 500 kda and thyroglobulin - af488 in water were 31% and 50% of the theoretically predicted value , respectively these low bees are most likely due to the large size of dextran 500 kda ( rh 15 nm ) and thyroglobulin ( rh 10 nm ) . addition of 3 wt % peg 8 kda significantly increased the bee of fitc - dextran and thyroglobulin - af488 in these vesicles , consistent with condensation of the dextran and thyroglobulin structures via macromolecular crowding.(15 ) although the error is large , it appears that with peg 3% , the luvs are essentially encapsulating even these very large solutes at cin = cout . bee was increased without chemically modifying the vesicle or solute to utilize specific attractive interactions . in contrast to the gv encapsulation data , improvements in standard deviation were not observed in the luv experiments . this underscores the difference in the two measurements : the error in gv cin measurements indicates vesicle - to - vesicle variability within a batch , while variability in luv bee experiments reports on differences between entire batches prepared separately and as such may reflect variability in capture volume . we interpret this as the result of some cf loss from the vesicle interior during centrifugation and/or other handling , which is commonly reported in submicrometer vesicles.(69 ) these data are exciting , because while the majority of reports published on bee utilize specific interactions between liposome / solute , this method is quite general and should apply to a variety of solutes that can be condensed under conditions of macromolecular crowding . although the absolute bee in these experiments was low , we have demonstrated that addition of 3 wt % peg provides a means of improving encapsulation for large solutes such that cin = cout for luvs prepared by freezethaw / extrusion . increasing the amount of lipid used in a batch has been shown by others to increase cv from < 1% to as high as 50% ; which would lead to an increase in bee , as previously reported.(20 )", "our results indicate that the addition of the polymeric coencapsulants peg 8 kda and dextran 500 kda as macromolecular crowding agents during passive encapsulation of biomacromolecular solutes can substantially increase mean encapsulated concentration and in some cases homogeneity for otherwise poorly encapsulated high mw proteins in giant vesicles . the addition of a crowding agent also reduced vesicle - to - vesicle encapsulation variability for the lower mw aat , which was already encapsulated efficiently ( i.e. , with cin = cout ) in dilute solution . the mechanism for increased encapsulation is general , such that crowding agents need not be varied with the identity of the solute . this work is important to those using gvs as bioreactors , where knowledge of encapsulated concentration is critical to quantifying the progression of products formed during a reaction . measurements of bulk encapsulation efficiency ( bee ) in luvs in the presence of peg suggest that this approach works similarly in these smaller vesicles as for gvs , bringing the internal solute concentration closer to the external concentration such that the bee is determined by capture volume . the approach used here should be effective for a wide range of membrane / macromolecular solute combinations .", "l--phosphatidylcholine ( egg pc ) , 1,2-dioleoyl - sn - glycero-3-[phospho - rac-(1-glycerol)](sodium salt ) ( dopg ) , 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc ) , cholesterol , and 1,2-dioleolyl - sn - glycero-3(phosphoethanolamine - n - lissamine rhodamine b sulfonyl ) ( ammonium salt ) ( rhodamine - dope ) were purchased from avanti polar lipids , inc . carboxyfluorescein , fluorescein isothiocyanate ( fitc)-labeled and unlabeled dextrans and poly(ethylene glycol ) ( peg ) were obtained from sigma . alexa fluor 488 and fitc labeling kits along with fibrinogen - alexa fluor 488 were purchased from molecular probes , inc . a fluorescein nucleic acid labeling kit was purchased from mirus bio ( madison , wi ) . a 173 nucleotide single - stranded oligomer with the sequence for influenza b was purchased from integrated dna technologies , inc . water used in these experiments was deionized to a resistance of 18 m with a nanopure diamond water system from barnstead int . silanized borosilicate glass culture tubes , 12 75 mm , were used to prevent biomolecules from sticking to the glass surface during formation ( kimble chase , vineland , nj ) . we first prepared a chloroform solution of lipids containing a 3:7 molar ratio of egg pc : dopg at a concentration of 0.26 mg / ml with 0.05 mol % rhodamine - dope . lipid solutions were dried under ar ( g ) to form a thin , uniform lipid film , and then , the vials were vacuum - desiccated for 2 h to remove any residual organic solvent . then , an aqueous solution containing a fluorescent biomolecule with or without 3 wt % unlabeled peg or dextran heated to 37 c was added along the wall of the tube , and the lipids were hydrated 48 h at this temperature . we chose 3 wt % peg 8 kda based on prior work with dextran 500 kda condensation , in which we observed increased encapsulation efficiency in gvs up to 10 wt % peg , with the bulk of the increase achieved by 3% and decreased average gv diameter for 10 wt % peg.(15 ) following incubation , the solutions were allowed to cool to room temperature . the contents of the vials , which remained undiluted , were removed with a micropipet and transferred to a microscope slide silanized with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane ( gelest , inc . , morrisville , pa ) for analysis . coverslips were also coated with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane to prevent the adhesion of fluorescently labeled biomolecules to the glass surface . the concentrations of crowding agents are referred to in wt % , since this is the common notation used for crowding agents in the macromolecular crowding literature ; the 3% peg 8 kda used here corresponds to 3.75 mm . concentrations for fluorescently labeled solutes , which are considerably lower than for the crowding agents , are given as molarities . a fluorescence line scan was taken across each vesicle to determine the fluorescence intensity of the alexa fluor 488 , fitc , or fluorescein inside the vesicle ( ii ) and in the undiluted bulk solution ( io , outside the vesicle ) , as reported previously . the concentration of biomolecule inside ( ci ) and outside ( co ) the gv was determined directly from the intensities ( ii , io ) by a calibration curve of the labeled biomolecule free in solution . from the calibration curves , it was determined that the bulk concentration ( co ) was equal to the initial concentration used to hydrate the vesicles . imaging was performed using an lsm-5 pascal laser scanning confocal microscope from carl zeiss , inc . ( oberkochen , germany ) with a plan - apochromat 63 oil immersion objective ( 1.4 na ) , and pascal software as previously reported . only vesicles that were greater than 3 m in diameter and appeared to be uni- or oligolamellar were analyzed . dppc / cholesterol vesicles ( 80:20 mol ratio ) were prepared using a modified technique provided by the distributor to yield large unilamellar vesicles by extrusion ( luvet ) as previously reported . briefly , dppc and cholesterol were combined with chloroform in a round - bottom flask and evaporated for 4 h. following this , 5 ml of the solute to be encapsulated was added in either water or 3 wt % peg 8 kda to yield a final lipid concentration of 0.34 mg / ml . the lipid mixture was allowed to incubate in this solution at room temperature for 30 min . then , the flask was subjected to five freeze / thaw cycles to promote entrapment of water - soluble compounds into the vesicles.(71 ) the mixture was then extruded through a 0.2 m polycarbonate membrane for a total of 20 passes ( mini extruder kit , avanti polar lipids , inc . , excess solute was removed from the vesicle suspension by washing with a 10 000 mw dialysis cassette in di water for 48 h ( slide - a - lyzer , thermo scientific , rockford , il ) and then centrifuging at 16 100 g , removing the supernatant , and replacing it with a solution free of fluorescent molecules . vesicle diameter was confirmed using dynamic light scattering ( zetasizer nano s , malvern instruments , worcestershire , u.k . ) . after centrifugation , the vesicles were lysed in 0.5% triton - x 100 , and the encapsulated concentration was determined via fluorescence measurements on a jobin yvon horiba fl3 - 21 fluorimeter ." ]

[ "we collected all possible literature from the year 1972 ( when kfd was first described ) to the present day conducting a medical search on kikuchi fujimoto disease ( kfd ) in english language . we included publications in authorized medical journals including original and review articles / theses , editorials , case reports and brief communications .", "kfd is an extremely uncommon disease but cosmopolitan with higher japanese and asiatic prevalence.1,2 however , sporadic cases have also been reported from europe . in asian woman kikuchi s disease is rare and benign cause of cervical lymphadenopathy.3 the disease , most often , happens to occur in young adults below 40 and seldom in children.4 at first , little female predominance was considered , but the recent literature regards it as male to female ratio 1:1.1,5 a current study on the patients with kfd conducted in taiwan reports the average age of 21.5 cardiac , hepatic and pulmonary involvement raises the morbidity .", "however , role of viruses ( epstein - barr virus and others ) in the pathogenesis of kikuchi s disease is controversial and unremarked.1 on the other hand , unger and coworkers are in favor of viral etiology as kfd manifests certain viral features ie , atypical lymphocytosis , certain histologic features , flulike respiratory prodrome and no response to antibiotic therapy.7 also , kikuchi s disease has been reported in patients with aids.8 like systemic lupus erythematosus ( sle ) , lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.9 it has been opinioned that in genetically susceptible individuals , kfd may belong to exuberant t - cell mediated immune response provoked by variety of stimuli.1 even though course of cell death in kfd needs to be studied and emphasized , ohshima and his associates remarked apoptotic cell death might be involved in the pathogenesis of kfd.10 regarding to this study , proliferating cd8 t - cells may kill or be killed in the apoptotic process of this disease using fas and perforine pathways .", "kikuchi s disease begins as an acute or sub - acute condition , developing over two to three week period . tender cervical lymphadenopathy is the characteristic feature ( 56%98% ) of kfd , predominantly involving the posterior cervical triangle . size of the enlarged lymph nodes ranges from 0.5 cm to 4 cm ( occasionally 6 cm ) . 59% patients represent painful lymphadenopathy and 1%22% patients undergo generalized lymphadenopathy.4,7,9,11 kfd , more or less , rarely involves mediastinal , peritoneal or retroperitoneal regions of the body.2 fever ( 30%50% ) associated with upper respiratory symptoms , sore throat , night sweats , weight loss , headache , rash , nausea , vomiting , and leukopenia ( about 50% ) are the other manifestations of the disease . 12,13 atypical lymphocytes have been reported in the peripheral blood film of patients with kfd . extranodal involvement is rare ; however , skin , eye and bone marrow affection has been reported.1 nevertheless , kfd is linked to sle and autoimmune conditions as lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.1,14 additionally , extranodal involvement in kfd is associated with frequent systemic symptoms . anecdotal reports bring to light the unusual features of kfd like haemophagocytic syndrome and carcinoma15 along with fatal multicentric disease . nervous system involvement ( septic meningitis , acute cerebellar ataxia , and encephalitis ) rarely happens to occur.16 regarding joint involvement , a case of 14 year old boy with kfd is in the record.17", "in patients with kikuchi fujimoto disease , an excisional biopsy of the involved lymph nodes is the investigation of choice . coagulative necrosis with ample karyorrhetic debris in paracortical areas of the involved lymph nodes is the characteristic histologic feature of kfd . other baseline investigations are reported unaffected . nevertheless , laboratory results of some kfd cases have reported anemia , little rise in esr and even leukopenia . one third individuals with kfd have shown atypical lymphocytes in their peripheral blood films.5 predominantly , t - cells ( cd8 + t - cells ) are found in kfd . however , neutrophils are found absent and scarce plasma cells may or may not be present . according to kuo , histopathologic features can be classified in three stages : proliferative , necrotizing , and xanthomatous.18 proliferative stage expresses various histiocytes , plasmacytoid monocytes and lymphoid cells containing karyorrhetic nuclear fragments , and eosinophilic apoptotic debris . necrotizing stage shows a degree of coagulative necrosis while xanthomatous stage is predominantly stuffed with foamy histiocytes . it must be born in mind that in the individuals with kfd , atypical reactive immunoblastic component is common and can be mistaken for lymphoma.4 histiocyte - associated antigens ( lysozyme , myeloperoxidase and cd68 ) are also expressed by histiocytes in kfd .", "kfd is an extremely rare disease and the differential diagnosis can be established on the basis of enlarged lymph nodes which are associated with many other disorders . it is necessary to born in mind the differential diagnosis of kfd as its treatment dramatically differs from other disorders . lymphoma ( non - hodgkin s lymphoma ) , tuberculosis , sle , plasmacytoid t - cell leukemia , kawasaki s disease , and myeloid tumor are included in the differential diagnosis of kfd.1 sometimes , because of similar clinical and histological features , it becomes problematic to differentiate kikuchi fujimoto disease from lymphadenitis associated with systemic lupus erythematosus ( sle ) . however , it has been reported that kfd is associated with sle . in order to exclude sle , all the necessary investigations of sle ( c3 , c4 , anf , anti - sm , and le cells ) are required . early recognition of kikuchi fujimoto disease is of prime importance to save the patient from undergoing extensive investigations related to malignant lymphoma and other related disorders.8 histopathologic features like presence of abundant reactive histiocytes and absence of reed - sternberg cells favor kfd . sometimes , kfd may express histiocytes resembling with signet - ring cells and can be confused with signet - ring carcinoma . however , metastaic adenocarcinoma contains cells with atypical nuclei and mucin debris instead of cellular debris .", "kikuchi fujimoto disease ( histiocytic necrotizing lymhadenitis ) is a self - limiting condition that resolves spontaneously within 1 to 4 months of period . however , studies reveal recurrence of the disease in 3%4% of the patients.9 additionally , sle may happen to occur some years later . no hereditary risk has been documented in kfd.8 most of the time symptomatic relief is offered for the local and systemic complains of the disease . lymph node tenderness and fever is treated with analgesics , antipyretics , and nsaids . sometimes , but rarely , steroids can be used temporarily , especially in severe extranodal involvement or generalized clinical course.19 in order to run an excisional biopsy of the enlarged lymph nodes , surgical consultation may be prerequisite . individuals with kikuchi fujimoto disease should be examined systemically and they must be under regular follow - up in order to monitor the manifestations of sle . the course of cervical lymphadenopathy is benign and resolves spontaneously . very few cases have been reported as fatal . however , no standard or specific treatment of kfd has been recommended .", "kikuchi fujimoto disease is an idiopathic , extremely rare , more or less worldwide , and often under - diagnosed condition ; predominantly involving the posterior cervical lymph nodes . early recognition of kikuchi s disease is of prime importance to avoid extensive and expensive investigations related to malignant lymphoma other related disorders . in order to avoid misdiagnosis , awareness of this disease" ]

[ "does it have any bearing on outcome in the icu or during the post - icu period ? although not evidence based in any prospective , controlled , randomized clinical trials , it is unlikely that anyone would dispute that under - feeding eventually leads to death . this parallels the lack of controlled studies on the use of parachutes when jumping out of aeroplanes at high altitude . nevertheless , a short period of starvation appears to be a part of clinical treatment in many icus . this is highlighted by a report from colleagues in the netherlands presented in this issue of critical care . intensive care medicine is a comparatively young speciality , and within our arsenal of treatments the amount of evidence - based knowledge is often embarrassingly low . an illustration of this is seen in the guidelines for the surviving sepsis campaign , in which the evidence for different treatment modalities was evaluated in a formalized way . when we come to icu nutrition , there is little evidence at hand in terms of prospective , randomized , controlled clinical trials ; this was highlighted in recently published canadian guidelines . . some authors even call it ' poisonous nutrition ' and ban it from use in the icu . most investigators and authorities in the field would advocate enteral nutrition before parenteral , merely on the grounds that it is the natural way to feed and is cheaper . the evidence in favour of enteral over parenteral nutrition , or the other way around , is weak ; furthermore , it is obsolete because it was generated more than 10 years ago , with obscure indications for parenteral nutrition and with no blood sugar control . in studies comparing enteral and parenteral nutrition in patients whose attending physician is unsure regarding which modality will be optimal , the results demonstrate a very low feeding success rate with enteral nutrition and that complications are related to the duration of nutritional treatment , regardless of the mode of administration . in general terms it has repeatedly been shown that when patients are given only enteral nutrition , the success of feeding is below 70% . successful feeding may be defined in terms of the percentage of prescribed calories , number of days with at least 80% of prescribed calories delivered , or any other measure . the study from the netherlands presented in this issue adds further evidence to a number of publications that demonstrate that clinical practice is equivalent to delivering as little as 50% of the prescribed kilocalories . does this make any difference ? probably , a very large study population would be required to demonstrate that patients do worse when they are administered only 50% of the prescribed dose of antibiotics . the safety margin for bacterial kill when administrating antibiotics is such that reducing doses to half would require 500010,000 patients to show a difference in a prospective study . still no - one would advocate such a regimen . however , the side effects of antibiotics create renal insufficiency , liver insufficiency and drug fever , and predispose to development of multi - resistant bacteria . nevertheless , no - one would deliberately give only 50% of the prescribed dose , stating that it does not matter whether 50% or 100% of the dose is administered . when it comes to nutrition , however , many of us do this . in the study presented in this issue , the post hoc analysis shows that any action that reflects an interest in nutrition , such as placing the tube in any other position than the stomach , using some type of enhanced formula , or having a percutaneous feeding device , increases success rates with nutrition . these specially treated patient groups were small , and findings in these groups should perhaps not be used as a basis for clinical recommendations , but it is clear that an enhanced interest in nutrition and delivery of nutrition will increase success rates . so , the main reason for the systematic under - feeding that is practiced in icus is probably a lack of interest by the attending physician . binnekade and coworkers do not give any information on success rates of feeding in relation to outcome . this is a difficult area , and studies that try to link nutritional practice to outcome must be designed carefully . there are examples in the literature of investigators jumping to conclusions based on insufficient information . nevertheless , in any patient in whom a huge energy deficit is built up , resulting in malnutrition , increased risk for complications can be predicted . in addition , evaluation of nutritional protocols in terms of success rate , nutrition related complications and outcome must be encouraged . systematic under - feeding of icu patients may be used as a marker of suboptimal care . wherever it is considered a human right not be hungry , the burden of producing proper evidence should be imposed on anyone who suggests that half the feed is good enough .", "", "" ]

[ "recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) . because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele . previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) . the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig . consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) . this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of contaminating foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig . we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig . these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays . no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) . ( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice . although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive . thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity . we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells . because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) . the resulting foxp3 foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3 cd4-cre mice . the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard , it was proposed that the autoimmunity previously reported in foxp3 cd4-cre mice ( 15 ) is due to cd4-cre mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells . to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig . 2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool . we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig . foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig . 2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3 foxn1 was confirmed by genomic pcr ( not depicted ) . when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) . foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes . foxn1 mice remained as healthy as foxp3 foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig . in contrast , foxp3 cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) . thus , foxp3 gene ablation in thymic epithelium does not result in autoimmunity . disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome . ( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma . thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) . right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp cd4-cre ( solid line ) mice . ( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes . ( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3 foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3 cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) . lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice . ( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm ( ; n = 4 ) or foxp3 wild - type nude bm ( ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm ( ; n = 5 ) or foxp3 wild - type nude bm ( ; n = 6 ) . these results were further supported by our experiments using transfers of t cell depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients . all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) . although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm . in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies , we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells . as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) . thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells . in these experiments , foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig . in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig . the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease . lack of autoimmunity in foxp3 nude foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) . major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig . these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice . thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets . ( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice . ( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets . ( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice . in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon , we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice . the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig . 3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates . no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig . 4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3 foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients . the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig . together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation . foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice . ( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice . collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells . this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology . thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .", "recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) . because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele . previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) . the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig . consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) . this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of contaminating foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig . we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig . these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays . no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) . ( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .", "although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive . thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity . we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells . because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) . the resulting foxp3 foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3 cd4-cre mice . the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard , it was proposed that the autoimmunity previously reported in foxp3 cd4-cre mice ( 15 ) is due to cd4-cre mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells . to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig . 2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool . we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig . foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig . 2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3 foxn1 when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) . foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes . foxn1 mice remained as healthy as foxp3 foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig . in contrast , foxp3 cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) . disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome . ( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma . thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) . right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp cd4-cre ( solid line ) mice . ( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes . ( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3 foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3 cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) . lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice . ( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm ( ; n = 4 ) or foxp3 wild - type nude bm ( ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm ( ; n = 5 ) or foxp3 wild - type nude bm ( ; n = 6 ) . these results were further supported by our experiments using transfers of t cell depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients . all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) . although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm . in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies , we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells . as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) . thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells . in these experiments , foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig . in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig . the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .", "lack of autoimmunity in foxp3 nude foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) . major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig . these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice . thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets . ( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice . ( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets . ( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice . in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon , we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice . the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig . 3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates . no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig . 4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3 foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients . the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig . together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation . foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice . ( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice . collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells . this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology . thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .", "foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background . b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains . bm chimeras were constructed using 7 10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 . experimental mice were age and sex matched and housed in specific pathogen - free conditions . disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) . all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington . 510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 . thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) . after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) . thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer .", "foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background . b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains . bm chimeras were constructed using 7 10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 . experimental mice were age and sex matched and housed in specific pathogen - free conditions . disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) . all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .", "510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 . thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) . after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) . thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti ", "s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer ." ]

[ "human parvovirus ( hpv ) b19 , being first discovered and introduced in 1975 , is a non - enveloped single - stranded dna virus from the parvoviridae family . the virus is transmitted by respiratory droplets and the prevalence is estimated to be high since most of the individuals are infected by the age of 15 . the clinical syndrome associated with hpv b19 strongly depends on the host ; for instance those suffering from hemolytic disorders , including sickle cell disease , hereditary spherocytosis ( hs ) , autoimmune hemolysis , thalassemias , and paroxysmal nocturnal hemoglobinuria ( pnh ) are susceptible to aplastic crisis . the virus has a predilection for infecting the erythroid progenitor cells of the bone marrow resulting in their lysis and aplastic anemia . thus , the bone marrow in these patients appears without erythroid precursors but with normal myeloid series . the hpv b19 induced aplastic crisis can unmask several hereditary hematological disorders that have been normally compensated . among these conditions , when hpv b19 infects the bone marrow erythroid cells of these patients , decompensation occurs and thus the patient presents with signs and symptoms of abrupt onset severe anemia . several similar sporadic cases have been reported by now , but familial hpv b19 induced aplastic crisis leading to the diagnosis of hs in all family members is a very rare condition being only reported three times in the literature . we herein , report hpv b19 induced aplastic crisis in an asymptomatic and undiagnosed family of three with hs demonstrating pancytopenia on peripheral blood .", "a 3.5-year - old girl , the youngest child of a 4-member family , was presented with severe pallor and high fever without localizing sign with 5 days duration . on physical examination , she had jaundice in the sclera and the spleen was palpable about 3 cm below the costal margin . the following day , her 14-year - old brother presented with high fever of unknown origin and severe pallor . on physical examination , he was febrile ( 39 c ) and had a mild prominence of frontal and maxillary bones and jaundiced sclera . physical examination revealed an enlarged spleen with the tip of spleen palpated about 3 cm below the costal margins . the hematological indices of the three patients admitted to our department with severe pallor and fever of unknown origin according to the family history and positive findings on physical examination ( jaundice and splenomegaly ) , a work - up for hemolytic anemias , including hb electrophoresis , osmotic fragility , and autohemolysis test was performed for each patient . the results were consistent with the diagnosis of hs . knowing hpv b19 as the most common causative agent in the development of aplastic crisis in hemolytic anemias ; specifically hereditary spherocytosis polymerase chain reaction ( pcr ) for hpv b19 , dna was performed which was positive in all three patients ( figures 1 and 2 ) . bone marrow aspiration of the girl revealed normal marrow cellularity with mild erythroid hyperplasia and clusters of erythroid nests heralding the recovery of the erythroid series already affected by parvovirus b 19 infection . in contrary , cellularity was severely decreased in all three lineages in the boy implicating suppression of all hematopoietic lineages . both siblings received intravenous immunoglobulin ( ivig ) in a dosage of 1 gr / kg along with blood transfusion , twice for each . the girl recovered after 3 days with reticulocytosis of 16% , while the boy recovered 8 days later with reticulocytosis of 8% . their mother also had to receive blood transfusion because of having hb of 3.6 gr / dl . pcr for parvovirus b19 . from left to right ; the siblings , their positive and negative control , and the ladder .", "these family members were typical examples of the occurrence of aplastic crisis due to hpv b 19 complicating hs simultaneously in a family . only three previous reports have shown that hpv b19 can induce aplastic crisis and unmask the hs in a family . previously , green et al . in 1984 reported an adult sibling pair with hs who developed aplastic crisis after a febrile illness , which was further diagnosed to be hpv b19 infection . the diagnosis of hpv b19 was developed based on specific igm antibody in their sera , as pcr was not available . they also found that the children of one of the patients also developed hpv b19 induced aplastic crisis , which was resolved with supportive care . these two adult patients were treated by blood transfusion and supportive care and were discharged after 6 - 8 days of hospital care . in a similar report , mclellan and rutter in 1987 reported hpv b19 induced aplastic crisis in two teenage sisters leading to the diagnosis of hs . they also had a history of splenectomy in their mother at the age of 11 years . they both were diagnosed to have hpv b19 and underwent splenectomy and supportive care until recovery . in 1962 , chanarin et al . reported aplastic crisis in 3 members of a family ( 2 sisters and the father ) . the 10-year - old girl presented with 7 days history of fever of unknown origin , jaundice , and dark urine . the same illness was reported in 2-year - old sister and 32-year - old father who both had hs . the 10-year - old girl was treated with repeated transfusions , splenectomy , and supportive care for 92 days . based on laboratory investigations , the authors first assumed that the megaloblastic hematopoiesis accompanied by urinary excretion of large amounts of urocanic acid following oral dose of histidine hydrochloride was responsible for aplastic crisis in these patients with hs . however , the authors reached the conclusion that hpv b19 was responsible for the aplastic crisis in this family with hs . as hpv b19 was first discovered in 1975 and this family was reported in 1962 , thus the etiology was misdiagnosed . in the present study , we described a family with hs that all three members developed aplastic crisis secondary to hpv b19 infection leading to the detection of asymptomatic hs . those patients with hs , who remain undiagnosed , usually have mild hemolysis and the disease remains undiagnosed until the compensation is interrupted because of some environmental stressors such as infections . the distinguished feature of our report is the occurrence of some degrees of leukopenia , neutropenia and thrombocytopenia in two of our patients ( the mother and his son ) . there are few reports of mild transient pancytopenia due to hpv b19 infection and recent report on persistent severe aplastic anemia in a previously healthy adult . however , none of them occurred in members of a single family and one of them was persistent in a healthy subject . one of the theories behind the etiology of transient pancytopenia in hpv b19 infections is that the virus could be responsible for the temporary arrest of hematopoiesis that leads to aplastic crisis in persons with chronic hemolytic anemia . the other hypothesis is the occurrence of hpv - associated hemophagocytosis leading to pancytopenia . among our three patients , two showed transient pancytopenia with more severity in the 14-year - old boy ; nevertheless , they did nt show any evidence of hemophagocytosis in bone marrow aspirate . according to available reports in the literature indicating probable efficacy of ivig in the treatment of patients with hpv b19 infections and its effect on replication on virus however , we can not draw any conclusion on whether the fast recovery of the patients was a positive impact of ivig or spontaneous recovery of hpv b 19 that is expected in hs patients , albeit happened very soon in our patients .", "hpv b19 induced aplastic crisis in a family leading to the diagnosis of hereditary hs is a very rare reported event in the literature . the distinguished feature of this report is that all affected members of a family developed some degrees of transient pancytopenia , not only anemia , all simultaneously in the course of their disease . however , it was transient and the recovery happened very soon in all of them within 10 days , which was attributed to the usage of ivig ." ]

[ "primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et al . , 2012 ) . no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et al . , 2011 ) . importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain . additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et al . , 2000 ; sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus . sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et al . , 2012 ) . the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein . known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et al . , 2013 ) . sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor . for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et al . recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice . this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et al . , 2011 ) . here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et al . , 2010 ) . sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et al . , 2009 ) . mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et al . , 1999 ) . most of the patients carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele . the second knockout allele in this animal model is functionally null ( yang et al . , 2010 ) . while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by exercise intolerance and associated with ubiquitous cox deficiency .", "to test the effects of persistent increase of the nad pool on the sco2 mouse , we first crossed it with a constitutive parp1 mouse ( de murcia et al . , 1997 ) , which shows increased levels of nad in skeletal muscle ( bai et al . , 2011 ) . the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure s1 available online ) . the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4 weeks starting at 2 months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure 1a ) . biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants , these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure 1b ; table s1 ) . accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure 1c ) . the activities of complex iii and iv ( figure 1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure 1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n = 4/group ) , as a food admix ( 400 mg / kg ) ( cant et al . , 2012 ) for four weeks . metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in vivo . nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure 2a ) , which showed no difference to each other . these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et al . , 2012 ) . accordingly , the nad / nadh ratio was significantly increased ( figure 2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure 2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals . we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure 2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure 2d ) . tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment . as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 . accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure 2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure 2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals . the histochemical staining for cox reflected the biochemical results ( figure 2 g ) . in both c. elegans and mammalian cells , nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et al . we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure 2h ) . no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) . next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp < 1 nm ; pirinen et al . , 2014 , in this issue of cell metabolism ) at 50 mg / kg as a food admix for 4 weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure s3 ) . weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure 3a ) . the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure 3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure 3c ) , indicating activation of sirt1 by mrlb-45696 . similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure 3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure 3f ; table s3 ) . histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure 3 g ) . again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) . in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure 4a ) , and respiratory chain activities in the brain ( figure 4b ; table s4 ) . the intensity of cox staining was increased as well ( figure 4c ) . similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) .", "the nad pool is set by the balance between de novo and salvage biosynthetic pathways and utilization by nad - consuming enzymes . nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et al . , 2010 ) . these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) . nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle . for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt . parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) . ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis . these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et al . , we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals . notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs . increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6 months ) ( pirinen et al . , 2014 ) . in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4 weeks ) compared to that of other studies ( 12 weeks ) . this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much earlier than stimulation of mitochondrial proliferation and increase in mtdna content . likewise , prolonged nr supplementation up to 6 months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et al . , 2013 ) . while we observed hardly any effect of nr in our 4 week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice . this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood . our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease . nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy . our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et al . , 2013 ) or complex i deficiency ( karamanlidis et al . , 2013 ) initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et al . , 2013 ) . several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et al . , 2013 ; tutt et al . , however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects .", "mouse tissues were homogenized in 15 volumes of 10 mm potassium phosphate buffer ( ph 7.5 ) . mitochondrial - enriched fractions were collected after centrifugation at 800 g for 10 min in the presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen . aliquots , 70 g each , were run through a 12% sds - page and electroblotted onto a nitrocellulose membrane , which was then matched with different antibodies . carlo besta neurological institute , in accordance with guidelines of the italian ministry of health . mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12 hr light / dark cycle and free access to water and food . a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et al . series of 8 m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) . muscle quadriceps samples stored in liquid nitrogen were homogenized in 10 mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et al . , 2004 ) . note that in all panels the activity of cii has been multiplied by 10 for visualization clarity . nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) . tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) . mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et al . , 2011 ) ." ]

[ "irinotecan is commonly used in combination with oxaliplatin as a component of folfirinox chemotherapy for several gastrointestinal malignancies . the purpose of this case report is to describe a patient who developed acute paralysis and aphasia while receiving her initial infusion of irinotecan .", "a 67-year - old woman with newly diagnosed metastatic pancreatic adenocarcinoma presented for her first cycle of folfirinox chemotherapy . during her infusion of irinotecan , she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate . this episode was self - limited and resolved within 2 h. prior to subsequent infusions she received intravenous repletion of potassium and had no recurrence of symptoms .", "in selected cases , coadministration of irinotecan and oxaliplatin may result in severe generalized weakness and aphasia , which may be triggered by underlying electrolyte disturbances .", "irinotecan is a topoisomerase i inhibitor derived from camptothecin , an alkaloid compound extracted from deciduous trees indigenous to eastern asia . its antineoplastic activity is mediated by its inhibition of double - stranded dna replication through stabilizing the cleavage complexes of topoisomerase i. its most common use is in the treatment of colorectal cancer . however , since 2010 , it has been combined with 5-fluorouracil , leucovorin , and oxaliplatin in the regimen known as folfirinox , which has been utilized as an effective therapy in patients with metastatic pancreatic cancer as well as other cancers . the most commonly cited adverse effects of irinotecan include late - onset diarrhea and bone marrow suppression , with clinically significant neutropenia and thrombocytopenia . a less frequent , acute cholinergic syndrome with resultant symptoms of diaphoresis , hypotension , anxiety , and abdominal cramping with diarrhea may result in severe discomfort and dehydration that can be life - threatening . atropine is commonly administered with the chemotherapy infusion both for the prevention and treatment of this syndrome . oxaliplatin is a third - generation platinum derivative that has shown to be an effective therapy in several malignancies , most commonly gastrointestinal cancers . side effects of oxaliplatin include a dose - limiting severe peripheral sensory neuropathy that is chronic in onset . less commonly , acute sensory disturbances that may be modulated by cold temperatures may occur . these effects are thought to be mediated by an interaction with voltage - gated sodium channels in peripheral nerves . here we report the case of a patient with a rare complication of combination therapy with irinotecan and oxaliplatin , i.e. severe generalized weakness , paralysis , and aphasia , and provide a synopsis of the current literature as well as a proposed therapeutic approach .", "a 67-year old asian woman with a history of poorly controlled diabetes presented with newly diagnosed metastatic pancreatic adenocarcinoma . in november 2013 , she received her first cycle of palliative chemotherapy using folfirinox ( oxaliplatin 85 mg / m , irinotecan180 mg / m , leucovorin 400 mg / m iv , and 5-fu 2,400 mg / m iv by continuous infusion over 48 h without a bolus , with dexamethasone 10 mg and ondansetron 12 mg iv as premedication ) . prior to beginning therapy with folfirinox , her electrolyte levels were checked , which revealed mild hypokalemia ( 3.5 meq / l , normal values 3.55.0 ) with normal serum sodium and calcium ( na 137 mmol / l , ca 8.7 mg / dl ) . halfway through the irinotecan infusion , she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate . throughout this , she was awake , alert , and aware of her surroundings , with stable vital signs . at this time , the infusion was stopped and she was given a second dose of atropine 1 mg as well as an iv bolus of 1 l normal saline . she was monitored carefully and observed to return close to her baseline status within 12 h. neither the infusion of irinotecan nor the 5-fu infusion was restarted , and she was discharged to her home with appropriate follow - up . she returned 2 weeks later for cycle 2 of her folfirinox therapy and felt completely well . prior to initiating the infusion , her electrolyte levels were checked , which showed a low - normal potassium level of 3.7 she received 20 meq iv kcl supplementation leading to an improvement in the potassium level to 4.4 meq / l immediately preceding the infusion . she was then given her second cycle of folfirinox with identical doses of the drugs as in the first cycle and was able to complete all of the therapy without recurrence of the symptoms previously experienced at her initial infusion . she was able to continue on therapy without event for a total of 3 cycles before she transferred her care to an institution closer to her home and was lost to follow - up .", "to date , there have been 9 reported cases of significant central nervous system toxicity during or following the administration of irinotecan , both with and without concurrent oxaliplatin administration . all of these cases involved the development of dysarthria , with 2 of them leading to a complete motor aphasia and 1 case with associated ataxia [ 1 , 2 , 3 , 4 , 5 ] . in each case , these symptoms developed with the initial infusion of irinotecan and completely resolved with time . the duration of symptoms ranged from as little as 15 min to as long as 8 h. the duration of symptoms appeared to be related to the dose of irinotecan , with doses < 200 mg / m having a quicker return to baseline ( 1545 min ) in comparison to larger doses of > 200 mg / m ( 28 h ) . irinotecan and its primary active metabolite , sn-38 , bind strongly to plasma proteins and tissues resulting in high plasma distribution . in animal models , irinotecan and its metabolites have been found to cross the blood - brain barrier into the central nervous system . in 2 patients in whom neurologic symptoms developed during irinotecan administration , hamberg et al . examined the pharmacokinetics of irinotecan and sn-38 and found both of these values to be within the normal range . the acute onset of symptoms shortly after beginning irinotecan infusion also suggests that the manifestation of these symptoms is not dose or duration dependent . therefore , it is unlikely that altered systemic clearance of irinotecan mediates the presence or absence of neurologic symptoms in these patients . the degree of severe generalized weakness seen in our patient following administration of irinotecan has not previously been reported in the literature . though no imaging of the brain was performed in our patient , ct and mri performed in prior similar cases have failed to show any evidence of stroke or other acute cns abnormalities to explain the clinical presentation . our patient was found to have mild hypokalemia ( 3.5 meq / l ) prior to chemotherapy ( which may have been even lower following hydration and drug administration ) , which may have contributed to her profound weakness and inability to move and speak while maintaining a normal sensorium . one case of acquired fanconi syndrome ( characterized by proximal tubular dysfunction resulting in electrolyte wasting of potassium , calcium , phosphate , and uric acid ) has been reported following combination therapy with capecitabine , irinotecan , and bevacizumab , but a single offending agent was not identified . correcting our patient 's electrolytes prior to the subsequent infusions and ensuring continued stability of these values throughout the infusion was the only change in the treatment plan and resulted in no recurrence of symptoms . we postulate that close monitoring and repletion of potassium was the factor that prevented further neurologic symptoms . as irinotecan alone has not previously been implicated in electrolyte - based neurologic complications , we also consider that the findings in our patient were not caused by irinotecan alone , but rather by combination therapy with oxaliplatin as administered in folfirinox . two prior case reports have noted the acute onset of severe neurologic deficits ( including generalized weakness , limb weakness , dysarthria , ophthalmoparesis , and coma ) during and shortly after the administration of oxaliplatin , which our patient received prior to initiation of irinotecan [ 8 , 9 ] . in both reported cases , mmol / l ) and much more severe in the case reported by basso et al . ( k 1.7 meq / l ) , which appears to correlate with the severity of symptoms . in these cases , symptoms developed during the oxaliplatin infusion or within 15 min of completion . our patient tolerated her oxaliplatin therapy without complications during the infusion , but it is possible that the coadministration of oxaliplatin and irinotecan may have increased the risk of developing these reversible neurologic effects . if this is the case , those patients receiving combination folfirinox therapy may be at significantly higher risk of developing neurologic side effects than those receiving folfox therapy alone . from our experience in this patient , and from our review of the literature , we conclude that in selected cases , the coadministration of irinotecan and oxaliplatin ( as is used in folfirinox ) may result in severe generalized weakness and aphasia . we believe that a careful correction of these abnormalities into the upper ranges of normal as needed , both prior to beginning and during the infusion of oxaliplatin / irinotecan , may prevent this reaction . while the symptoms may be transient and self - limited , they may equally be severe . without knowledge of their etiology and the appropriate therapy , patients may be denied further treatment or may be reexposed to a serious adverse reaction ." ]

[ "alcohol dependence is the most prevalent substance use disorder that can lead individuals with the disorder to serious health - related and social problems . according to a report by nutt and colleagues ( 2007 ) , the level of potential harm and risk of alcohol use and misuse was among the top five with heroin and cocaine . the 12-month prevalence of alcohol dependence is reported to be approximately 2% to 6% in the general populations . in part reflecting the high potential harm and high prevalence of alcohol dependence , there have been a number of studies on how chronic alcohol use or alcohol dependence interacts with the structure of the brain of animals and humans . overall brain atrophy , including the lesser volumes of gray matter and white matter with increased cerebrospinal fluid , in patients with alcohol use disorders , has consistently been reported . deficits in the prefrontal cortex , temporal cortex , cerebellum , striatum , hippocampus and amygdala have also been reported in patients with alcohol dependence . neuropathological studies have shown that the alcohol - related neuronal and glial loss would preferentially involve the prefrontal cortex among cortical regions . functional neuroimaging studies demonstrated altered metabolism or activation of the frontal cortex in association with the deteriorated neuropsychological functioning . cortical thickness analysis , a reliable and valid method , can capture important information on cortical structures . durazzo and colleagues exhibited that alcohol - dependent adults had thinner cortical regions in the left anterior cingulate cortex , bilateral frontal cortex , and bilateral insula than healthy controls . other researchers found that alcohol - dependent adults had reduced cortical thickness in the widespread brain regions of the superior frontal , precentral , postcentral , middle frontal , middle and superior temporal , middle temporal , and the lateral occipital cortex than healthy comparison participants . in the report by momenan and colleagues , participants with alcohol dependence exhibited thinner cortical regions that encompass the medial superior frontal cortex , insula , precentral and the precuneus of the right hemisphere as well as the superior frontal gyrus of the left hemisphere , in comparison with the healthy controls . previous studies have reported widespread cortical deficits without covarying out the effects associated with the global atrophy of the brain . among these widespread regions that show alcohol - related atrophy , we wanted to localize the cortical regions that may be particularly vulnerable to alcohol consumption . we therefore undertook the cortical thickness analysis adjusting for the hemispheric average cortical thickness , in 21 detoxified alcohol - dependent patients and matched 22 healthy comparison subjects . the objective of this study was to identify brain regions with cortical thickness alterations that exceed the level of global alterations in alcohol dependence . we used the whole brain - wise cortical thickness analysis , which is validated histologically and with manual outlining method . we also investigated whether the magnitude of deficits are correlated with the alcohol use - related variable . given the studies that suggest the preferential involvement of prefrontal cortex among brain structural and functional alterations in alcohol dependence , we hypothesized that patients with alcohol dependence would have thinner prefrontal cortex in comparison with healthy comparison subjects , after correcting for the global cortical thinning associated with alcohol dependence .", "patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea . age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period . inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists . exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants . additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders . t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner . repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 . t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) . t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) . smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel . for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used . non - cortical areas of medial wall and corpus callosum were excluded from the model building . hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence . 5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 . clusters with the size that would pass the family - wise error rate correction were considered significant . thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses . pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed . local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t ) as additional covariates . computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) .", "patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea . age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period . inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists . exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants . additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders .", "t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner . repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 . t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) . t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) . smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel .", "for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used . non - cortical areas of medial wall and corpus callosum were excluded from the model building . hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence . 5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 . clusters with the size that would pass the family - wise error rate correction were considered significant . thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses . pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed . local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t ) as additional covariates . computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) .", "there were no significant differences in age and sex between diagnostic groups ( table 1 ) . patients with alcohol dependence drank alcohol more frequently and more heavily than healthy comparison subjects ( table 1 ) . patients with alcohol dependence had general cortical thinning ( left hemispheric average cortical thickness ( mm ) : 2.440.07 [ healthy comparison subjects ] vs 2.240.27 [ patients with alcohol dependence ] , t=3.35 , p=0.002 ) ( right hemispheric average cortical thickness ( mm ) : 2.450.07 [ healthy comparison subjects ] vs 2.250.23 [ patients with alcohol dependence ] , t=3.77 , p<0.001 ) . in order to identify regionally specific cortical deficits in patients with alcohol dependence compared to healthy comparison subjects , hemispheric average cortical thickness was added as a covariate in the whole brain vertex - wise analysis . in the glm model that includes age and average cortical thickness as covariates , significant cortical thickness deficits in patients with alcohol dependence , compared to healthy comparison subjects , were noted in the left superior frontal cortex , after correcting for multiple comparisons with the permutation method ( fig . 1 ) ( cluster size=1489.5 mm ; number of vertices in the cluster=2,035 ; talairach coordinates = x [ -15.3 ] , y [ 61.8 ] , z [ 5.0 ] ; cluster p value=0.024 ) . given that different scanning parameter may influence on the cortical thickness variations , analysis was re - run with scan parameter as a covariate . when analysis was repeated covarying for comorbid depression that may be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant ( p=0.001 ) . when analysis was repeated covarying for comorbid anxiety that may also be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant in the left superior frontal cluster ( p=0.017 ) . post hoc correlation analysis between cortical thicknesses in the left superior frontal cluster of significant group difference and duration of alcohol use in alcohol dependence group demonstrated the significant association ( r=-0.55 ; p=0.02 ) ( fig . cortical thicknesses in the left superior frontal cluster was also correlated with the clinical institute withdrawal assessment for alcohol scores ( r=-0.548 , p=0.028 ) . otherwise we found no significant results between cortical thicknesses in the left superior frontal cluster and audit scores ( r=0.048 , p=0.859 ) ; and calculated alcohol use ( alcohol dose x frequency x duration ) ( r=-0.193 , p=0.509 ) .", "in the current study , we have identified the brain region with altered cortical thickness in patients with alcohol dependence . the region of cortical thickness deficits in patients with alcohol dependence encompassed primarily the superior frontal cortex , after adjusting for the effects on the global cortical atrophy induced by alcohol dependence ( fig . this is consistent with prior reports indicating that chronic alcohol use may have the most detrimental effects on prefrontal cortical regions . the level of n - acetyl aspartate , the viability marker of neurons , was decreased in prefrontal brain regions in chronic alcohol abusers . neuronal and glial loss has consistently been noted in the prefrontal cortical regions , particularly in the superior frontal cortex . alcohol use has been associated with the decreased performances in executive and attention tasks that are important function of the prefrontal cortex . pre - existing vulnerability in these regions may predispose individuals to alcohol dependence , since the function of these regions are to executively control over drug craving and seeking . these regions may be associated with the compulsive substance - related behaviors , which is also in line with our finding that shows the association between duration of alcohol use with the magnitude of the cortical deficits in this region . the current findings do not provide information regarding whether the pre - existing prefrontal cortical deficits have rendered individuals vulnerable to alcohol dependence , whether neurotoxicity of chronic alcohol use , including the oxidative stress , have damaged the cortex , or whether both processes have contributed to the observed findings . supporting evidence for the neurotoxicity as the cause of prefrontal deficits may come from the reports that show patients with longer abstinence have lesser deficits . it has also been suggested that subjects with alcohol dependence may have prefrontal cortex that are less recuperative from toxic effects and may undergo vicious cycle after initial exposure to substances . considering that alcohol dependence can be divided into two subtypes , type 1 more environmentally influenced , and type 2 more genetically induced , a comparison between type 1 and type 2 alcohol dependent patients in a study with larger sample with balanced composition of type 1 and type 2 alcohol dependence may provide an opportunity to approach this question . longitudinal brain imaging study that follows up patients with alcohol dependence would also provide important insights . this study alone does not provide direct information as to cellular level mechanisms that may underlie the observed deficits of the cortical thickness . however , there is a vast literature describing the impact of chronic alcohol on the brain . miguel - hidalgo and colleagues ( 2002 ) reported , in their study with the postmortem brain of alcohol - dependent patients without wernicke or korsakoff syndromes , that the glial pathology of reduced size and density was the most characteristic finding . kril and colleagues ( 1997 ) have shown selective loss of non - gabaergic pyramidal neurons . selective dendritic retraction , rather than cellular death , has been suggested as main pathology related to the volume loss of the gray matter . there is a study that examined the cortical thickness differences in subjects with fetal alcohol syndrome or prenatal alcohol exposure , compared to control subjects , which demonstrated deficits in prefronto - temporo - parietal regions . however , few studies have examined cortical thickness in alcohol - dependent adults . the cortical thickness analysis has been reported to provide information on an important aspect of the gray matter structure , complementary to the conventional volumetry or the vbm . for example , highly folded regions could have high gray matter density in vbm analysis since there would be more voxels of gray matter within a fixed radius , but thinner cortical thickness . this is the first study that used cortical thickness analysis in patients with alcohol dependence excluding comorbid substance use , which showed regionally specific cortical thickness deficits in alcohol dependence . large age range , though covaried in the statistical model , can be another weakness of the study . with the current study of a cross - sectional design , information on whether the observed cortical deficits would be progressive and nicotine dependence , which is highly comorbid with alcohol dependence , has also been reported to be associated with cortical atrophy , particularly that in frontal cortex . now that we neither were able to exclude participants with co - morbid nicotine dependence nor delineate the effects of alcohol dependence from those of nicotine dependence , the current results may have been confounded by the effects of chronic nicotine use . intellectual ability has also been reported to be associated with the prefrontal cortical development . the fact that the intelligence quotient was not included in the statistical model for comparisons of cortical thickness measures , is also an important limitation of the current study . future studies in larger samples with narrow age range and with neuropsychological assessments for the frontal lobe function , would provide valuable information . although there could be challenges in following up patients with alcohol dependence , long - term prospective longitudinal study is also warranted ." ]

[ "nutrient intakes , health behaviors and quality of life are interrelated and are of particular importance among rural residents . rural areas exhibit a variety of dietary habits and food - intake patterns ( lee et al . , 2002 ; ministry of health & welfare , 2002 ) with food intake and food selection being lower , consequently nutrient intakes being lower than urban areas . dietary diversity is a fundamental concept of dietary guidance internationally ( katanoda et al . , 2006 ; kennedy , 2004 ; who , 1996 ) , since increasing the diversity of foods is thought to ensure an adequate intake of essential nutrients and to promote good health ( ruel , 2003 ) . a low dietary diversity is a particularly severe problem among vulnerable populations of the poor ( ruel , 2003 ) and elderly ( bernstein et al . , 2002 ; adequate nutrient intake is essential to health promotion . hence increasing nutrient intakes as well as the dietary diversity of rural koreans is a potential way to improve their nutritional , and consequently , their health status and quality of life . ( 1987 ) reported that variety among and within major food groups was associated with diet quality assessed by the mean adequacy ratio ( mar ) of 11 nutrients . evaluating the dietary diversity score ( dds ) and mar are , therefore , a good way to predict quality of diet and the entirety of dietary habits . in this study , we examined the dietary intakes and health behaviors of asan residents in order to improve health status and consequently the quality of life of asan residents , especially those who live in a rural area . asan is a medium - sized city with the unique feature with three distinctive regions since there are some areas with various kinds of industrial factories such as semi - conductor factories or automobile , air - conditioner and refrigerator manufacturing factories . additionally , other areas are mostly composed of rice farms or other kinds of rural farms in the boundaries of asan . some areas with a largely populated urban area ( on - yang si ) are also within the district of asan . therefore , asan can be easily differentiated into three distinctive regions of rural , factory and urban areas . the purpose of this study was to examine and compare nutrient intake , dds , mar and health behaviors of asan residents by the three distinctive regions .", "volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent . a total of 930 subjects ( 351 men and 579 women ) , aged 61.4 13.8 ( mean sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants . information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires . dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 . detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids . food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) . all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake . the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) . dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed . information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) . the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) . nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation . differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova ) was conducted to detect true differences among regions after controlling age and income levels as covariates .", "volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent . a total of 930 subjects ( 351 men and 579 women ) , aged 61.4 13.8 ( mean sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants .", "information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires . dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 . detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids .", "food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) . all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake . the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) . dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed . information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) . the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) . nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation .", "differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova ) was conducted to detect true differences among regions after controlling age and income levels as covariates .", "the fundamental characteristics of the participants and comparisons by regions are presented in table 1 . for the total participants , mean age was 61.4 13.8 years , with the men being older ( 64.8 11.7 years ; p<0.001 ) than the women ( 59.3 14.6 ) . for the men , factory area residents were significantly older than those living in other areas whilst for the women , urban residents were significantly younger than those living in other areas . the education levels were significantly different by regions ( p<0.001 ) , with more college graduates in the urban area . forty - five percent of the participants earned less than 500,000 won per month , which is less than half the korean average wage . current drinkers were significantly more in the urban area while never - drinkers were more in the rural or factory areas ( p<0.001 ) . however , proportions of current , past and never smokers were not significantly different by regions . physical activity levels were significantly higher in rural or factory areas than in the urban area ( p<0.001 ) , whereas urban residents exercised more frequently than both rural and factory area residents ( p<0.001 ) . there were more people who consider themselves less healthy than others with similar age in the rural and factory areas ( p<0.001 ) . on the other hand , more urban residents felt more stress than the rural or factory area residents ( p<0.001 ) . energy intakes of the participants were 1679 523 kcal for men and 1467 436 kcal for women ( table 3 ) . energy intake of the total participants was higher in urban area than those in the rural or factory area , but after ses variables such as age and income levels were controlled as covariates in ancova model , regional differences were no longer significant . meanwhile , energy intake of women in the rural area was significantly lower than that in the urban area residents and the results were not different after ses variables were controlled . the energy - adjusted intakes of protein , riboflavin and calcium were lower in the rural area than in factory or urban areas for the total participants and after adjustment of ses variables , the results showed same significant differences ( p<0.05 ) . energy - adjusted intakes of fat , vitamin c , and thiamin were significantly higher in the urban area than in the other two areas ( p<0.05 ) by anova , but ancova models did not show any significant differences . however , for vitamin e and cholesterol , both anova and ancova models showed same results of significantly higher intake in the urban area than in the other two areas ( p<0.05 ) . vitamin a intakes were significantly different among all three regions , being lowest in the rural area , significantly higher in the factory area and highest in the urban area by both anova and ancova ( p<0.05 ) . zinc intake of the factory area residents was significantly higher than that of the urban area by anova ( p<0.05 ) , but by ancova , the difference was no longer significant . carbohydrate , niacin , folic acid , vitamin b6 , iron , sodium and fiber intakes did not show any regional differences by both anova and ancova models . the result of sodium intake after adjustment of ses variables , however , showed highest intake in the factory area . just for the male participants , differences of fat , vitamin c , vitamin e , thiamin , riboflavin , iron and cholesterol intakes were not significant among regions while protein , vitamin a and calcium intakes were higher in the urban area and lower in the rural area . after ses variables were controlled in ancova model for male participants , protein , vitamin a and calcium intakes remained lowest in the rural area . for the women , regional differences of energy - adjusted nutrient intakes were similar to those of the total participants except for protein and niacin by both anova and ancova . energy - adjusted protein and niacin intakes of the women were higher in the factory area ( p<0.05 ) . sodium intake also showed significantly higher intake in the factory area after adjustment with ses as the case of the total participants . 1 presents dds of the participants by regions and by sex before and after adjustment with age , income and energy intake levels . dds were higher in the urban area for total participants and women ( p<0.05 ) , but for men , dds was not different by regions before ses variables and energy were adjusted . only male showed different dds ( higher in the factory area ) while total and female participants did not show significant differences . 2 shows mar of the participants by regions and by sex before and after adjustment with age , income and energy intake levels . however , mars of rural participants were lowest for total , men and women after adjustment with age , income and energy intakes ( p<0.05 ) .", "previous studies showed that selected health outcomes are dependent on adequate nutrient intakes , which rely highly on dietary quality ( foote et al . , 2004 ; haines et al . , 1999 ; kim et al . , 2003 ) , dietary diversity ( guthrie & scheer , 1981 ; hatloy et al . , 1998 ; kant et al . , 1991 , 1993 ; krebs - smith et al . , 1987 ) or both parameters ( bernstein et al . , 2002 ; drewnowski et al diet quality varies widely depending on the target population , since dietary habits are complex in nature and influenced by many factors . the korean rural population exhibits a distinct pattern of food choices ( ministry of health and welfare , 2002 ) and korea shows a unique pattern in dietary intake in terms of a lower proportion of energy from fat compared to both developed and developing countries ( lee et al . , 2002 ) . in this study , we described the nutrient intakes , dietary diversity ( dds ) , dietary quality ( mar ) and health related behaviors of asan residents . this population is unique since asan has regionally unique features so that the results of the current study may not be applicable to others residing in urban communities or geographical locations . our results showed that social economic status ( ses ) of asan residents was significantly different by different regions . as korea becomes industrialized , urbanization is more prompt and the tendency of urbanization is not different in asan . the younger urban resident showed higher education and income levels . about 50% of the participants of the present study were older than 65 years , and their education and income levels were lower than the korean averages . sixty - four percent of the total participants were educated up to the elementary school level only , with this increasing to 88% among those who are older than 65 years ( data not shown ) . therefore , the general ses of asan residents is lower than the korean average , with urban residents being the youngest and highest in education and income levels compared to rural or factory areas . rural and factory area residents did not show much difference in education and income levels although there were more elderly residents in the factory area . the energy intake and quantitative dietary diversity for the one - day diets was not high in this population even though all foods from mixed dishes were included after decomposition . only urban women showed the similar energy intake level compared to older women ( aged from 50 to 64 y ) living in big cities ( chung et al . , 2005 ) . however , diet quality ( mar ) of urban residents went up to 1.00 while other area residents showed mar being 0.86 for rural and 0.95 for factory residents . the difference of mar became significant for all participants regardless of sex after the ses factors were controlled . therefore , the true regional differences in quality of diet exist in the population of asan , and public health strategies to improve diet quality of rural residents should be considered . the proportion of carbohydrate contributing to energy intake was very high ( 69% , data not shown ) and that of fat was very low ( 15% vs. 19% korean national average ; ministry of health and welfare , 2002 ) . this high proportion of energy intake from carbohydrate may have limited the selection of foods from other groups in our population . the 1998 korean national health and nutrition survey found that the 30 most - consumed foods were similar in all areas except for rural areas , where three different kinds of kimchi were included in the top-10 most - consumed foods ( ministry of health and welfare , 2002 ) . many participants in the present study , especially older women from the rural area , reported that they consumed rice and kimchi almost exclusively , with few animal products and fresh fruits ( data not shown ) . the limited food choices resulting in a low dietary diversity may be related to the insufficient intake of essential nutrients in the korean rural population including the rural and factory area participants of this study . especially , after the ses factors were controlled , significantly low dds of rural men reflect the poor quality of diet among this population . the current major concerns in korea as in many other countries are over nutrition and excess intakes of certain nutrients and foods rather than under nutrition ( ministry of health and welfare , 2002 ) . however , our results demonstrated that insufficient intakes of essential nutrients such as protein , vitamin a and e , riboflavin and calcium are still a problem in the rural area of asan even after ses was controlled . therefore , korean nutrition concerns should focus on dietary quality including both the concepts of nutrient deficiency and over nutrition . especially , significantly low dietary diversity of rural men and low quality measured with mar of rural men and women are of particular concern other health behaviors such as drinking , physical activity and perceived stress levels were much better among rural or factory residents compared to the urban residents , although exercise habits and perceived health were better in urban residents . ( 2005 ) with korean national data also reported that self - rated usual activity showed higher in the rural area while higher exercise rate in the urban areas . therefore , the urban area residents of asan showed better nutrient intakes , dietary quality and exercise habits , which may result in the better perceived health compared to the rural or factory area residents of asan . the rural or factory area residents of asan showed much less perceived stress , however , even though nutrient intakes and dietary quality were not as good as urban residents . urban residents reported higher perceived stress even if they exercised more , ate better diets and showed better perceived health . in conclusion , ses and quality of life measured by diet intakes and perceived health were lower in the rural area residents of asan . therefore , in order to achieve better quality of life and health status of the rural residents of asan , improving diet quality is essential . for the urban resident , however , despite of higher ses and diet quality , their perceived stress was higher . therefore , health promotion education to reduce the stress levels for urban residents of asan should be recommended ." ]

[ "the treatment of chronic hepatitis c ( chc ) has evolved in the last 15 years from monotherapy with interferon alpha ( ifnalpha ) to the combination treatment with pegylated ifn ( peg - ifn ) plus ribavirin for 24 - 48 weeks . up to now , several viral , host and drug - related reactions in response to ifnalpha - based therapy have been identified . recent studies suggest that liver inflammation in chc is controlled by several mechanisms , including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity . it is well known that cutaneous side effects of treatment with ifnalpha alone or ifnalpha plus ribavirin in patients with chc have been widely reported , beyond the fact that the virus itself can cause skin lesions . however , the cutaneous side effects during therapy of chc are of inflammatory type with local erythema , edema and , much less frequently , necrosis at the injection skin sites . by contrast , skin side effects of such drugs have few data available in the literature , although the number of reports has been increasing in the last years , including cosmetic filler site injections . roughly one half of these patients had cutaneous findings , either alone or with systemic involvement . more recently there have been reported dermatological diseases in patients receiving the combination of ifn / ribavirin or ifn / ribavirin / amantadine for the treatment of chc .", "a 44-year - old man with chc genotype 1a and a viral load of hcv > 800,000 iu / ml , a2-f1 on liver biopsy according to metavir criteria , was treated for the hepatic disease . peg - ifnalpha-2b 1.5 g / kg weekly and 1,000 mg of ribavirin daily was introduced . in the 40th week the patient reported sudden intense pain , pruritus , erythema and skin hypertrophy in the left deltoid area over the seahorse tattoo and in a scar on his face ( fig . 1 ) . a skin biopsy on the scar and tattoo showed granulomatous dermatitis ( fig . chest x - ray was normal and the level of angiotensin enzyme converter was 50 iu / ml ( range 18 - 55 iu / ml ) . we intend to call attention especially to dermatologists to the possibility of a granulomatous tattoo reaction as a side effect during chc treatment with peg - ifnalpha-2b plus ribavirin . until now , eight cases of sarcoidosis as a complication of ifn therapy in chc have been reported in the english language literature . the most relevant topic in this case is the occurrence of disease on a tattoo area . six months later retreatment using peg - ifnalpha-2a 180 g once a week plus ribavirin 1,000 mg daily was tried . in the 6th week , cutaneous symptoms relapsed in an unbearable way . the patient did not accept to continue therapy , since hydrocortisone cream was prescribed again but no satisfactory pruritus and pain improvement was noticed . a prompt regression of skin reaction occurred after stopping therapy .", "sarcoidosis is a granulomatous disorder of unknown etiology and whose epidemiology suggest a genetic tendency face to infectious agents being supposed to result of immune system deregulation leading to non - caseating granulomas as an immune reaction to an unknown persistent antigenic stimulus . ifn has been linked to pulmonary macrophage activation , a characteristic feature of sarcoidosis which has been assumed an exaggerated t helper 1 ( th-1 ) immune response to a variety of exogenous antigens . it seems very likely that a potent immunoregulatory protein for th-1 response such as ifn may induce the disease . in january 2003 the eighth case of ifn - related sarcoidosis was reported with a review of the literature . chest x - ray revealed hilar lymphadenopathy in three patients , with reticulonodular shadows in another three , while the patient with only cutaneous involvement had a normal chest x - ray . the case we report presented with cutaneous sarcoidosis during chc therapy and spontaneous regression of the lesions was noted with treatment discontinuation . we intend to emphasize , especially to dermatologists , the risks of a granulomatous tattoo and other cutaneous sites during chc treatment with peg - ifnalpha-2b plus ribavirin . thalidomide has been also shown to have specific activity to the inflammatory mediators of sarcoidosis and to be an alternative beneficial therapy ." ]

[ "pleomorphic carcinoma is an aggressive tumour initially described in the lung by nash and stout . in the 4th edition of the world health organization ( who ) classification of tumours of the lung , pleomorphic carcinoma is defined as a poorly differentiated ( squamous cell carcinoma or adenocarcinoma ) or undifferentiated carcinoma in which at least 10% spindle and/or giant cells are identified , or as a carcinoma constituted purely of spindle and giant cells . primary sites of occurrence of extrapulmonary pleomorphic carcinomas have been described in other organs such as the stomach , thyroid , gallbladder , pancreas , kidney , liver , bladder , and colon . to the best of our knowledge , only one report described a case of giant cell carcinoma of the colon resembling that observed in the lung . based on the recommendations of the nomenclature committee on cell death , mitotic catastrophe is defined as a tumour - suppressive mechanism occurring during or after aberrant mitosis leading to cell death or cell senescence . histological patterns of micro- and multinucleation have been used as morphological markers for the detection of mitotic catastrophe [ 5 , 6 ] . a high frequency of multinucleated giant cells is characteristically found in pleomorphic carcinomas . in this report , we present an additional case of primary pleomorphic carcinoma of the colon , and we underline similarities to and differences from pulmonary pleomorphic carcinoma . the possibility of the pleomorphic component as morphological expression of mitotic catastrophe is finally discussed .", "a 65-year - old caucasian woman with a medical history of hypercholesterolaemia , chronic obstructive pulmonary disease , and paroxysmal tachycardia was admitted to the surgery unit of the university hospital g. martino ( messina , italy ) in 2013 for abdominal obstructive symptoms ( colicky pain to the right iliac fossa as well as borborygmi ) , change in bowel habits ( diarrhoea ) , asthenia , and severe weight loss since 6 months . there was no nausea or vomiting . on physical examination , a globular and tympanic abdomen , painless at superficial and deep palpation , was evident . she reported a history of adequate food intake with regular bowel habits , alcohol consumption ( < 20/30 g daily ) , and smoking . there was no family history of gastrointestinal malignancy , although familiality for bladder carcinoma was reported . abdominal ultrasonography highlighted an exophytic / stenosing mass at the level of the ascending colon . afterwards , colonoscopy also revealed an exophytic / stenosing malignant neoplastic mass in the caecum , subsequently confirmed through histological evaluation of biopsy . carcinoembryonic antigen ( value for smokers < 10.0 ng / ml ) and carbohydrate antigen 19 - 9 ( value < 35 iu / ml ) were within normal limits . a contrast - enhanced computed tomography scan showed no evidence of liver or distant metastases .", "routine haematoxylin - eosin sections were made from formalin - fixed , paraffin - embedded tissue . sections were also stained with periodic acid - schiff and alcian blue at ph 2.5 . additional sections collected on silanized , coated slides were used for the immunohistochemical stains , using the dako envisiontm flex , high ph detection system together with autostainer instruments . the commercial source , clone , and dilution of the primary antibodies are detailed in table 1 .", "macroscopic examination of the resected colon revealed an endophytic / ulcerative lesion measuring 7 cm in its greatest dimension , invading the caecum nearly to the ileocaecal valve . microscopically , the tumour was a poorly differentiated adenocarcinoma with a grade 4 pleomorphic component that occupied up to 10% of the whole tumour section . the pleomorphic component showed trabeculae and nests of neoplastic cells possessing eosinophilic cytoplasm , as well as irregularly shaped vesicular nuclei with a single large eosinophilic nucleolus . giant cells containing multiple nuclei , micronuclei , and prominent eosinophil nucleoli were also found ( fig . the micronuclei appeared as round chromatin fragments with a diameter less than one third of the diameter of the nucleus . 1b ) and large geographical necrotic areas with sharp boundaries with respect to the viable tumour tissue ( fig . only 1 out of the 44 harvested perivisceral lymph nodes presented carcinomatous metastasis ( pt3 , pn1 , clinical stage in accordance to the 2009 ptnm system ) . the histochemical stains were negative for periodic acid - schiff and alcian blue in all of the pleomorphic neoplastic cells . on the immunohistochemical stains , these cells were diffusely and strongly positive for ceap , ckae1/ae3 , ck7 , and vimentin , but they were negative for ck20 , smooth muscle actin , desmin , synaptophysin , and -human chorionic gonadotropin . 2b ) , whereas p53 labelling was shown in more than 90% of the tumoural nuclei ( fig . ", "we described a rare case of pleomorphic carcinoma of the right colon showing opposing clinicopathological features . although it was classified as a grade 4 tumour , it was characterized by a pushing growth pattern and presented only one lymph node with metastasis . these data confirm that classic grading of colorectal carcinoma is insufficient to predict a prognosis , and that further parameters such as growth patterns are needed . accordingly , several recent studies have shown that colorectal carcinomas with pushing margins are associated with superior disease - free survival rates when compared with tumours with infiltrative margins . the present tumour had some of the morphological features described in giant cell carcinoma of the lung , such as pleomorphic tumour cells , malignant giant cells , atypical mitoses , and coagulative necrosis . however , pleomorphic carcinoma of the lung has been reported to have a poor prognosis , whereas in our case , the patient has been well without evidence of recurrence or metastases 2 years after the operation . many studies suggested that pleomorphic tumours are a heterogeneous group of neoplasms arising in different anatomical sites ( e.g. lung , uterus , breast , central nervous system ) characterized by variable morphological features and prognoses [ 8 , 9 , 10 , 11 ] . in particular , a certain percentage of patients affected by them were found to be long - term survivors despite the anaplastic morphology of their primary neoplasms [ 8 , 10 , 11 ] . the differential diagnosis of pleomorphic carcinoma includes adenocarcinoma with a choriocarcinomatous component as well as mixed adenoneuroendocrine carcinoma [ 7 , 12 , 13 ] . a choriocarcinomatous component usually is characterized by immunoreactivity to -human chorionic gonadotropin , which was not found in our case , while the latter was excluded by the lack of expression of neuroendocrine markers such as synaptophysin and chromogranin [ 12 , 13 ] . in our case , multinucleated tumour cells contained not only abnormally large giant nuclei but also those that are abnormally small , in the form of micronuclei . the presence of micronuclei is usually overlooked in the reports of pleomorphic carcinoma in the current literature . micronuclei derive from chromosomes and/or chromosome fragments that have been irregularly distributed between daughter nuclei after abnormal mitosis . besides micronucleation and multinucleation , we interpret these findings as a morphological expression of mitotic catastrophe , a particular type of cell death occurring in tumour cells after aberrant mitosis [ 5 , 6 , 14 ] . after mitotic catastrophe , tumour cells may continue to divide and thus develop polyploidy and/or aneuploidy . ki-67 is an antigen of cell proliferation frequently used in routine histopathological diagnostics as a prognostic factor in more malignancies . specifically , it is an epitope of a nuclear and nucleolar protein of 360 kda only expressed in nuclei of cells in active proliferation , i.e. during cell cycle phases g1 , s , g2 , and m , but not in the quiescent phase , i.e. in cell cycle phase g0 [ 17 , 18 ] . normally , p53 acts as a guardian of the genome , protecting cells against cancer via two main ways : by determining cell cycle arrest at g1 and g2/m and by inducing cellular apoptosis [ 19 , 20 ] . nuclear p53 immunohistochemical positivity in tumour cells suggests mutation in the p53 gene [ 21 , 22 ] . thus , immunohistochemical overexpression of p53 and ki-67 constitutes further evidence of mitotic catastrophe in the pleomorphic component of our case of colonic neoplasm . ionizing radiation and different classes of cytotoxic agents induce cell death through mitotic catastrophe [ 15 , 18 ] . histological recognition of mitotic catastrophe could be useful to predict an eventual pharmacological modulation ( induction or inhibition ) of tumour cell death . therefore , mitotic catastrophe has recently gained attention as a potential therapeutic target in neoplasms [ 23 , 24 , 25 ] . in summary , we reported a case of colorectal carcinoma with clinicopathological features partially similar to pleomorphic cell carcinoma of the lung , a neoplasm associated with a bad prognosis . in particular , this tumour presented only one lymph node with metastasis , and the patient is still well 2 years after the operation . this case presented morphological and immunohistochemical features compatible with mitotic catastrophe , a form of non - apoptotic cell death due to aberrant mitosis . the inclusion of mitotic catastrophe as part of a microscopic evaluation may be useful for understanding the pathogenesis of this rare entity and for new cancer treatment modalities .", "no further ethical approval was necessary to perform histology and immunohistochemistry in the case included in this report .", "" ]

[ "management of suspected scaphoid fractures with normal x - rays at emergency department ( ed ) presentation is a common clinical challenge . there has long been concern that failure to identify and immobilise a scaphoid fracture might result in avascular necrosis or non - union with significant functional impact for the patient and potential medico - legal risk for the practitioner . traditional management would see the patient immobilised in a plaster cast for 714 days followed by clinical examination and re - x - ray to identify fractures not visible on the initial films . that approach can been challenged on two fronts : whether cast immobilisation while awaiting clinical review is justified and the role of alternative imaging modalities at or near the time of initial presentation . the aim of this project was to characterise current management of adult patients with possible occult scaphoid fracture in australasia .", "this was an internet - based survey of directors of emergency medicine training ( demt ) throughout australasia as identified from the australasian college for emergency medicine web - site ( www.acem.org.au ) . demts are the designated supervisors of specialist training at hospitals accredited for training , both metropolitan and rural ( one / hospital ) . as such , they are easily identifiable , experienced specialists with a good understanding of clinical practice within the ed in which they work . participants were sent an invitation to participate in the confidential on - line survey by e - mail ( where one could be found ) or by post . data were collected using internet - based survey software ( www.surveymonkey.com ) and included the most common management strategy used for patients with possible occult scaphoid fracture ( from a range of options , see table 1 ) and whether there was a written ed guideline regarding management of such cases . the outcome of interest was the most common management strategy for possible occult scaphoid fractures . table 1management options and reported frequencymanagement strategyfrequency ( n , % ) backslab ( half - cast ) with assessment and re - x - ray at 714 days23 , 38%plaster cast ( full ) with assessment and re - x - ray at 714 days19 , 32%backslab with early ct ( within 7 days)6 , 10%backslab with early bone scan ( within 7 days)5 , 8%backslab with early mri ( within 7 days)2 , 3%support bandage with early mri ( within 7 days)1 , 1.7%support bandage with early ct ( within 7 days)1 , 1.7%plaster cast ( full ) with early ct ( within 7 days)1 , 1.7%plaster cast ( full ) with early bone scan ( within 7 days)1 , 1.7%support bandage with re - assessment at 1 week , with further tests only if symptoms or signs persist1 , 1.7%support bandage with assessment and re - x - ray at 714 days0plaster cast ( full ) with early mri ( within 7 days)0support bandage with early bone scan ( within 7 days)0othersame / next day ct1 , 1.7% management options and reported frequency this project was approved by western health under the nhrmc quality assurance guidelines .", "the most common management reported was immobilisation in a backslab ( 23 , 38% ) or full cast ( 19 , 32% ) with clinical assessment and re - x - ray in 710 days . ct scan within 7 days was used by 9 ( 15% ) , bone scan within 7 days by 6 ( 10% ) and mri within 7 days by 3 ( 5% ) . eighty - three percent of sites reported not having a written guideline / protocol for this condition .", "the rate of occult scaphoid fractures in cohorts of patients with clinical evidence suggestive of scaphoid fracture but normal initial x - rays is reported to be 016% [ 24 ] , weighted average 3.7% ( 95% ci 2.55.6% ) . in the subgroup who prove to have a fracture , the rate of delayed / non - union is low ( 1020% ) . traditional treatment for suspected occult scaphoid fracture is cast immobilisation with clinical re - assessment and re - x - ray at 710 days . that approach , however , has been suggested to be over - treatment , with a very low rate of significant fractures at the cost of significant time lost from work or school [ 57 ] . it has been suggested that symptomatic treatment with follow - up within 2 weeks for these cases is sufficient [ 6 , 8 , 9 ] . we found that most australasian eds continue to use plaster immobilisation for this patient group ( backslab or full cast , 70% ) and that uptake of alternative imaging modalities is only moderate ( 30% ) . the high use of immobilisation is at odds with reported uk practice , where only 46% of eds reported routinely using plaster immobilisation for x - ray - negative suspected scaphoid fracture . ct , mri and bone scan have all been shown to be effective in the diagnosis of occult scaphoid fractures , but to date no studies have shown additional benefit from early advanced imaging for hard clinical outcomes . at present , the logical role for advanced imaging is for patients with persistent symptoms and normal follow - up x - rays . there is a clear need for further research comparing combinations of the various immobilisation and imaging strategies and reporting clinical outcomes ( including time off work and time immobilised ) and cost - effectiveness outcomes . there is no evidence that lack of access to advanced imaging impacts adversely on clinical outcome . the weight of evidence would suggest that this is over - treatment and that a simple bandage is sufficient . as this was a survey , the management strategy reported may not be that actually used .", "the traditional approach to management of possible occult scaphoid fracture of immobilisation with re - x - ray at 710 days remains the most commonly used in australasia , despite evidence that this is probably over - treatment with significant consequences for patients ." ]

[ "women 's reproductive health is a major area of concern , especially in developing countries . maternal complications and poor perinatal outcome are highly associated with nonutilization of antenatal and delivery care services and poor socioeconomic conditions of the patient . more than half a million women die annually worldwide because of pregnancy - related complications . migration is the most observable and impressive fact in the growth of cities , and it is also considered as an essence of urbanization in the globe . in india , major cities have noticed an increase of around 75% population due to migration . migrants face numerous constraints , including lack of political representation ; inadequate housing and lack of formal residency rights ; low - paid , insecure , or hazardous work ; limited access to state - provided services such as health and education ; and discrimination based on ethnicity , religion , class , or gender . migrants face denial of basic entitlements including access to subsidized food , housing , drinking water , sanitation and public health facilities , and education and banking services and often work in poor conditions devoid of social security and legal protection . internal migrants in india constitute a large population : 307 million internal migrants or 30% of the population and by more recent estimates 326 million or 28.5% of the population . out of the total internal migrants , 70.7% are women . marriage is given as the prominent reason for female migration in both the rural and urban areas migration of women has been acknowledged as a demographic factor which affects the reproductive health of women . the migrant women , who move to the urban areas , face many challenges in relation to access to health care . a study showed that female migrants are vulnerable to environmental , social , and institutions forces that may affect their health - seeking behavior . women of socially disadvantaged groups , migrant , and/or from ethnic minority groups have been recognized to be less likely to receive early prenatal care and the necessary care during pregnancy , childbirth , and postnatal period . empirical evidence has pointed out that migrant groups face several barriers in accessing national maternal health services . maternity care is classed as essential care and so can not be refused due to inability to pay . there is no scope for special care for such vulnerable population in india , at present . women suffer more because though antenatal care ( anc ) services are available , they could not reach them . hence , assessment of present situation of migrant women with reference to anc services utilization and institutional deliveries becomes essential . thus , this study was conducted with following objectives among interstate female migrants . \n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women .", "\n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women .", "a cross - sectional community - based survey was conducted among migrant women in reproductive age group in an urban slum of population ( 30,000 ) , which is the field practice area of a medical college . sample size was calculated using formula , where p is prevalence of the event , q is probability of nonoccurrence of event , and l is allowable error , the prevalence of anc services utilization according to the national family health survey-3 is 77% , and considering 10% allowable error , sample size comes approximately 120 . if there are no women fulfilling inclusion criteria in a randomly selected household , then next household was selected . if there is more than one woman in a household fulfilling inclusion criteria , then only one woman was selected by lottery method from that household . the national sample survey organization 2008 defined migrant as a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household . in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more . full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets . \n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent was included in the study . interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared . initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed . their household was visited with the female social workers , and meetings conducted by social workers were attended . while interacting with the participants , one representative from the social workers was always present . data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 . chi - square test was used for evaluating association between anc utilization and categorical variables .", "the national sample survey organization 2008 defined migrant as a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household . in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more . full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets .", "\n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared . initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed . their household was visited with the female social workers , and meetings conducted by social workers were attended . while interacting with the participants , one representative from the social workers was always present . data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 . chi - square test was used for evaluating association between anc utilization and categorical variables .", "table 1 shows that 77% ( 93/120 ) of migrants were under the age of 29 years . sixty - two percent ( 74/120 ) of migrants were married before the age of 18 years though marriage before the age of 18 years is punishable by law . sixty - four percent ( 77/120 ) of migrants were belonged to lower socioeconomic class . sociodemographic profile of migrant women with their association with full antenatal care package utilization table 2 shows that almost three - fourth of migrants were hailing from uttar pradesh followed by bihar , i.e. 11.7% ( 14/120 ) . thirty - three percent ( 40/120 ) of migrants visited their native place at least once in 6 months . migration details of female migrants table 3 depicts that even after availability of reproductive and child health ( rch ) services in slum area , still 10% ( 12/120 ) of mothers did not register for anc services . registration percentage in the first trimester is less , i.e. , 21% ( 25/120 ) . these women could not be educated about various health topics such as nutrition , immunization , and family planning . nearly 63.4% ( 76/120 ) of mothers visited at least three times to health - care facility . thirty - seven percent ( 44/120 ) of mothers were not able to visit health - care facility at least three times . fourteen percent ( 17/120 ) of mothers did not consume a single tablet of ifa . ten percent ( 12/120 ) of mothers did not receive a single injection of tt . antenatal care services utilization by migrant women table 4 shows that 12% ( 15/120 ) of deliveries occurred in home . even after implementation of rch program with janani suraksha yojana which emphasizes on institutional delivery delivery practices of migrant women table 5 shows that most of the participants , i.e. , 30% ( 36/120 ) mentioned that too far location of government health facility was the most common reason for full nonutilization of anc services . they did not want to bear expenses toward traveling to health center , and visit to health center leads to loss of their daily wages . nearly 25.8% ( 31/120 ) migrant women did not think it is necessary to utilize full range of anc services . elder members in family did not allow 20.8% ( 25/120 ) anc mothers to go for regular anc visits as they did think it is necessary . they expect that their children should follow the same practice as they practiced at their time . poor quality service at health center 17.5% ( 21/120 ) was one of the important reasons mentioned by migrants . staff working at health center was not cooperative , spoke arrogantly , investigations and necessary medicines were not available are the reasons included in poor quality services . 15.8% of migrant women said they have not utilised anc services fully as they were not having any information ( lack of knowledge ) about this services .", "a study conducted in urban slums of delhi showed that majority of migrants were hindus ( 61% ) , aged 2429 years ( 44% ) , with a literacy rate of 38% . majority of migrants were muslims ( 73% ) , aged 1823 years ( 39% ) . twenty - seven percent were illiterates , and anc care utilization was significantly associated with religion and type of family . a cross - sectional study conducted in tribal blocks of maharashtra showed that anc registration in the first trimester is 63.8% , 82% received tt injections , 68.5% consumed ifa tablets , and 72% paid at least three visits to health centers . as compared to this study , in the present study , the percentage of anc registration in the first trimester is very low , i.e. , 21% . most of the anc migrants registered in the second trimester . almost 91% of migrant women received at least one tt injections . ifa tablets are consumed by all , but 100 tablets were consumed by only 28% of migrants . therefore , in the present study , ifa consumption and no of anc visits to health center are very less . these figures clearly point out toward the inadequate utilization of anc services by migrants women . a cross - sectional study was carried out among recently delivered women residing in tea gardens of darjeeling district of west bengal . it mentioned that the major barrier toward utilization of these services was ignorance followed by distance to the health - care center . according to the district level household and facility survey 2007/2008 , the percentage of institutional delivery and home delivery in urban area is 87.5% and 12.5% , respectively . delivery at home conducted by skilled health personnel was 2.8% . in the present study , a community - based cross - sectional study conducted in urban area in nainital district mentioned that high proportion of home deliveries even in urban area despite proximity to hospitals is a result of many factors including weak demand , minimal outreach services , weak community - provider linkages , and timings that often do not suit the daily - wage - earning urban poor . in the present study , out of 15 home deliveries , 11 deliveries reason mentioned was lack of transport facility to hospital . maternity care is an essential care which has to be made acceptable , accessible , and available at a cost affordable to women , but in case of migrant women , it is available but not acceptable and accessible due to above - mentioned reasons .", "the present study revealed low utilization of pregnancy - related health - care utilization among the study population . health - care workers and influential people from muslim community should be made aware and trained in explaining necessity of maternal and child health ( mch ) care . for this purpose , during health awareness session , elder members of the family specially mother - in - laws have to be involved on a priority basis and educated about importance of full anc care utilization and institutional delivery . if necessary , one - to - one counseling should be provided to convince them about correct practices . in the present study , the percentage of anc registration in the first trimester is very low , i.e. , 21% . moreover , migrant woman feels comfortable to conduct delivery by dai if she is available . it means that the message of importance of institutional delivery is not reached or convinced to beneficiaries . auxiliary nurse midwife , accredited social health activist , and anganwadi workers are not reached to them . then , it becomes necessary to train presently working and close to population dais also so that they will conduct deliveries following proper aseptic precautions , i.e. , to follow all 7 cs - clean hand , clean blade , clean cut , clean cord , clean tie , clean towel , and clean bed . poor quality service by health workers is one of the reasons for nonutilization of anc services . health - care workers should give sufficient time to counsel patients , or one counselor should be posted for a primary health center ( phc ) area ( six subcenters ) so that he / she should visit each subcenter under the phc area at least for a day in a week and educate groups or counsel patients on various health topics with proper feedback mechanism . frequency of outreach activities should also be increased for migrant women staying at a far distance from health center in slums which does not come under municipal corporation . this study can provide new insight for policy makers to devote resources for achieving the best possible quality of mch services .", "", "" ]

[ "phytoestrogens , naturally occurring hormone - like compounds with a unique diphenolic structure found in several plants , have received much attention as dietary components as they are known to promote better health . phytoestrogens have been also associated with reduced incidence of various cancers , cardiovascular disease , and osteoporosis and lower cholesterol level [ 25 ] . some studies suggest that the dietary intake of phytoestrogens decreases the risk of breast cancer in humans [ 6 , 7 ] . the most extensively studied phytoestrogen in vitro and in vivo is genistein which is found richly in soybeans . however , biological activities of many other naturally occurring isoflavones have not been studied in detail till now . the phytoestrogen daidzein is an isoflavone present as a glucoside in many plants used in human diets . daidzein is especially concentrated in soy and soy - based products used for human consumption [ 8 , 9 ] . a higher incidence of breast cancer in western population is seen as compared to asians who consume diet containing relatively high levels of low - fat , high - fibre , high - soya content . asian women show protection against breast cancer in comparison to women living in usa or britain [ 11 , 12 ] which is believed to be lost upon immigration and exposure to western lifestyles within a few generations . these studies suggest that exposure to phytoestrogens at an early stage is extremely important , in order to gain their cancer - preventive effects . the female mammary gland undergoes cell division during puberty , and throughout adult life there is cyclical proliferation and involution during the estrous cycle . during early development , rising endogenous estrogen promotes mammary duct branching which ends in highly proliferative structures , terminal end buds ( tebs ) . at postnatal day ( pnd ) 21 , the number of tebs is maximal , and at puberty the onset of estrous cycles results in changing progesterone and estrogen levels that promote differentiation of tebs to the less proliferative alveolar buds or lobules [ 1517 ] . highest number of tumours per animal was observed when carcinogen exposure occurred in rats at postnatal day 4050 , a time when mammary gland exhibits a high density of the highly proliferative tebs . the incidence of carcinomas is positively correlated with the number of tebs in mammary gland of the young virgin rat at the time of carcinogen exposure [ 16 , 17 ] . administration of the phytoestrogen genistein to neonatal and prepubertal rats reduced tebs by promoting teb differentiation [ 18 , 19 ] . a study on sprague dawley rats ( mammary tumor model ) shows that daidzein has been an effective inhibitor of dmba - induced mammary tumor . chemoprevention study from our lab has also shown a decrease in tumor burden and incidence by prepubertal daidzein administration . although daidzein also has the ability to bind to the ers , the precise mechanism of action by which it shows a chemoprevention against breast cancer has not been clearly elucidated . considering the above literature , it could be hypothesized that daidzein could also affect the differentiation and signaling pathways as a mechanism of chemoprevention . hence , to explore the mechanism of actions of daidzein , its effect on cell proliferation , mammary gland differentiation , and the expression of ers in a prepubertal mammary tumour model was carried out .", "daidzein , estradiol benzoate ( eb ) , and hoechst 33258 were bought from sigma aldrich co. ( st . louis , mo , usa ) , while dimethyl sulphoxide ( dmso ) and carmine were bought from qualigens ( india ) . , santa cruz , calif , usa , and secondary antibodies , diaminobenzidine ( dab ) , avidin - biotin complex , and normal goat serum ( ngs ) were purchased from bangalore genei ( india ) . female sprague dawley rats , kept in animal facility in a 12 hr day : 12 hr night cycle , and maintained in the animal house of the university were used for the present study . the studies were conducted according to the ethical guidelines of committee for control and supervision of experiments on animals , government of india , on the use of animals for scientific research . experiment 1a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500 g / g body weight daidzein and 500 ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection , 6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter . the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500 g / g body weight daidzein and 500 ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection , 6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter . the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . \n however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . all mounts of fourth pair of the mammary gland of the animals were prepared . mammary glands were dissected at the time of sacrifice , spread on the microscope slide , and then placed in neutral buffered formalin for 8 h ( 22-day - old animals ) and overnight ( 50-day - old animals ) . glands were defatted in acetone for 4 h or overnight , placed in 70% alcohol for 30 min , hydrated in water ( 15 min ) , and stained in alum carmine for overnight . after staining , glands were run through a series of graded alcohols ( 30100% ethanol ) and placed in xylene to clear the tissue . glands were then compressed between two glass slides for 24 h , released and allowed to expand for at least 8 h , and finally mounted using a glass coverslip and dpx . all mounts were then evaluated via light microscopy using the criteria established by russo and russo [ 16 , 17 ] and murrill et al . . all mounts were evaluated for the number of terminal end buds , terminal ducts , and lobules type i by the help of the software imagepro . the mammary glands dissected were fixed in 10% neutral buffered saline , and sectioning was done by cryostat at 20 microns . sections were treated with 10% normal goat serum ( ngs ) for 2 h for blocking and then incubated in the primary antibody for 3 days at 4c . the antibody was diluted in 0.1 m pbs containing 5% ngs and 0.5% triton - x-100 . after being washed in pbs , the sections were incubated in biotinylated goat antimouse igg at 1 : 200 for 18 h at 4c . following this secondary antibody step , the sections were washed and placed in preformed avidin - biotin peroxidase complex ( abc ) at 1 : 400 dilutions for two hours at room temperature . for visualizations of the reaction sites , the sections were treated with the chromogen diaminobenzidine ( dab ) and hydrogen peroxide for 2 - 3 min . the sections were rinsed with distilled water dehydrate in ethanol and coverslipped with distyrene plasticizer xylene ( dpx ) mounting medium . the nuclei were stained with hoechst 33258 ( sigma ) ; 10 m sections were mounted on gelatin - coated slides and washed in pbs for 10 min ( figure 6 ) . they were incubated with hoechst 33258 ( 1 g / ml ) for 30 min in dark at room temperature . after washing twice with pbs ( 5 min each ) and once with distilled water ( 5 min ) , the slides were coverslipped with 10% glycerol and stored in dark . the slides were viewed in a fluorescent microscope under the blue filter ( excitation wavelength = 343 nm , emission wavelength = 483 nm ) . since hoechst is membrane permeant , the procedure does not require membrane permeablization . immunostaining was evaluated by examination of slides under a bright field microscope ( carl zeiss axioscop mot 2 ) at magnification 400x , and images were captured through a digital camera for measurement of intensity and counting of cells . intensities of immunostained cells were estimated by densitometry using morphometric software scion image . in order to count the number of er , bcl2 , bax , caspase-3 positive cells and hoechst stained apoptotic nuclei , magnified views ( 400x ) of sections were captured . the total cells ( unstained ) and immunostained cells were counted with the help of the software imagetool ( uthscsa ) . values were compared using one - way anova ( sigma stat , jandel scientific ) .", "differentiation of mammary gland was studied by observing the branching of mammary gland and the terminal end buds and ducts and lobular count at two different stages ( i.e. , pnd21 and pnd50 ) after a prepubertal exposure of daidzein to the animals . an increase in the count of tebs , tds , and lobules has been seen in the daidzein - treated rat mammary glands at pnd21 , whereas there is a decrease in the teb and td count and further increase of lobule i count at pnd50 as compared to the control , as shown in figure 1 . a similar significant increase of tebs , tds , and lobules has also been seen in the eb - treated animals at pnd21 , and a decrease is seen in tebs and tds and an increase in mammary gland lobules at pnd50 . immunohistochemistry results at pnd21 in daidzein - and eb - treated mammary glands showed that the percentage er+ cell count decreased significantly , that is , 41.92 1.0% , p < 0.001 and 39.21 0.8% , p < 0.05 , as compared to the control ( 45.7 0.5% ) , whereas intensity of daidzein - and eb - treated epithelial cells decreased up to 80.9% , p < 0.001 , and 82.8% , p < 0.001 , respectively , as compared to the control ( 100% ) . at pnd50 , daidzein - and eb - treated mammary gland epithelial cells in both the count and intensity were shown to be increased significantly . the percentage count of er+ cells increased to 39.2 0.7% , p < 0.001 , and 41.54 0.7% , p < 0.001 , in daidzein - and eb - treated epithelial cells respectively , from control ( 33.65 0.57% ) , and intensity increased to 121.5% , p < 0.001 , and 125.4% , p < 0.01 , in daidzein - and eb - treated epithelial cells , respectively from control ( 100% ) significantly . in 21-days - old mammary glands , the bcl2 + cell count and intensity were found to be increased significantly in daidzein - treated animals ( 40.26 0.38% , p < 0.001 ; 111% , p < 0.01 ) as well as in the eb - treated animals ( 44.16 1.02% , p < 0.001 ; 122.4% , p < 0.001 ) from control ( 35.9 0.6% , 100% ) , whereas the cell count and intensity of bax+ cells were decreased significantly in daidzein - treated animals ( 19.37 0.22% , p < 0.05 ; 82.0% , p < 0.001 ) as well as in eb - treated animals ( 19.7 0.68% , p < 0.05 ; 83.6% , p < 0.05 ) from control ( 23.61 0.91% , 100% ) . in 50-days - old mammary glands , bcl2 + cell count and intensity were decreased significantly in daidzein - treated animals ( 31.3 0.64% , p < 0.001 ; 82.3% , p < 0.01 ) as well as in the eb - treated animals ( 29.11 0.65% , p < 0.001 ; 79.5% , p < 0.01 ) from control ( 40.4 0.65% , 100% ) . simultaneously , the cell count and intensity of bax+ cells were increased significantly in daidzein - treated animals ( 40.65 0.67% , p < 0.001 ; 114% , p < 0.05 ) and in the eb - treated cells ( 39.0 0.84% , p < 0.001 ; 116.3% , p < 0.01 ) from control ( 28.05 0.37% , 100% ) . bcl2/bax ratio was found to be enhanced in mammary gland at pnd21 but not at pnd50 . caspase-3 + cell count percentage was shown to be increased significantly at 50 days in mammary glands treated with daidzein ( 20.57 1.07% , p < 0.05 ) and eb ( 23.39 1.21% , p < also the percentage apoptotic nuclei count increased in daidzein ( 17.94 0.46% , p < 0.001 ) and eb ( 18.82 0.71% , p < 0.001 ) , compared to the control ( 13.28 0.42% ) , at 50 days , significantly ( figures 25 ) .", "our data clearly demonstrates , the prepubertal exposure to daidzein by the female sprague dawley rats produces an enhancement in the differentiation of the mammary gland as well as influences apoptosis and the er expression . the highest susceptibility of a rat mammary gland to a carcinogen occurs in the postpubertal virgin females around the age of 50 days , which correlates well with the higher number of tebs and the high cell proliferation , and a low incidence of mammary carcinoma is related to the loss of tebs and the low cell proliferation activity . there was an increase in the count of tebs and tds and lobules by the prepubertal administration of daidzein in pnd21 , which shows an increased proliferation in the cells , which is probably necessary for the developing of mammary gland at a prepubertal stage . lobules are more stable structures than the tebs and tds , hence , by the increase in the count of lobules , it could be interpreted that the undifferentiated tebs and tds are progressing gradually towards the formation of more differentiated lobules , which could be due to the influence of daidzein . in a similar kind of study with genistein , it was observed that prepubertal genistein exposure increases the count of tebs and lobules at pnd21 [ 19 , 23 ] . at pnd50 , the decrease in the count of tebs and tds indicated a decreased proliferation , and coupling this with an increase in count of lobules at the same time suggests further enhancement in the differentiation of mammary glands at pubertal stage , in daidzein - treated mammary glands . earlier genistein had shown a similar potential to increase the mammary gland differentiation , along with reduction of teb count and increase in the lobules , at pnd50 [ 19 , 23 ] . some of the xenoestrogens such as diethylstilbestrol ( des ) , o , p-ddt , aroclor 1221 , aroclor 1254 , and 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) also showed an enhancement in the mammary gland differentiation when administered prepubertally . since the development of mammary gland is controlled by female reproductive hormones like estrogen , it is likely that exogenously the administration of the estrogen agonist estradiol benzoate ( eb ) will show an enhancement in the mammary gland development , which has also been reported earlier . here , the eb - treated mammary glands also showed an increase in the tebs and tds at pnd21 and a decrease in the tebs and tds count along with the increase in lobules at the old mammary gland pnd50 . a similarity in the results of both eb - and daidzein - treated mammary gland differentiation data suggests that daidzein might have a similar mode and magnitude of action as that of eb , when administered to rats during the prepubertal stage . thus , the overall results of the mammary differentiation data reveals that shortly after exposure to daidzein , there was a rapid development of the mammary gland , yielding more differentiated structures ( lobules ) and fewer undifferentiated structures ( terminal end buds ) . tumorigenesis experiments done earlier in our laboratory had also shown a decrease in tumour incidence and burden by treatment of daidzein to sd rats in the prepubertal stage . hence this chemopreventive effect of daidzein can be correlated to the increased differentiation rate of mammary gland caused by the prepubertal exposure of compound . immunohistochemical data of er- indicated that there is a decreased expression of er- in response to daidzein , seen in the mammary epithelial cells at pnd21 . normally , the percentage of er--positive nuclei varies according to the developmental state of the mammary gland . decreased expression of er- may also reflect proliferation , as proliferating epithelial cells of the mammary glands of young virgin rats do not express the receptor [ 27 , 28 ] , while its increase after 50 days suggests that er- expression might not be somewhat necessary for proliferation but may be required for the differentiation of mammary glands . this is consistent with our previous results which indicated that prepubertal daidzein treatment resulted in a proliferative mammary gland at pnd21 , which led to a more differentiated structure evident at pnd50 . a similar result in the expression of er- in response to daidzein and eb may suggest a common molecular mechanism of action . there has been a link established between the proliferating epithelial cells and the expression of er- in developing rodent mammary gland . the increase in the count and intensity of bcl2 at pnd21 further supported the result that there might be an increase in proliferation of cells which might have resulted in the increased count of the unstable proliferating structure , tebs , accordingly . later at pnd50 , there is a decrease in the bcl2 expression which indicates decreased proliferation . this can be related to the enhanced differentiation of the mammary glands in the daidzein - treated rats , where there was an increase in the lobule count along with a decrease in the count of teb at pnd50 . and since the differentiation of the cells has increased and proliferation has slowed down , the count of bcl2 has also decreased in 50-day - old daidzein - treated rats , significantly , as compared to control . furthermore , a decrease in the level of antiapoptotic protein bax at pnd21 and its increase in pnd50 show that apoptosis is not seen in pnd21 rather it increased in pnd50 as evaluated by the simultaneous expression levels of bcl2 and bax . the process of apoptosis is integral to normal mammary gland development , and the morphogenesis of ducts in the development of mammary gland is dependent on the selective death of epithelial cells to form mammary acini [ 29 , 30 ] . nuclear condensation and fragmentation because of degradation of dna into oligonucleosome fragments , which is a characterized feature of apoptosis , was seen to be increased in pnd50 , whereas no significant change in the apoptotic level was observed at pnd21 . this was further confirmed when the apoptotic marker protein , caspase-3 , which is also known to be one of the principal intracellular effectors of the apoptotic cascade , simultaneously increased in the daidzein - and eb - treated cells along with the increase in apoptotic nuclei , at pnd50 . since daidzein and eb decrease the expression of er- in prepubertal epithelial cells along with the increase in the proliferation and differentiation of cells at the same time , it can be said that the estrogen receptor expression and the mechanism for the proliferation of cells may not be directly correlated , and both proteins might have been influenced by the test compound by separate pathways . both processes may be independent of each other , but both might be required for the enhancement of differentiation of the gland .", "the phytoestrogen daidzein has a potential to regulate the differentiation of mammary gland by maintaining a balance between proliferation and apoptotic death of the cells which is critical for its normal development . perturbations in this balance can contribute to the development of various disorders in mammary gland including cancer . conditions that upregulate cell proliferation or downregulate apoptosis can allow the accumulation of mutations that contribute to the subsequent development of breast cancer . daidzein enhances the differentiation of mammary gland in a controlled manner at all the developmental stages . although the influence of daidzein on some key proliferative and apoptotic proteins has been discussed here , investigations on other cellular pathways integral to the differentiation process can be explored to have a better understanding of the mechanism of actions of daidzein . thus , it can be concluded that daidzein , in a similar mechanism of action as that of eb , influences the differentiation of mammary gland by affecting cell proliferation proteins and the er- expression . since the more differentiated the mammary gland is , the less susceptible it is to cancer incidence , dietary intake of daidzein from an early developmental stages may be beneficial to women folk in particular , which can be further confirmed through clinical trials ." ]

[ "acute appendicitis , one of the commonest surgical emergencies , affects nearly 7% of the world 's population and accounts for about 1% of all surgical operations . faecoliths formed by mineral deposits layered with faecal debris and lodged in the appendix are called appendicoliths . the prevalence of faecoliths in the general population is 3% , and appendicoliths are seen in 10% cases of acute appendicitis .", "a 25-year - old male presented to us with pain in the right flank for 1 day . urinalysis was normal , and abdominal ultrasound raised a suspicion of small calculi in the right kidney . non - contrast computed tomography ( ncct ) abdomen revealed a few subcentimetric calculi in both kidneys without hydronephrosis or hydroureter . [ figure 1c and d ] ( mineral core ) , composed mainly of calcium and struvite on spectral analysis [ figure 1a and b ] . he underwent laparoscopic appendectomy using a 10-mm supra - umbilical camera port and two 5 mm ports at the supra - pubic region and the left iliac fossa , respectively . a pre - ileal appendix was found hidden in flimsy adhesions posterior to the terminal ileum . an incidental meckel 's diverticulum was also found [ figure 1f ] , which was not pathological and was left alone . the mesoappendix was cut using harmonic scalpel ( johnson and johnson make ) and the appendix was cut at the base between ligatures [ figure 1e ] . it was removed using a glove bag after enlarging the supra - umbilical incision because of the large stone size 2.5 cm 3.0 cm [ figure 1 g and h ] . ncct abdomen ( a and b ) the ct spectral analysis of the appendicolith shows the highest peak of the histogram corresponding to struvite - calcium ( c ) multiplanar reconstruction ( mpr ) coronal image showing appendicolith 2.50 cm size ( longitudinal ) ( d ) axial image showing appendicolith 2.10 cm size ( transverse ) ( e - h ) laparoscopic appendectomy , procedure , specimen and appendicolith ( e ) multiple ligations of base of appendix ( f ) meckel 's diverticulum ( g ) size of appendicolith 3 cm on longest axis ( h ) size of appendicolith 2.5 cm on the perpendicular axis", "acute appendicitis was first reported by fitz in 1886 , and wangensteen and bowers proposed the theory of an obstructive component as a causative factor in 1937 . other proposed aetiologies include lymphoid hyperplasia , constipation , trauma , diet , genetic predilection , hypersensitivity and mucosal ulceration . appendicolith on plain abdominal x - ray is a reliable sign of appendicitis ( 70% ) . however , computed tomography ( ct ) is more sensitive , detecting even non - calcified faecoliths . on ct , appendicoliths appear as laminated bodies with gas in centre or homogenous opacity . when symptomatic , they carry 90% probability of acute appendicitis and 50% higher risk of perforation and abscess formation . some authors have found good correlation ( 65 - 100% ) between faecoliths on ct and appendicitis , while others have not . in a retrospective review by lowe et al . , an appendicolith detected on ct had a sensitivity of 65% , specificity of 86% , and positive predictive value of 74% for the diagnosis of appendicitis . despite appendicoliths being common , a giant appendicolith ( > 2 cm ) is extremely rare and only sporadically reported . others include a 2.1 cm appendicolith reported by garg and a 2.2 cm stone by kaluarachchi . our case is probably the largest documented stone ( 3 2.5 cm ) and the first whose chemical composition has been determined preoperatively by spectral analysis . the authors have not received any resources from a third party , directly or indirectly , to complete this work .", "", "the authors have not received any resources from a third party , directly or indirectly , to complete this work ." ]

[ "it is commonly treated with conservative surgery ; prognosis is rapidly improved by performing concomitant chemotherapy after the initial surgery.1,2 gliomatosis peritonei is the metastatic implantation of mature glial tissue within the peritoneal cavity of patients with ovarian teratomas.3 as second - look or secondary debulking surgery is not generally performed for immature teratoma , as gliomatosis peritonei is not usually discovered in a patient who has completed chemotherapy and shows no evidence of disease.4 this is a rare case of a patient with ovarian teratoma recurrence in the contralateral ovary . the first and later occurrences were treated with conservative surgery , using local resections or cystectomies .", "gravia 0 , para 0was referred to us because of a lower abdominal mass ; magnetic resonance ( mr ) imaging revealed a tumor 20 cm in size . tumor marker tests revealed cancer antigen 125 = 279 u / ml , ca19 - 9 = 130.6 u / ml , squamous cell carcinoma = 3.9 ng / ml , and alpha - fet protein = 50.6 ng / ml . after providing informed consent , she selected conservative surgery . we performed a right salpingo - oophorectomy , partial omentectomy and peritoneal biopsy , and found lint - like scattered mass lesions 2 mm in size at the vesi - couterine pouch and the douglas pouch . the tumor is a mass with cystic regions of many sizes , with solid parts and adipocyte - laden parts ( fig . the diagnosis was stage ii c(a ) immature teratoma grade2 of the right ovary with gliomatosis peritonei . pathologically , the mass contained squamous epithelium , adipose tissue , bone and cartilage tissue , and immature neuroectodermal tissue , and was therefore classified as a grade 2 immature teratoma ( fig . peritoneal lesions , which were composed of mature glial cells , were diagnosed as gliomatosis peritonei using hematoxylin and eosin ( h&e ) stain ( fig . immunohistochemically , the glial cells were positive for glial fibrillary acidic protein ( gfap ) , which is expressed by numerous cell types of the central nervous system , including astrocytes , ependymal cells and glial cells ( fig . sixteen months after the first operation , the patient underwent cystectomy of a left ovarian tumor and a douglas pouch biopsy . pathological diagnosis was mature cystic teratoma of left ovary and gliomatosis peritonei ( fig . sixty months after the first operation , mr imaging revealed a left cystic ovarian tumor with solid parts 7 cm in size . pathologically , the mass contained immature neuroectodermal tissue , and so was classified as a grade 1 immature teratoma and gliomatosis peritonei ( fig . 2f and g ) . 71 months after the first operation , cystectomy of a left ovarian tumor5 cm in size and biopsies of the douglas pouch and left external iliac lymph node8 mm in size were carried out . the pathological diagnosis was endometrial cyst of left ovary , gliomatosis peritonei and glial implant in lymph node ( fig . although this patient has never conceived a child , she has maintained a regular menstrual cycle , despite multiple surgeries for ovarian teratoma and chemotherapy .", "immature teratoma of grades 1 and 2 are considered ovarian germ cell borderline tumors , according to the world health organization s classification . as they occur predominantly in women in their teens and 20 s , conservative surgery is performed as a standard ; concomitant chemotherapy after the initial surgery has led to rapid improvements in prognosis.1,2 jefferys et al produced a retrospective study of 47 patients with malignant ovarian germ - cell tumors who were treated by conservative surgery and adjuvant chemotherapy . during chemotherapy , 61.7% ( 29/47 ) of patients developed amenorrhea but 91.5% of these women resumed normal menstrual function upon completion of chemotherapy . twenty patients ( 42.6% ) attempted conception after chemotherapy , 19 of whom ( 95% ) were successful ; 14 healthy live births were recorded , with no documented birth defects.5 in patients with ovarian germ - cell tumors , a recurrence in the contralateral ovary still could be treated by a local resection or cystectomy followed by chemotherapy if fertility was desired , thereby preserving some normal ovarian tissue , if present.5 however , there is little information about recurrence in the contralateral ovary in patients with immature teratomas . in the present case , we re - performed conservative surgery for contralateral recurrence of an ovarian immature teratoma . as such cases are too uncommon to offer evidence for or against repeated fertility - conserving surgery , such surgery should be performed only after obtaining informed consent . histological grading of peritoneal implants , from grade 0 to grade 3 , is essential for therapeutic and prognostic considerations . grade 0 is defined by tumor consisting of only mature tissue with no mitotic activity . gliomatosis peritonei is the metastatic implantation of the mature glial tissue in the peritoneal cavity of patients with ovarian teratomas.3 the prognosis for gliomatosis peritonei should be very good and no further therapy should be necessary . peritoneal implants consisting of embryonic tissue and metastatic immature teratomas imply a poor prognosis.6 if no other teratomatous elements or malignant glial tissue can be found in the implants , the mature glial implants can be ignored and the methods of therapy should be judged only by the stage and grade of the primary ovarian teratoma.7 progression of the peritoneal implants may be as follows : ( 1 ) transforming fibroblastic and eventual disappearance ; ( 2 ) transformation to malignant tissue ( glial or teratomatous ) ; and ( 3 ) persistence without morphological changes.8 in the present case , gliomatosis peritonei was initially classified as grade 0 . because the peritoneal implants were not malignant and shrank over the 8 years , we thought them to be of the first stage ( 1 ) described above . although the gliomatosis peritonei shrank , a lymph node glioma was detected in the fourth surgery . presence of glial tissue in lymph nodes is rare , with very few reported cases . patients with intraperitoneal and lymph node metastases of mature glial tissue do not need therapy for such metastases . the prognosis for these patients is excellent , but they require long - term follow - up ." ]

[ "cesarean delivery rates have been increasing in a fast manner throughout the world within past few decades ( 1 ) . although reasonable cesarean rates have been proposed as 510% by who , cesarean delivery rates across the world varies between 0.4% and 41% ( 2 ) . while cesarean birth rates had reached 28% in usa , 21% in canada , it is around 37% in brazil , 39% in mexico , 40% in china ( 2 , 3 ) . cesarean birth rates are also increasing in turkey in parallel to the developments in the world . while 6.0% of all births were realized by cesarean section in 1998 , this rate has risen to 48.1% in 2013 ( 4 , 5 ) . the birth and postpartum processes can imply significant risks for both mother and infant health . one of the most important conditions , which have effect on these risks during the birth process is the way of delivery . these complications may include staying at intensive care unit , postpartum depression , infection , thrombosis , hysterectomy , bleeding , blood transfusion and internal artery ligation related to maternal health , and iatrogenic prematurity , pulmonary hypertension , inability to breastfeeding , fetal respiratory syndrome in relation to newborn ( 68 ) . cesarean delivery rates are influenced by many non - medical factors such as cultural factors , personal characteristics of the woman and socioeconomic features ( 9 ) . the purpose of this study was to determine cesarean birth rates and to find out social factors affecting the cesarean birth in primiparous women .", "this study was conducted in burdur province , turkey between the dates of 1 jan 201231 dec 2012 . there are 78 family health units and four general hospitals including three public , and one private sector in the province of the study . pregnancies and pregnancy outcomes ( abortion , stillbirth and live birth ) are reported to provincial public health directorate by the hospitals and family physicians . a total of 737 childbirths were realized between the dates of 1 jan31 mar 2012 . the universe of this cross - sectional type study was formed by 223 primiparous women . ninety - six percent of the universe was reached ( 214/223 ) . the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) . data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight . the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence . the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate . the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection . the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses , chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated . the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses .", "the universe of this cross - sectional type study was formed by 223 primiparous women . the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) .", "data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight . the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence .", "the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate . the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection .", "the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses , chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated . the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses .", "when the table is monitored , it is seen that there is statistically significant relationship between the delivery mode and the place of residence , women s age , women s family type and her husband s employment status and presence of health insurance . when it is examined in terms of birth and infants characteristics , a significant relationship was shown between the delivery mode with doctor s influence in taking decision and the place of birth ( table 2 ) . sociodemographic features of women birth and infant features 3 data is missing in cesarean group the factors affecting caesarean deliveries are seen in table 3 . accordingly , when public hospitals are taken as reference , caesarean deliveries were increased by 11.2 fold in the event of the delivery in private hospital ; 6.1 fold in the event of the delivery in university hospital . again with reference to the conditions of woman s husband work on her own account , working in private sector or public sector increases 2.2 fold the caesarean birth . doctor s guidance increases the caesarean 4.0 times with reference to those who say doctor had no impact on the delivery mode . when the deliveries made between 17:0007:59 are taken as reference , caesarean deliveries are increased by 7.1 times between 08:0016:59 .", "women s place of residence , age , presence of health insurance , family type , husband s job , the place of birth , the birth hour and doctor s intervention to delivery mode have been identified as factors affecting the caesarean section delivery in univariate analyses performed . meanwhile in multivariate analyses , the place of delivery , time of the birth , doctor s effect , and husband s employment status have been identified as factors affecting the caesarean delivery among these factors . caesarean deliveries realized 11.2 times more in private hospitals and 6.1 times more in university hospitals in primiparous with reference to public hospitals in the study conducted . it was demonstrated that caesarean deliveries were increased by 12.7 times in university and top level hospitals with reference to deliveries of primiparous women in second - line hospitals in a study conducted ( 3 ) . caesarean deliveries are particularly among the preferable methods in terms of avoiding the complications associated with the childbirth . caesarean deliveries are preferred especially for pregnant women at risk due to possible complications in terms of mother and infant during delivery , cephalopelvic disproportion or high birth weight infants . in such cases , if pregnant women are followed at 3rd step health institutions or are guided for delivery by 2nd step institutions , then it may increase caesarean deliveries in these institutions . one of the surprising aspects of the study was the 11.2 fold increase in caesarean delivery rates at private hospitals in spite of its equivalent adequacy with 2nd step health institutions in terms of the equipment with reference to public hospitals . caesarean delivery rates in private hospitals are significantly more than public hospitals in the studies performed ( 10 , 11 ) . this increment is rooted in social and economic factors as well as medical reasons . in our study , working female ratio is around 50% both in case and control groups . and a very small portion of the husbands are unemployed . the employed status of the husband regardless of private or public or his own account , means the woman is also under the insurance coverage . on the other hand , employed status of the husband caesarean deliveries are more in the women with any health insurance compared to those without insurance in all studies conducted ( 3 , 12 , 13 ) . this situation should be considered in conjunction with the finding of caesarean deliveries in private hospitals are , more which is another finding of the study . caesarean deliveries within working hours are 7.4 times more with reference to out of working hours ( 18:0008:00 ) in our study . caesarean operations increased on fridays and between 06:00 a.m. and 06:00 p.m. with respect to primaparous ( 14 ) . meanwhile in another study , a relationship was shown between the time of caesarean section and insurance status . while the time of caesarean shows dispersed within a day in kaiser type insured women , there was an increase between 4 in the morning and 6 in the evening in other type insured women having caesarean . the caesarean section rate is the lowest between 10 at night and 6 in the morning in all insured groups ( 15 ) . caesarean deliveries are also the operations performed with the patient s consent in line with physician s clinical evaluations and conviction just like in all other medical procedures . physicians can offer particular options about delivery mode even though a specific preference is expressed for the patient . if the caesarean delivery is planned , caesarean delivery will take place , however if the plan is vaginal delivery then the delivery may be realized through vaginal or caesarean section ( 16 ) . elective caesarean elective caesarean deliveries are estimated to be around 418% of all caesarean sections and 2% of all deliveries ( 17 ) . 31.1% of the women who gave birth through caesarean section have decided caesarean delivery without doctor s influence in the study . on the other hand , caesarean delivery rates increase by 4.03 times with reference to those without doctor s effect for the determination of delivery mode . we believe that the increase with doctor s effect in caesarean deliveries occurs in two ways . even if the woman herself decides the delivery mode , beforehand women are informed by physicians about benefits or risks of caesarean delivery ( 16 ) or physicians directly affect the patient s decision - making process in caesarean practices due to the knowledge asymmetry between the patient and the physician ( 14 ) . caesarean deliveries are preferred because of physicians habits , caesarean deliveries bring more income and vaginal births take more time and defensive medicine application due to the fear of malpractice and complication during birth or pregnancy ( 18 ) .", "although the results do not show all of the factors affecting the caesarean delivery in primiparous , they also reveal that medical reasons are not the only reason in this increase trend . health policy makers and health professionals are required to identify the causes of this increase and to take measures . the strong side of the study is taking only the women who gave first birth in the study and reaching the complete universe of the study . on the other hand , indetermination and non - exclusion of caesarean sections with medical causes in study questions", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ." ]

[ "las fracturas por fragilidad asociadas a la osteoporosis suponen un gran coste econmico y personal para la sociedad . la ingesta de calcio farmacutico o diettico es necesaria para aumentar la densidad mineral y prevenir fracturas por fragilidad . aunque los productos lcteos son una buena fuente de calcio , los pacientes que no pueden digerir la lactosa tienden a evitarlos y tienen un mayor riesgo de fractura que la poblacin general . informes anecdticos indican que las personas que no digieren bien la lactosa , al consumir leche cruda , tienen una gran reduccin de sntomas en comparacin con la leche pasteurizada . el mecanismo de la reduccin notificada en los sntomas , de ser verdad , se desconoce . el propsito del estudio actual era hacer una encuesta a las personas que beben leche cruda para determinar sus motivaciones relacionadas con la salud para consumir leche cruda , sobre todo en relacin con la mala digestin de la lactosa .", "153 de 1527 miembros de una comunidad que compra leche cruda complet una encuesta online en relacin a la leche cruda .", "el motivo principal que los encuestados citaron para beber leche cruda fue que crean que era ms sano ; el 30 % notific ciertas molestias gastrointestinales al beber leche pasteurizada , aunque casi todos ( 99 % ) notificaron consumir leche cruda sin molestias . a pesar de los informes de molestias gastrointestinales , un profesional mdico haba diagnosticado intolerancia a la lactosa en solo el 5 % de los encuestados , y solamente al 1 % se le haba diagnosticado intolerancia a la lactosa mediante el mtodo de referencia de prueba de hidrgeno en el aliento .", "el motivo principal para beber leche cruda es su valor saludable percibido , no su digestibilidad . aunque la leche cruda parece ser digerida ms fcilmente que la pasteurizada en la muestra de nuestra encuesta , se desconoce el mecanismo de digestibilidad .", "the popular media occasionally present anecdotal stories regarding the benefits of consuming raw dairy products . yet we found no reports in the literature supporting the anecdotal reports that lactose maldigesters are able to consume raw milk without discomfort . also unreported or under debate in the literature is the mechanism that allows maldigesters of pasteurized milk to consume raw milk without discomfort . if the enzyme is present in raw milk , it is very heat labile and would be denatured during the milk pasteurization process . naturally occurring lactobaccili and lactococci , which are killed during pasteurization , another proposed mechanism of lactose maldigestion is that milk from cows with the a1 -casein variant ( commonly european breeds ) contains a peptide called -casomorphin-7 that reportedly can cause an immune response , lactose intolerance symptoms , and other health - related issues . these claims are not well substantiated in the literature and are controversial . further complicating the issue , raw milk is often produced and sold from small family farms that typically use jersey or guernsey cows and pasture their cows on grass . jersey and guernsey cows typically have the a2 -casein variant , whereas holstein cows ( primarily used in large industrial dairies ) have the a1 variant . so what are perceived by some to be the benefits of raw milk may have as much to do with the type of cow ( a1 vs a2 ) and husbandry ( pastured and grass - fed vs confined and ration - fed ) as with the manner in which the milk is pasteurized ( or not ) . we intend to clinically evaluate the digestibility of raw milk vs pasteurized milk , but to guide the design of our study , we need more background information . therefore , we surveyed consumers of raw milk to determine the health - related motivations for consuming raw milk , especially as they relate to lactose maldigestion .", "we developed an online survey of 16 questions , which was available from december 7 , 2012 , until january 31 , 2013 . the director of a raw milk buying consortium in maryland sent an email to its 1527 members , inviting them to complete our online survey . because trafficking in raw milk intended for human consumption is against the law in maryland , survey responses were anonymous . this study was approved by our institutional review board , and respondents were informed that completing the survey served as informed consent . most of the 153 respondents ( response rate of 10% ) were females between 30 and 50 years of age ( table ) . of the 153 respondents , 74% reported drinking at least one glass of raw milk per day . results of survey of raw milk drinkers although instructed to choose only one response , 11 respondents chose one of the letter selections , then added a comment under other ( please specify ) . we sought to differentiate those who consumed raw milk because they considered it more healthful than pasteurized milk from those who consumed it because they found it easier to digest . the concept of easy to digest was based on the absence of bloating , gas , diarrhea , and cramping . we queried on the digestibility of drinking milk under three conditions : ( 1 ) the reaction to drinking pasteurized milk before switching to raw milk ; ( 2 ) the reaction experienced currently when drinking raw milk ; and ( 3 ) the reaction of drinking pasteurized milk after having switched to raw milk . we also asked if there was a transition period at the onset of drinking raw milk to identify if some colonic adaptation had occurred as a result of drinking raw milk . we sought to identify subjects who had been diagnosed by a medical professional as lactose intolerant and if the diagnosis had been confirmed by a hydrogen breath test ( hbt ) . we asked about the digestibility of other dairy products , namely yogurt , cheese , and ice cream . it has long been known that in parts of the world where prevalence of lactose maldigestion is high , those populations nevertheless consume large quantities of yogurt . fermented products such as cheese and dairy products with live cultures ( yogurt ) are more easily digested than other dairy because of the presence of microbial -galactosidase . in an attempt to learn more about the potential role the -casein variant ( a1 vs a2 ) has on milk digestibility , we asked from what breed of cow the raw milk came and what type of diet the cows were fed , if the participants knew . lastly , we sought to assess the importance of calcium in the motivation for consuming raw milk . lactose intolerance is related to reduced milk consumption and therefore reduced calcium intake , which is associated with an increased risk for osteoporotic fractures . we analyzed differences in reported discomfort from drinking raw vs pasteurized milk and in drinking pasteurized milk before and after drinking raw milk for significance ( p<.05 ) using mcnemar 's test .", "of the 16 questions on the survey , only four were answered by all respondents , but all were answered by at least 148 ( 96.7% ) ( table ) . of the 153 respondents , most ( 86 ) cited that they consumed raw milk because they believed it to be more healthful and easier to digest . : 12 said raw milk tasted better than pasteurized milk ; 8 cited social / environmental concerns such as supporting local farmers , it is better for the cows , etc ; 6 cited that they had allergies to pasteurized milk ; two claimed it cured unspecified ills ; and six cited individual reasons such as i grew up on it or [ i ] do n't eat processed food . 71 respondents reported no discomfort from drinking raw or pasteurized milk . two reported discomfort from drinking raw and pasteurized milk , and one commented that he / she does not consume cow milk but instead drinks goat milk . fifty - nine respondents claimed no discomfort after drinking raw milk but discomfort from drinking pasteurized milk . one reported no discomfort from drinking pasteurized milk but did report discomfort from drinking raw milk . eighteen consumed raw milk without discomfort but reported they do no drink pasteurized milk . considering the number of respondents who cited discomfort drinking milk , it was surprising that only 5 ( 3% ) respondents were diagnosed as lactose intolerant and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt . in terms of symptoms between before and after beginning to drink raw milk , responses included , ( 70 ) ; do not have discomfort drinking pasteurized milk ( 41 ) ; and still get discomfort drinking pasteurized milk ( 28 ) . twenty - six respondents reported no discomfort from drinking pasteurized milk , but nevertheless they do not drink pasteurized milk . four reported no previous discomfort but did report discomfort since beginning to drink raw milk . five respondents reported previously having trouble drinking pasteurized milk but no symptoms since drinking raw milk . two respondents reported not drinking pasteurized milk but symptoms when they did drink pasteurized milk . there was no significant reduction in discomfort of the respondents between the before and after drinking raw milk periods .", "in the current study , survey results suggest that the motivation for raw milk consumption is complex . we hypothesized that one motivation for raw milk consumption was to avoid symptoms associated with lactose maldigestion . yet almost half of the respondents indicated that they had no ill effects after drinking pasteurized milk . despite the number of respondents indicating some gastrointestinal ( gi ) discomfort when having previously consumed pasteurized milk , only 5 ( 3% ) respondents were diagnosed as lactose intolerant , and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt . these findings suggest that the symptoms of lactose intolerance were mild enough to not warrant the respondents ' seeking a medical diagnosis or that the respondents self - diagnosed and sought alternatives to pasteurized milk . diagnosing lactose maldigestion and the severity of the maldigestion would seem straightforward using the hbt . it is intriguing that a sizable percentage of those exhibiting lactose maldigestion symptoms have not been diagnosed by a medical professional as being lactose intolerant . it would be interesting to determine the hbt diagnosis of those who report gi discomfort . some indicate that what is self - diagnosed as lactose intolerance / maldigestion may be a misinterpretation of gi dysbiosis or irritable bowel syndrome . it may also be a self - fulfilling diagnosis because those who consider themselves to be lactose maldigesters tend to avoid dairy , which causes a change in the gut microbiota , making the bacteria less adept at decomposing lactose . others have found that those diagnosed as lactose maldigesters could ingest modest amounts of milk per day without incident and that symptoms associated with lactose maldigestion were misattributed . the underlying mechanism by which lactose in raw milk may be more readily digested , if in fact that is the case , is more difficult to determine . the predominant bacteria in raw milk are lactococcus lactis ( strains of which are used commercially as starter cultures in cheese making ) , but the bacteria are virtually eliminated when milk is pasteurized . it is likely that these native bacteria account for the alleged enhanced digestibility of raw milk over pasteurized milk , but we found no report in the literature of a direct clinical comparison . in one study , yogurt with active bacterial colonies fed to maldigesters was more readily digested than pasteurized yogurt or milk with active cultures . therefore , yogurt maintains the stomach at a higher ph than milk , which protects the bacteria . in one study , heating yogurt and reintroducing live bacteria up to 10 bacteria / ml concentrations was not as effective as standard yogurt with 10 bacteria / ml in reducing h2 in lactose maldigesters . furthermore , certain strains of active bacteria appear to be more efficient than others at digesting lactose . exposing the intestinal microflora to lactose allows the lactose - fermenting non hydrogen - producing organisms to thrive . the resulting colonic adaptation results in lactose being metabolized to short - chain fatty acids and lactate instead of hydrogen , which is the main component in flatus . this colonic adaptation may confound the study results , which is why we included the question regarding discomfort from drinking pasteurized milk after the respondent had been consuming raw milk . our results did not indicate any evidence of colonic adaptation , but our results may be skewed because most respondents , after drinking raw milk , reported never drinking pasteurized milk , so it is impossible to know how they would react to pasteurized milk consumption . other investigators have suggested a placebo effect instead of colonic adaptation because clinical symptoms of maldigestion ( eg , bloating , diarrhea ) decreased in lactose - treated and control groups . yet objective analysis of h2 excretion decreased significantly in the lactose - exposed group compared with controls , suggesting some adaptation . in our survey , 95% of the respondents reported eating yogurt with active cultures at least once a month . our survey did not ask if the yogurt with active cultures consumption was similar before and after raw milk consumption , so it is unknown what role yogurt might have in colonic adaptation in our survey group . however , after the survey , we learned that some of the respondents made their own cheese and ice cream from raw milk . therefore , it is unknown what percentage of responses was associated with pasteurized commercial cheese and ice cream compared with raw - milk cheese and ice cream . when asked about drinking raw milk , almost 99% of respondents indicated no ill effects . this result suggests that , in the 20% to 30% who initially reported gi discomfort drinking pasteurized milk , the symptoms abated when consuming raw milk . although these findings might argue the case that raw milk is more easily digested , it is unlikely that the result would apply to all who experience gi discomfort when consuming pasteurized milk because of a selection bias in our data set . it is unlikely that those who would experience gi discomfort from drinking raw milk would be part of a raw dairy buying club . thus , there is likely an overestimate of those lactose maldigesters who would benefit from raw milk . that 99% of the respondents could drink raw milk without incident is high but consistent with the published finding that 82% of patients diagnosed as lactose intolerant after ingesting pasteurized milk were capable of ingesting raw milk without incident . the patients in that survey were self - reporting their diagnosis by a healthcare professional , but the definition of healthcare professional was unclear . nevertheless , the information contained in that report is the best currently available for comparison . the current finding suggests that , at least among the group of survey respondents , milk is an important source of calcium . one might surmise that for those who are not able to digest milk easily , milk as a source of calcium may be eliminated from their consideration . in terms of osteoporosis prevention , it should be noted that calcium absorption from milk was not affected by lactose maldigestion status . the reduction in calcium intake in patients with lactose maldigestion more than 75% of the respondents reported consuming at least one glass of milk per day . the size of the glass was not specified , so it is unknown what magnitude of nutrients would be available for ingestion on a daily basis . finally , the role of a1/a2 -casein variant in cow genetics and the role of husbandry on digestibility remain elusive . almost 90% of the respondents reported that their milk came from grass - fed cows . although these results are consistent with expectations of the cows and husbandry that are associated with raw milk for human consumption , it is unknown how reliable the reporting of this information is . more than half of the respondents readily admitted not knowing from which breed of cow their milk came ." ]

[ "the ultrastructural localization of a molecule requires that a certain degree of electron - density , resulting in adequate electron scattering , be present in sufficient amount . binding of heavy atoms can be accomplished easily with regards to proteins , nucleic acids and other cellular components , in some cases with a high degree of specificity . however , the sub - cellular localization of ions is a challenge , since in many cases ions are diffusible and not easily kept in place after the procedures of sample preparation for electron microscopy ( em ) . many ions can offer enough scattering in spite of not being heavy metals , such as manganese , while others necessitate of blocking or precipitating techniques to be visualized . in particular , calcium ions are easily diffusible inside the cell , and even when protein - bound are not easily detectable . the pioneering paper by tandler et al . demonstrated the presence of calcium ions by em via pyroantimonate precipitation , with a good yield of the final product . the end product of the reaction , however , is probably overrated , since the technique is capable of stabilizing and precipitating calcium and other bivalent cations , such as magnesium , as proved by electron spectroscopic imaging ( esi ) analysis . since their introduction by tsien and coworkers , calcium studies have used specific ca - sensitive fluorochromes , such as fura-2 , indo-1 and fluo-4 , coupled to wide - field digitized video fluorescence microfluorimetry and confocal microscopy to image acute elevations in intracellular ca concentration ( [ ca]i ) with high spatio - temporal resolution . cytosolic and nuclear ca signals regulate a multitude of vital cellular processes , such as gene transcription , cell cycle progression , cytoskeletal and chromatin remodelling , and migration . however , proper ca handling within the endoplasmic reticulum ( er ) , the most abundant intracellular ca reservoir , is also crucial for cell - fate decisions . the steady - state er ca level ( [ ca]er ) does not only control synthesis , folding , export and trafficking of proteins , but it also fuels mitochondrial bioenergetics thereby suppressing autophagy . low affinity ca indicators , such as mag - fura 2 , and genetically - encoded probes , such as the bioluminescent protein aequorin and the gfp - based fluorophores camgaroos , pericams , and cameleons , have been devised to assess er ca levels .. nevertheless , these sophisticated tools do not provide any reliable information on ca distribution within the heterogeneous structure of the er . we propose in this paper a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques . maranto and sandell and masland have proposed the possibility of photoconverting fluorochromes into an electrondense end product . the technique utilizes the photo - oxidation of diaminobenzidine ( dab ) in presence of the emission of an excited fluorochrome , and it has been shown to be efficient on different dyes and conditions . this method combines the specificity of available fluorophores to the high spatial resolution offered by transmission em to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles . the present manuscript sought to exploit dab photo - oxidation of mag - fura 2 for visualizing er ca distribution in hela cells , which are among the most widespread model cell lines used for studying intracellular ca signaling .", "hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence , cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) . the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition . the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 . all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) . intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m . cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy . an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software . instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st . louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs . thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel , some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above . moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature . once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) . ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .", "hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence , cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) . the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition . the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 . all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .", "intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m . cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy . an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software . instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st . louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs . thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel , some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above . moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above .", "fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature . once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) . ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .", "calcium labeling with mag - fura 2 results in highly fluorescent staining mainly localized in the cytoplasm . after incubation of the cells with 20 mm ca for 1 h , the signal is present in large vesicles in the cytoplasm and around the cell nucleus , as well as in elongated structures probably related to mitochondria ( figure 1a ) . the faint signal visible in the nucleus is very probably due to the superposition of the cytoplasmic layer surrounding it . when the cells are incubated in pss , the signal is lower ( figure 1b ) and mostly present in the perinuclear vesicles , while the cytoplasmic signal far from the nucleus somehow disappears . in the last condition , without calcium and in the presence of egta , some labeling is still visible , although to a much reduced level ( figure 1c ) . this continuous presence of ca represents very probably the last ions the cell can not allow to lose , a sort of surviving threshold concentrations . supplementary figure 1 confirms that altering extracellular ca concentrations results in a consequent change in er ca homeostasis , which may have important consequences also on cytosolic ca dynamics . at em level , the controls did not show any electron dense precipitates , both in the absence of the fluorochrome or after aldehyde blockade ( not shown ) ; consequently , the end product obtained in the mag - fura 2 stained cells is due to the fluorochrome photoconversion . the dab photoconversion is an efficient way to stabilize the ions and to visualize them . it must be underlined here that we observe a thin section , 60 - 80 nm thick , corresponding , consequently , only to a fraction of the fluorescent signal . after pss incubation , the cells show a distinctive positivity ( figure 2a ) . in some cases it is possible to see the presence of precipitates without the direct association with smooth or rough er ( ser or rer , respectively ) membranes , likely related to unbound ca ions ( figure 2b ) , and electron dense material inside the vesicles is also positive ( figure 2c ) . a possible suggestion of ca internalization or extrusion is shown in figure 2 d , e , where the deposits are present on the outer membrane . when the cells are incubated without calcium and in the presence of egta , the deposits are extremely reduced , although still present on the outer cell membrane , suggesting the presence of an extruding mechanism ( figure 2f ) . on the contrary , the diffuse , low density end product is almost absent from the cytoplasm . at higher ca concentration in the medium ( figure 3a ) , the dark precipitate is detectable on the inner membrane of cytoplasmic vesicles possibly pertaining to ser , and in discrete foci with no apparent association with vesicles or membranes . the deposits are present within electron dense vesicles ( figure 3b ) and electron lucent structure , in some cases associated , and suggesting the idea of passage of ions from one compartment to another . in other cases , dab - positive material is present on the membranes of complex systems such as multivesicular bodies ( figure 3c ) or on the outer membranes of cytosolic vesicles ( figure 3d ) . interestingly , calcium deposits are detectable on the outer cell membrane ( figure 3e ) in spots , localized and separated , suggesting the presence of calcium channels . in figure 3f , the end product of the dab photoconversion is a speckled , dense precipitate of varying size . however , another abundant end product is present as small sized dots , showing a markedly lower electrondensity ( figure 3f , inset ) and a widespread distribution .", "we show in this paper that mag - fura 2 photoconversion via dab oxidation is an efficient way for localizing ca ions at em level . moreover , this could be considered also as a means of stabilizing their presence , thus limiting the loss of ions . as for the method , photoconversion is easily carried out and reproducible , can be obtained on a good amount of cells , since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp . the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . in these conditions , the blackness of the product stands out from the background . in our conditions , in fact , ca ions are immediately detectable within the vesicles and the tubules of the ser , while it is much more difficult to see their presence when in the cytosol , due to their low electrondensity . this is a clear advantage over the conventional imaging of mag - fura 2 fluorescence that must be removed by the cytosol in order to study truly er signals . this is achieved by permeabilizing the plasma membrane with brief applications of antibiotics or detergents , but this procedure may destroy intracellular signaling pathways , dilute cytosolic modulators of er ca release / sequestration , and disassemble the cytoskeletal elements that maintain the contact between cell membrane and er . alternatively , the whole - cell configuration of the patch - clamp technique could be exploited to remove the dye , but this experimental approach is rather expensive and challenging , requires highly skilled electrophysiologists and has a quite low throughput . the final yield of photoconverted mag - fura 2 is in direct relation with the amount of ca present in the sample considered . in our condition , the ca - enriched medium gave a higher end product staining than the normal physiological conditions or than ca deprivation . it is interesting that in several cases , dense precipitates could be found in areas suggesting the local accumulation of ca nearby the mouth of plasmalemmal ca channels . accordingly , such precipitates are evident when extracellular ca concentration is increased , which is consistent with the notion that constitutive ca inflow from the extracellular milieu is enhanced . this unexpected outcome of dab photoxidation is extremely relevant in light of the master role served by local ca microdomains generated by extracellular ca influx , which regulate cell functions as diverse as cycle progression , proliferation , migration , nitric oxide production , and adenylyl cyclase activity . one thing that should be underlined here is that we could not visualize the ser tubule filled with the end product . this could be due to three different causes : i ) ca can be dispersed ( lost ) in the conditions used here ; ii ) local intraluminal ca concentration is less than expected ( which is less probable as it is unlikely to be lower than 100 m ) ; or iii ) dab photoconversion is not efficient enough for mag - fura . by comparing fluorescence images with em micrographs , it is clear the difference in the signal coming from vesicles and the elongated filamentous structures , the latter being much less fluorescent . the relative low yield when comparing fluorescence images is definitely related to the extremely lower amount of end product which can be found within a 60 - 80 nm thin section in relation to the fluorescence originating from the whole cell . in addition , albeit [ ca]er fluctuates between 100 and 500 m , ca accumulates in specific regions of the er that may be missed in our sections . moreover , releasable ca is actually trapped by oligomers of the ca - binding proteins calnexin and calreticulin : these form a concealed ca source that is not in rapid equilibrium with the [ ca]er , but is easily accessible to ca - releasing channels upon extracellular stimulation . thus , the lack of ser tubules or cisternae filled with the end product of the photo - conversion , i.e. , ca , is not surprising . finally , the photoconversion process seems to be efficient , and gives an end product more reasonable than the precipitation technique utilized , for instance , by tandler et al . in the latter case , an important amount of precipitates were found in the cell and especially in the nucleus ; our method gives a much finer end product , and this allows a superior resolution and localization . in conclusion , this method is a reliable and efficient technique for stabilizing and visualizing ca ions at em . it gives a high resolution end product thus allowing the fine structural ca localization . in principle , this approach could provide important qualitative information about the amount and sub - cellular distribution of calcium ions both in cultured cells and , more importantly , in ex vivo samples , which are far less amenable to high resolution ca imaging of er ca levels ." ]

[ "nearly , 90% of metastatic tumors occur in jaw bones , especially premolar - molar region of the mandible , whereas metastases to soft tissues are extraordinarily rare and account for 0.1% of all oral malignancies . most metastatic tumors to the orofacial region are seen in patients aged between 40 and 70 years . metastasis to the soft tissues mostly involves gingiva and alveolar mucosal sites ( 54% ) followed by tongue ( 30% ) . in some cases , oral metastatic tumors are found to be the first sign of metastatic spread and in some instances ; they are an indication for unidentified primary tumor of the distant site . the primary site differs according to oral site colonization . in men , primary tumor of the lung followed by the prostate gland and kidney the breast is the most common primary tumor site affecting the jawbones and soft tissues ( 41% and 24.3% , respectively ) , followed by the adrenal and female genital organs . clinically , these metastatic lesions appear nonaggressive , mimicking reactive or benign lesions or even simple odontogenic infections . the clinical features of these lesions include a bony swelling with tenderness , pain , ulcer , hemorrhage , paresthesia and pathological fracture . it is important to diagnose the origin of the tumor as the management will depend significantly on the histological type . although infrequent , oral metastases are rarely encountered as the first sign of occult cancer . hence , this article emphasizes on detailed dentoalveolar examination and early diagnosis for finding the primary focus of metastatic tumor .", "a 60-year - old male patient presented with a swelling in the right lower back teeth region of the jaw since 3 months [ figure 1 ] . the patient complaints of pain for 10 days , radiating to the right temporal region . the patient was a known hypertensive since 4 years . a well - defined solitary swelling present on the right side of mandible on extraoral examination , a well - defined , solitary swelling measuring 4 cm 5 cm was noted on the right side of the mandible , which was hard in consistency and fixed to the underlying skin . no other pathologic findings were noticed during physical examination . on intraoral examination , firm , nontender swelling with ill - defined borders measuring 3 cm 3 cm was noted in the right mandibular premolar - molar region [ figure 2 ] . there was no ulceration , paresthesia or pathological fracture . on intraoral examination , firm , nontender swelling with ill - defined borders measuring 3 cm 3 cm was noted patient was advised for oral pantamograph and computed tomography ( ct ) scan . ct scan of the face with three - dimension reconstruction revealed a well - defined osteolytic lesion involving the ramus and body of the right half of the mandible , measuring 54 mm 45 mm 50 mm ( anterioposterior transverse craniocaudal ) . it showed speculated periosteal lesion with multiple irregular calcifications and extended into the lingual and mandibular soft tissues [ figure 3 ] . based on the clinical presentation , a differential diagnosis of inflammatory swelling , odontogenic cyst , odontogenic tumor , soft tissue tumor or metastatic tumor to the jawbones was given . computed tomography scan of the face with three - dimensional reconstruction revealed a well - defined osteolytic lesion with multiple irregular calcifications that extended into the lingual and mandibular soft tissues incisional biopsy was done for the histopathological diagnosis . histopathological examination of the h and e stained tissue showed diffuse fibrous connective tissue stroma invaded by the tumor cells which were cuboidal to short columnar with eosinophilic cytoplasm arranged in alveolar / follicular pattern of varying sizes [ figure 4 ] . bony trabeculae were evident in some areas [ figure 5 ] . based on these features , the lesion was diagnosed as a metastatic malignant tumor , probably metastasizing from the lung . ( h&e stain , 100 ) tumor islands along with few bony trabeculae were evident in some areas . ( h&e stain , 40 ) x - ray chest posterior - anterior view revealed consolidation of the right lower zone [ figure 6 ] . the ct scan of the chest with contrast revealed dense soft tissue mass lesion with speculated margins and focal pleural thickening noted at the right posterior basal segment . x - ray chest posterior - anterior view revealed consolidation in right lower zone of the lungs treatment for the patient was palliative external beam radiotherapy on linac given to a total dose of 3000 cgy in 10 fractions to jaw by rl / ll for 12 days which was well - tolerated by the patient . chemotherapy included injection cisplatin ( 75 mg)-intravenous ( iv ) ; injection etoposide ( 150 mg)-iv ; tablet vomiset ( 8 mg ) ; tablet ultracet .", "the common primary sources of tumors metastatic to the oral region are the breast , lung and kidney . the actual incidence of these cancers is unknown since oral metastatic disease is usually a manifestation of advanced disease . the mean age of occurrence is 54 years with slight male predilection . in our case , also the patient was aged about the same age ( 60 years ) as supported in the literature . most of the patients are aware of primary tumors before the metastatic spread to the oral cavity . in our case , the most common primary sites for oral metastases were the lung , kidney , liver and prostate for men ; breast , female genital organs , kidney , and colorectum for women . in our case too , lung was found to be the primary site . the clinical presentation of the metastatic lesions differed between the various oral sites . in the jawbones , most of the patients complain of rapidly progressing swelling , pain , paresthesia , difficulty in chewing , dysphagia , disfigurement and bleeding . our patient presented with features such as firm , nontender swelling with ill - defined borders . there was no paresthesia , difficulty in chewing , dysphagia , bleeding and pathological fracture . pathogenesis of oral metastasis is unclear but thought to be a multistage process in which cells detach themselves from the primary tumor and get transported by lymphatic or blood vessels . recent studies state that cancer cells metastasizing to bone have shown to alter the physiologic balance between both bone resorption and bone formation . treatment of oral metastasis depends upon its presentation and the stage during which it has been identified . management includes chemotherapy , radiotherapy , surgical excision or a combination of the techniques under local anesthesia . an average survival time for lung cancer metastasis is 4 months to 1 year with a maximum survival rate of 5 years .", "", "" ]

[ "the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran . for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders . after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist . the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants , 19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group , finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group . the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group , none had a history of psychiatric disorders or developmental delay based on their parents report . exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines . students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study , all the students ( from both sexes ) with all adhd subtypes were participated as the study group . all cases in the study and the control group were matched based on age , sex and iq level . in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used . child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv . the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder . there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often . the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study , this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h . the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4 is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types . the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 . furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) . wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years . wisc - r gives two different scores for verbal and performance iq and a total iq scores . moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants . wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test , a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner . the pattern is a red triangle , green star , yellow cross and a blue circle . for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not . after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) . data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes . scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis .", "the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran . for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders . after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist . the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants , 19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group , finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group . the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group , none had a history of psychiatric disorders or developmental delay based on their parents report . exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines . students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study , all the students ( from both sexes ) with all adhd subtypes were participated as the study group . all cases in the study and the control group were matched based on age , sex and iq level .", "in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used . child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv . the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder . there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often . the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study , this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h . the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4 is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types . the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 . furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) . wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years . wisc - r gives two different scores for verbal and performance iq and a total iq scores . moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants . wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test , a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner . the pattern is a red triangle , green star , yellow cross and a blue circle . for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not . after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) .", "data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes . scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis .", "the study group consisted of 45 school aged children ; of whom , 27 were male and 18 were female . in the control group , there were 30 school aged children , of whom 18were male and 12 were female . other characteristics of the two groups ( age , iq , adhd subtype frequency ) are presented in table 1 . data analysis showed that adhd and normal group were significantly different in terms of perseverative responses , perseverative errors and total errors on wsct ( tables 2 and 3 ) as shown in table 4 , adhd subtypes were significantly different in terms of perseverative responses ( p 0/01 ) and perseverative errors ( p0/001 ) . based on scheffe s test , adhd - h was not different from adhd - i and adhd - c but there were significant differences between adhd - i and adhd - c in terms of perseverative responses and perseverative errors .", "the aim of the present study was to investigate the differences between adhd subtypes in terms of executive function profile . previous studies showed inconsistent findings in this regard . in an extended study , houghton et al . investigated the executive function in 94 children with adhd ( without any co - morbid disorder ) in terms of subtype and gender using five tests of the wisconsin card sorting test , the stroop color - word test , the matching familiar figures test , the trail making test , and the tower of london . they found that children with both types of adhd ( adhd - i and adhd - c ) differed from normal children in terms of perseveration and response inhibition , but this difference was significant in adhd - c , only ( 27 ) . the lack of any co - morbidity in children with adhd showed that the impairments in executive function were obviously found in adhd , particularly in the adhd - c , so providing support for barkley s theory of adhd ( 27 ) . consistent with this finding , in another study , klorman et al . found that children with adhd - c revealed more non - perseverative errors in wcst and solved fewer puzzles and also violated more rules on the tower of hanoi ( toh ) than adhd - i , but they were not different based on perseverative errors in wcst(28 ) . also , lawrence et al . their finding revealed that children with adhd were only different in set - shifting on wsct ( perseverative responses and errors ) ( 10 ) . in this respect , we found that children with adhd significantly differed from normal children in terms of total errors , perseverative errors and perseverative responses ( p 0.01 ) . also , we found that adhd subtypes operated differently in the executive function domain ( p 0.01 ) ; children with adhd predominantly combined type showed more perseverative responses and perseverative errors than children with adhd predominantly inattentive type . some researchers suggest that the two primary subtypes of adhd share similar neuropsychological weaknesses in inhibitory control , but there are subtype differences in response to success and failure that are contributed to a child s ultimate level of performance ( 36 ) . unlike the barkley s behavioral inhibition theory , song and hakoda found that both types of adhd ( adhd - i and adhd - c ) showed deficits in the inhabitation domain ( 24 ) . also , li et al . found noticeable cognitive impairments such as poor response inhibition , impaired working memory , dysfunction of planning and set - shifting in children with adhd , but there were no significant differences between the two subtypes of adhd in their study ( 12 ) .", "the first and most notable limitation of our study was that we only used one test to assess the executive function ; and the second limitation was that we did not directly evaluate the control group for psychiatric disorder and only relied on the parental report . our findings revealed that executive function patterns are different in children with adhd compared to normal children . our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition .", "our findings revealed that executive function patterns are different in children with adhd compared to normal children . our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition ." ]

[ "the visual environment provides a vast assortment of information to the sighted individual during the acts of daily living . for the visually - impaired individual , a need exists to compensate for the lack of this information , which has motivated the introduction of a wide variety of devices that transfer at least some visual information to another sense . however , the primary assistive technologies widely used today by the visually - impaired to navigate through the environment are essentially unchanged from those of twenty years ago , namely , white canes and guide dogs . although these two methods can facilitate the ability to travel safely in a variety of indoor and outdoor environments , neither provide the kind of assistance needed to straighten a picture frame on the wall or find a can of soup on a counter - top . electronic navigation aids are finding some acceptance , for example , ultrasonic canes ( ultracane from sound foresight technology , ltd . ) that provide tactile cues to objects beyond the tip of the cane , as well as portable computers with global positioning systems ( gps ) and electronic braille or speech interfaces . however , replacing the more general capabilities of vision to provide detailed information about objects in the environment has proven more difficult . there exists a need for a device that allows active interrogation and sensing of the 3d visual environment surrounding the operator while moving through everyday environments . there also exists a need for a device that not only allows the user to sense the environment but also provides control of specific aspects of the environment that have been detected . these abilities to interrogate and control the environment should not be limited to a specific , predetermined environment , such as those that already contain infrared ( ir ) transmitters . devices using a concept we call fingersight are intended to allow visually impaired users to remotely identify objects of interest in , and navigate through , natural environments , by acting as a sensory substitution system for the user 's sense of sight . there are already a number of technologies that monitor hand , eye , and body motion from a fixed or mobile camera to interpret gestures , commands , gaze direction , e.g. , . some of these may be used to track the operator 's interrogation of the environment , as with the commercially available eye - tracking glasses ( e.g. , tobii , tokyo ) , but more often they are used to interpret the user 's motions as commands . our original goal was , and largely remains , to serve the population who are visually impaired . most devices designed with this goal make use of sensory substitution systems , which transmit stimuli normally interpreted by one sense through another sense . sensory substitution systems generally consist of three components : a sensor that collects a signal , a coupling system that processes the signal , and finally an output transducer , which transmits the signal to a sense organ not normally used for that signal , . we specifically consider substitution for the visual system by the haptic system ( i.e. , one 's sense of touch ) . although hearing offers a potentially higher bandwidth than touch , it is crucial not to impede the existing use of hearing , which can be acutely well developed by those who lack normal vision , providing essential acoustic cues about the environment . as for touch , the hands offer the greatest versatility and sensitivity , but still we must not completely usurp their use , since they are essential for so many tasks in daily living . some employ tactile pin arrays , which feature a grid of small pins with adjustable heights . tactile pin arrays can be used for either passive stimulation of the fingers or during active exploration to simulate contact with surfaces and objects . existing systems capture an image and then display that image on a tactile screen that can be worn on a belt , for example . the fingers can then be used to interrogate the image depicted on the tactile screen in an effort to visualize the image , . some devices have been developed to directly convey a predetermined map from coordinates of physical space to a tactile spatial layout . an example is bach - y - rita 's brainport device , which uses an array of electrodes placed on the tongue to relay camera information . sensory substitution systems also exist that provide an audio output , such as text - to - speech devices that employ optical character recognition , canes that provide audio feedback to enhance the natural tactile forces detected at the tip itself , and the voice system , which maps image pixels into frequency and time . vibrotactile sensitivity is found in mechanoreceptors lying within the subcutaneous tissue , which respond maximally to frequencies of 250300 hz , as well as kinesthetic receptors . the cybertouch glove ( immersion corp . , san jose , ca ) uses optical tracking of each finger to determine when vibratory stimulators on that finger should be activated . some sensory aids , such as the optacon ( telesensory corp . , defunct ) , aim to simulate tactile exploration of virtual objects and have been developed specifically for reading the printed page . our proposed device uses a vibrotactile stimulator to provide visual information gathered from a color camera system . the information from the camera system can be processed into a single feature whose presence or absence is communicated through the stimulator . for example , the camera may provide a positive signal when an edge is detected in the field of view . our system is similar in some ways to that of lenay , who attached a photocell to the tip of a finger and used it to activate a vibrator held in the other hand in an all - or - nothing manner . another system , developed by burch and pawluk , is mounted on the same finger as the sensor , for interacting with graphical objects on a flat screen . this system senses a single rgb pixel at a short distance ( i.e. , to the screen that the subject is touching ) , and vibrates based upon the color sensed . our system goes beyond this basic idea by detecting not just the general directional presence of light or contact with a single light source , but rather actual images ( or a series of images over time ) using a miniature camera . like burch and pawluk we believe that this placement promotes an intuitive coupling between the scene pointed to by the camera mounted on the fingertip and the feedback felt by that finger . in general , it is imperative for designers of any sensory substitution system to consider not only what is technologically feasible , but also what is functional in the context of the sensory and cognitive limitations of the user . in a review of sensory substitution for the blind , loomis and klatzky pointed to the need to couple device design to the capabilities of the user for a given task . low bandwidth , for example , need not be a negative feature of a device if a task can competently be performed on the basis of the information provided . high bandwidth communication to the user is not an advantage if the target sensory channel can not process the information provided . furthermore , it should not be assumed that information - processing capacities of blind and sighted are equivalent ; for example , superior tactile sensory systems may be preserved in older braille readers relative to sighted populations of the same age , whereas tasks that draw on visual imagery may be impeded in a blind population without any experience of sight .", "we have developed a series of working prototypes , leading up to the one used in the experiments described in the present paper . as the progression is informative , we review them briefly here . our initial system permitted active interrogation of the visual surroundings with a miniature red laser attached to the fingertip ( see fig . 1 ) . the laser was modulated at 10 khz , allowing its reflection by objects in the environment to be detected by a non - directional phototransistor in the midst of other sources of light . thus , like the burch device , it sensed the light ( in this case reflected ) from a single point in space , but at a distance . moreover , it differed from that device by actively interrogating across the detected point for an edge . this was accomplished by means of a solenoid constructed from tiny magnets and a coil . regenerative feedback between the amplitude of the detected signal from the reflected laser and the solenoid caused the laser to vibrate vertically when the laser spot was located on any edge between light and dark objects . thus the system created a haptic stimulus ( vibration ) whenever a properly oriented visual edge was encountered by the laser . \n the laser - based system was limited to simple edges and had the disadvantage that the active visible light source could be disturbing to others . more importantly , limiting fingersight to what amounts to a single scanning line makes it extremely difficult to integrate more than the most rudimentary features in the visual field . a full video image captured at each pose of the finger offers many advantages , including immediate extraction of more complex features , identification of entire objects , and even determination of camera motion directly from the changing image . the recent availability of very inexpensive and small cameras , brought about largely by their use in cell phones , has led us to adopt a camera - based approach , the first version of which is shown in fig . 2 \n . a small black - and white camera ( supercircuits pc206xp 510492 ) was mounted on the subject 's finger along with a cell - phone vibrator . real - time analysis of the video signal from a miniature camera was used to control the cell - phone vibrator . the cell - phone vibrator has the advantages of low cost and size , but presents problems in that amplitude and frequency are inextricably linked together by the operating voltage . also , the frequency is low enough to cause noticeable vibration in the camera image , and the time for the motor to come up to speed is relatively long . \n two small speakers ( gui , inc . # gc0251k - nd , 1w , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 8\\omega$\\end{document } ) were converted into haptic vibratory stimulators , or tactors , by cementing a short wooden dowel onto the central cones of each speaker . the converted speakers were mounted on either side of the finger and held there , along with the camera , by a springed clip , leaving the palmar aspect of the fingertip bare to use for touching and grasping objects . we hoped , by including two such tactors , to be able to use the relative strength of the vibration from each tactor to convey a parameter such as location of an image feature along the horizontal axis . \n this model did not perform well for identifying the location of visual targets , primarily due to mechanical design issues . it proved problematic to mount the device securely on the distal phalanx of the finger . furthermore , given the influence of flexion / extension at the distal inter - phalangeal joint on the camera orientation , it was difficult for users to judge where they were aiming the device . in addition , there was low sensitivity to vibration the speakers mounted on the lateral aspect of the finger , as compared to the speaker on the medial aspect . collectively , these issues made any asymmetry in signal strength from the two tactors of little value to the user 's perception . the current version of fingersight uses the same speaker - based tactor shown in fig . 3 , but with only one tactor on the ( more sensitive ) medial aspect of the finger , along with a miniature color camera ( supercircuits pc208 , 510492 ) . the camera extended approximately 8 mm from the tip of the finger , and the speaker was attached to the side of the splint . the splint weighed 18.8 g and caused minimal interference with movement of the finger as a whole , though it did restrict flexion between the phalanges , thereby reducing uncertainty in camera orientation . covering the palmer aspect of the finger made it difficult to use the finger for other purposes , but we were willing to accept this for the present experiment . the camera and the speaker were connected to a computer system , which performed spatiotemporal edge detection ( described below ) and controlled vibrator output . the haptic feedback was provided by a low - frequency ( 20 hz ) audio signal sent to the speaker , the amplitude of which was adjusted to produce detectable vibrations . \n fig . 4.current fingersight device apparatus with camera and stimulator attached . \n for the purposes of experimental testing the area of the circle was divided across its center , one semicircle containing red and one containing green pixels ( see fig . 5 ) . the line across the center of the circle separating red from green pixels could be set to any desired orientation . the circular area was surrounded by white pixels and the images were projected on a screen , where the user could point the finger - mounted camera at it . \n 5.examples of edge stimuli , at 12 ( a ) and 4 ( b ) o'clock , respectively . \n software was developed to analyze the video image from the finger - mounted camera in real time and to control the vibratory feedback to that finger . the goal was to generate vibrations whenever the user 's finger was pointing at , or had just passed over , the boundary between the red and green areas , irrespective of the edge 's orientation . optical edge detection was performed with a novel spatiotemporal kernel , designed to detect edges both in the spatial domain ( when the camera was resting on an edge ) and the temporal domain ( when the camera had moved completely past an edge between one video frame and the next ) . this design accommodated fast hand movements , which might cause the edge to never actually have been within the kernel in any given frame , as well as slower continual tracking along the edge . the spatiotemporal kernel accomplished both of these for edges of any orientation by comparing each pixel in one time frame with the corresponding pixel directly across the center of a circular kernel in the next time frame . if the two pixels differed , an edge was present or had just been crossed . it is important to note that unlike the general usage of the term kernel in image processing , especially for convolution , our spatiotemporal kernel was applied not at every pixel , but only at one location , the center of the image . just as the fovea of the eye ( the high resolution central region ) is constantly moving for human vision to function , our kernel was physically moved by the user 's finger along with the entire image , to interrogate the visual environment . the optimal size of the circular kernel varied depending on the scale of the edge features to be considered . for the particular experiment described here , the optimal radius of the kernel was determined experimentally to be 50 pixels . a detailed formulation of the algorithm follows : we define k as the set of pixels within the circular kernel at the center of the image . based on the particular red - green - blue ( rgb ) video image coming from the camera in real time , a color label \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c(x , y)$\\end{document } was created for each pixel in k , denoting whether the pixel at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } was predominantly red , green , or ambiguous.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ c\\left(x , y\\right)=\\cases{+1/2,&$ if~predominantly~red$\\cr-1/2,&$ if~predominantly~green$\\cr 0,&$if~ambiguous$\\cr}\\eqno{\\hbox{(1)}}$$\\end{document } for our purposes , predominantly indicates that the red ( r ) or blue ( b ) color value for the pixel in question was above a specific threshold , while the other two color values in the rgb pixel were both below their corresponding thresholds . appropriate thresholds for red , green , and blue were determined experimentally , so that red , green , and white pixels ( which registered as ambiguous ) responded appropriately . we also created a binary mask value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w(x , y)$\\end{document } for each pixel that had the value of 1 wherever c was non - zero , so that boundaries including the white area surrounding the circle could be excluded from generating edges.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ w\\left(x , y\\right)=\\cases{1&$if~c\\left(x , y\\right)\\ne 0$\\cr 0&$otherwise$\\cr}\\eqno{\\hbox{(2)}}$$\\end{document } for each pixel in k , we also considered another pixel in k directly across the origin ( center of the kernel ) at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document}. our measure of boundary strength was calculated by comparing the value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } in the current image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t$\\end{document } to value of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t-1}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document } in the previous image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t-1$\\end{document}. the mask values \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t-1}$\\end{document } were used to ensure that no comparisons were made using ambiguous pixels . thus a measure of edge strength \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was computed as \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ d\\!=\\!\\!\\!\\sum_{\\left(x,\\!y\\right)\\in k}\\!\\!{\\left\\vert c_{t}\\left(x , y\\right)\\!-\\!c_{t-1}\\left(-x,\\!-y\\right)\\right\\vert w_{t}\\left(x , y\\right)w_{t-1}\\!\\left(-x,\\!-y\\right)}\\eqno{\\hbox{(3)}}$$\\end{document } if the edge measure \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was above some threshold ( determined experimentally ) , the software reported that an edge had been detected , and triggered the haptic feedback in the form of vibration at the fingertip . the algorithm has the advantage that it measures equally well the conditions of sitting on an edge ( of any orientation ) for two successive timeframes and having passed completely over the edge from one timeframe to the next . for example , in 3d one would simply compare pixels directly across the center of a spherical rather than a circular kernel . twelve sighted subjects ( 10 males and 2 female ) were recruited from the university population , with an average age of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 29.4+/-{9.8}~{\\rm years}$\\end{document}. all gave informed consent . subjects took part in a series of trials , in which they identified the angle of an edge stimulus according to a closed set of alternatives . each edge stimulus , as described above , was projected on a display screen and consisted of a circular area 115 cm in diameter on a white background bisected with one half colored red and the other green . the circle was surrounded by white , so that the perimeter of the circle would not itself be interpreted as an edge ( as described above ) . subjects were fitted with earplugs , blindfolded , and seated with the right arm resting on a foam support 79 cm above the floor . the circles were projected ( epson powerlite 70c ) on a 154115 cm screen positioned 168 cm in front of the subject 's fingertip . the arm support was arranged so that the subject 's arm and finger were initially pointing perpendicular to the center of the projected image . during each trial , the subject freely explored the display edge with the fingersight device as long as desired . generally , subjects maintained their arm on the rest and moved their forearm and wrist . angle set 1 consisted of six angles equally spaced over a 180 range in 30 steps ( from positive vertical , 90 , to just short of negative vertical , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { -}{60}^{\\circ}$\\end{document } ) , corresponding to clock - hours from 12 to 5 o'clock . angle set 2 consisted of seven angles equally spaced over a 90 range in 15 steps ( from positive vertical , 90 , to horizontal , 0 ) corresponding to the hours and half hours between 12 to 3 o'clock . subjects reported their responses using these clock face labels . within each set ,", "tables 1 and 2 show the confusion matrices for all subjects for angle sets 1 and 2 , respectively , plotting reported angle vs. stimulus angle . a confusion matrix shows correct measurements along the diagonal , with increasing error as one moves off the diagonal . overall , subjects had an accuracy rate ( proportion correct ) of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.77\\pm 0.05$\\end{document } on angle set 1 and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.52\\pm 0.09$\\end{document } on angle set 2 ( 95% confidence intervals ) . these levels are well above chance in this six - alternative choice task \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm chance}=0.16)$\\end{document}. \n table iconfusion matrix for set 1reported angle ( deg)90603003060stimulus angle ( deg)9071131011060168252003008843100101081313000068196040003260confusion matrix for angle set 1 . each entry is the number of occurrences of the associated stimulus / response pair . \n table iiconfusion matrix for set 2reported angle ( deg)0153045607590stimulus angle ( deg)0642532200151949242101301205519100450836391210600115234692750011533389900011112558confusion matrix for angle set 2 . each entry is the number of occurrences of the associated stimulus / response pair . \n the data were also analyzed by measuring information transfer \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , in order to determine the number of bits that can be transmitted by the device under the experimental conditions .\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i=\\sum_{j=1}^{k}\\sum_{i=1}^{k}{p\\left(s_{i},r_{j}\\right)\\log_{2}\\left({{p\\left(s_{i}\\vert r_{j}\\right)}\\over{p\\left(s_{i}\\right)}}\\right)}.\\eqno{\\hbox{(4)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ k$\\end{document } is the number of possible stimuli , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ s_{i}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ r_{j}$\\end{document } represent a specific stimulus - response pair . this statistic can be estimated from the confusion matrices according to equation 6.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i_{est}=\\sum_{j=1}^{k}\\sum_{i=1}^{k}\\left({{n_{ij}}\\over{n}}\\right)\\log_{2}\\left({{n_{ij}\\times n}\\over{n_{i}\\times n_{j}}}\\right)\\!.\\eqno{\\hbox{(5)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{ij}$\\end{document } is the number of joint occurrences of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } and response \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j , n_{i}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{j}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j$\\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document } is the total number of trials . relative to the 2.58 bits of information in the six - alternative choice task , angle set 1 ( angles with 30 increments ) had an information transfer of 1.62 bits , while angle set 2 ( angles with 15 increments ) had an information transfer of 1.12 bits .", "we have developed a new method of providing the finger with haptic feedback about visual targets in the general environment , by permitting the finger to scan the environment directly to locate those targets . we have developed this idea , which we call fingersight , through a series of prototypes , exploring the particular application of detecting edges . in the process , we have developed a new spatiotemporal \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document}-dimensional edge detection algorithm of potentially general interest , which is simultaneously capable of detecting the presence of a stationary edge as well as having passed over an edge between successive timeframes . we have conducted proof - of - concept experiments showing that under active exploration the fingersight device is capable of transmitting visual spatial information through haptic channels . sighted , blindfolded subjects were able to use the device to differentiate between angles separated by as little as 15. performance with the coarser angle set , where responses were separated by 30 showed that fingersight transmitted close to 2/3 of the available information in the stimulus , with most errors being near misses . the information transmission of the fingersight device is intrinsically constrained by basic sensorimotor abilities of motor control , kinesthetic sensing , and spatial cognitive processing . was found that when blindfolded , sighted adults tried to report the orientation of a raised line that was easily tracked by touch on the plane of a tabletop , their responses pulled the true value towards the sagittal axis by about 25% ( plus some constant error ) . since motor control was minimized in the task in , errors can be entirely attributed to the processes of kinesthetic sensing and building a spatial representation . the observed level of distortion is by itself substantial enough to lead to confusion errors in the present task . these observations suggest that performance with fingersight could be improved by features that support motor control and augment kinesthetic feedback . the present experiments measure only the performance of blindfolded sighted subjects who are novices using the device ; training would be expected to considerably improve performance . siegle and warren have shown that distal attribution , i.e. , the direct perception of objects in external space , can occur with similar finger - mounted sensory substitution systems after a period of training . however the device used in their experiments had the vibrotactile stimulator mounted on the subject 's back . it is possible that training time may be decreased with a device such as fingersight , where the stimulus is more strongly tied to the subject 's proprioceptive knowledge of the hand 's location in space . it is interesting to compare the accuracy of angular measurements by fingersight in 3d space with other researchers who restricted interrogation to a fixed plane . , explored the ability to recognize 90- or 180-degree rotation on raised - outline drawings directly touched by the blind or sighted - blindfolded subjects , but did not explore finer angular resolution . postma , et al . , demonstrated the role of visual experience in such haptic spatial tasks , including the description of angles between bars on a table , showing that blindfolded sighted subjects outperformed late blind subjects , who outperformed early blind subjects . they found that having experienced vision , even to a limited extent , helps in the interpretation of angle by touch alone . the worst performance in verbally judging the angle of the bars ( demonstrated by the early blind ) was 7.2 degrees , a good deal better than our errors , possibly because they were constrained to a tabletop . it would also be interesting to see if sighted individuals also have a similar advantage in learning to use fingersight over blind individuals , due to previous visual experience . also relevant to the question of angle and touch , with haptic computer mice , which are modified to have tactile pin arrays on their upper surface . significant lack of accuracy in the haptic position information , which is critical for individuals to haptically piece together a 2-d graphic . the inaccuracy is due to the fact that the tactile mouse ( or any normal mouse ) is a relative positioning device , dependent upon the speed of motion and orientation of the mouse to determine total displacement on the screen . in contrast , fingersight is inherently an absolute positioning device , given a stationary environment , and as such , fingersight may have an advantage . the system of burch and pawluk previously mentioned uses a fingertip photosensor and piezoelectric stimulator to scan specially created graphical displays , in which texture is added to enhance perception of edges and orientations . a single photosensor thus suffices for this purpose , only because preprocessing is performed to populate regions on either side of boundaries with differing textures . multiple photosensors on different fingers were found to improve results with this system , because the operator 's knowledge of the spatial relationship between the fingertips could be used to integrate the inputs . but still the approach relies on preprocessing the image to create textures , after which individual photosensors can be effective . with fingersight , we are exploring the unadulterated 3d environment , where depending on only a few photosensors is not sufficient . we benefit greatly by having a multi - pixel image at each timeframe . by further processing of the camera images , the fingersight device could be adapted to identify more complicated features than simple visual edges , perhaps even constituting an object recognition system for a blind operator . for example , it could be used to find a particular person in a crowd , or identify a door displaying a particular sign . the rapid advancement in computer vision algorithms , driven in part by the security and social networking industries , will provide ever more sophisticated capabilities . for example , determining camera motion and target depth from a image sequence could permit greater 3d integration of multiple perspectives and facilitate providing navigational cues to the blind operator , such as where the curbside is , whether the approaching stairwell goes up or down , or whether one is moving towards or away from the elevator . such analyses are not feasible for a single photosensor , but rather , they require an entire image . a common problem in any real - world computer vision application is the variability of lighting , and there are established techniques for solving this problem using such constructs as the illumination cone . for example , a recently developed time - of - flight ( tof ) 3d camera , the swiss ranger sr4000 ( mesa imaging , zuerich ) , can deliver a 176144 pixel image with each pixel reporting range up to 10 m with 1 cm accuracy . the present camera is roughly 7 \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm cm}^{3}$\\end{document } and is being integrated by at least one research group into portable devices for the blind . for some of these more sophisticated systems , it may become problematic to rely solely on vibrotactile feedback to the finger . the bandwidth of audio , especially combined with language , makes it an appealing option , though as noted above , one does not want to impede the natural use of auditory cues that are especially crucial for the vision impaired . however , intermittent use of verbal output by fingersight , and for that matter , verbal commands by the operator to the device , could prove extremely useful , while not impeding auditory cues form the environment . as noted above , it is imperative to test whether features added to the technology ultimately add to the functional capability of the user , given intrinsic limitations on human information processing . that said , one promising avenue is the incorporation of control capabilities into the fingersight device . one of our previous systems included the capability to control graphical objects on a screen . in this implementation , the location of a small white square on a screen is controlled by motion of the finger . the algorithm detects the square in the field of view of the camera , moving it on the screen to keep it constantly in the center of the camera image , while providing haptic feedback about whether the tracking system has locked onto the target . a variation on this system constrains the square to move along a straight line , simulating the action of a slide pot under the operator 's control . a further variation uses a small white triangle to simulate a knob , whose orientation is determined using standard computer vision techniques and subsequently controlled by rotation of the finger . clearly , such systems are not limited to actively controlling graphical objects on a screen , but could also identify inanimate objects such as a light switch or door latch . in such cases , remote control could still be achieved by motion of the finger once the target has been identified , using a separate control channel to turn on the light or lock the door . continued work on the control aspects of fingersight a final note is merited on the eventual miniaturization of a fingersight device such that it might be small enough to be worn like an artificial fingernail . the cameras themselves are almost small enough already , and the main considerations are power and communications . one can envision radio communication between a fingertip device and a pocket unit , much as wireless earphones and microphones communicate with cell phones now . such devices might be fully integrated into the everyday activities of the vision - impaired ." ]

[ "it negatively influences the child 's quality of life and is an important cause of absence from school and also a significant factor in health - related costs among children and adolescents . its prevalence varies greatly from 5.9 to 82% , depending on the definition criteria and the age of the patients . the international headache society classifies headaches into primary and secondary type in classification of headache disorders 2 edition ( ichd - ii ) . primary headache is not attributed to any other disorder . in the majority of patients with primary headache , general and neurological examinations are normal and primary headaches include migraine , tension - type headache , cluster headache , autonomic cephalalgias and other primary headache disorders . frequency of headache increases with age in this population . in the present study we aimed to determine epidemiologic characteristics and compare socioeconomic and some clinical factors related to primary headaches in children / adolescents ( 4 - 15 year olds ) presented to neurology clinics of tabriz university of medical sciences .", "the institutional review board at tabriz university of medical sciences approved the study protocol . after obtaining a written informed consent from parents , children with 4 - 15 years of age and with the diagnosis of primary headache ( migraine or tension - type headaches ) who presented to the neurology clinics affiliated to tabriz university of medical sciences , tabriz , iran from march 2009 to october 2011 were included in this cross - sectional study . a total of 190 patients fulfilled the criteria for enrollment in the study and their demographics were collected . we noted the type of headache , history of atopy , asphyxia during labor ( according to the history of admission in the neonatal intensive care unit after birth due to respiratory problems based on the parents report and review of the previous medical records ) and breast feeding , family history of headache , presence of headache triggers and the socioeconomic status of the family . diagnosis of migraine and tension - type headache was based on the international headache society diagnostic criteria for the primary headache disorders . socioeconomic status was determined according to the total monthly family income ( < 7,000,000 rl vs = 7,000,000 rl ) based on the index of statistical center of iran for grand cities in the years of study in addition to the highest level of education of the father ( < or = high school vs. > or = college ) . statistical analysis was conducted with spss , version 16.0 for window ( spss , chicago , il ) . data were expressed as meansd for quantitative variables and as numbers and percentages for categorical variables . statistical analysis was performed using the chi - square test for categorical variables and the student 's t test for numerical variables .", "eighty eight patients ( 46.3% ) had migraine and 102 patients ( 53.7% ) had tension - type headache . the distributions of gender and age groups in patients with migraine or tension - type headache are shown in table 1 . \n the distributions of gender and age groups in patients with migraine or tension - type headache data are presented as numbers no significant differences were seen in the distribution of gender between the patients with migraine and tension - type headache . migraine and tension - type headache had comparable frequency in different age groups of male patients ( p=0.1 and 0.07 , respectively ) . in female patients the frequencies of both migraine and tension - type headache were significantly higher in the age group of 10 - 15 years as compared to the age group of 4 - 9 years ( p=0.03 and 0.04 , respectively ) . there was no significant differences regarding the prevalence of migraine or tension type headache between different genders in the same age group . the most common clinical presentations of aura in patients with migraine are demonstrated in table 2 . the comprehensive data regarding the epidemiologic characteristics of patients with migraine or tension - type headache in our study group are shown in table 3 . \n the clinical presentations of aura in patients with migraine the characteristics compared between patients with migraine and tension - type headache as triggering factors for headache ; or : odds ratio ; ci : confidence interval history of asphyxia during labor was more prevalent among patients with migraine as compared to the tension - type headache ( p=0.007 , or = 3.24 ) . history of headache in parents was also significantly higher in patients with migraine as compared to tension - type headache ( p=0.001 , or = 3.19 ) . moreover , the prevalence of atopy was significantly higher in patients with migraine ( p=0.01 , or = 5.70 ) . prevalence of feeding with formula during infancy did not differ between the patients with either migraine or tension - type headache ( p=0.9 ) . the low socioeconomic status was more prevalent in patients with migraine headache ( p=0.003 , or = 2.38 ) .", "in our study primary headache was more common in older age group which is consistent with danold 's findings which showed that frequency of headache increases with age . tension type headache in patients was more frequent than migraine which is comparable with findings of kroner - herwig et al . the main finding in the present study was that gender can not predict the type of primary headache in younger children , as no gender differences were seen between the patients with migraine or tension - type headache in the age group 4 - 9 years . this finding is in contrast to previous report by krner - herwig et al which showed that being female was a significant predictor for three main subtypes of primary headaches a few other studies showed that the prevalence of migraine was higher in the males . our results also demonstrated that the frequencies of migraine and tension - type headache were comparable in different age groups of male patients , whereas female patients in the age group 10 - 15 years had higher frequency of both types of headache especially migraine as compared to the age group 4 - 9 years . this finding reveals that female adolescents had higher risk for developing primary headache specially migraine as compared to female children . these results agree with those reported by krner - herwig et al who showed that children with migraine were significantly older than children in the other groups . it could be due to hormonal effect as the trigger of migraine headache in the female adolescents . it was previously shown that primary headache in children is closely related to history of headache in their parents . our results demonstrated that when a parent showed combined headache , the risk of developing migraine in his or her child is more conspicuous as compared to the tension - type headache . this finding is consistent with previous reports by fallahzadeh et al and krner - herwig et al which showed a stronger association between family history of headache and being afflicted by migraine as compared to tension - type headache in children . our study also demonstrated that if the patients with history of peripartum asphyxia would develop primary headache it would more probably be of migraine type . these results partly agree with those reported by maneyapanda et al who found an association between chronic migraine headache and significant perinatal insult due to neuroplasticity and its induced hyperexcitability . however , it is recommended to design prospective studies to compare the frequency of primary headache ( either migraine or tension - type headache ) in patients with or without perinatal insults to elucidate the role of peripartum asphyxia as a risk factor for developing primary headaches . we also showed that atopy is more prevalent in patients with migraine as compared to tension - type headache . also ozge et al found an association between atopy and both migraine and episodic tension - type headaches . it has been shown that alteration in several interleukins occurs in migraine which supports an association between migraine and atopic disorders . although it was shown by pogliani et al that breast feeding has a protective role against primary headache disorders , our results showed that children and adolescents with history of formula feeding during infancy have comparable prevalence of developing either migraine or tension - type headache . our results showed that socioeconomic status of the patients could significantly influence the frequency of migraine in comparison to tension - type headache . ayzenberg et al in a study on 2725 adults in russia , showed that low socioeconomic status is associated with higher frequency of primary headache disorders . moreover , katsarava et al in another study on 1145 adult patients in the republic of georgia showed that low socioeconomic status and female gender are risk factors for development of migraine , not tension - type headache . moreover , the possible role of psychological factors and behavioral characteristics influencing primary headaches could have been considered .", "while some variables have been checked by the present report , further prospective studies with larger sample sizes are needed to provide more comprehensive data regarding associated factors in childhood primary headache .", "" ]

[ "a total of 30 mabs that were either developed or in - licensed by companies are currently undergoing evaluation in late - stage studies ( tables 1 and 2 ) . five of these ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) transitioned into phase 3 studies during the period from late 2012 to april 2013 . brodalumab ( amg827 , amgen ; khk4827 , kyowa hakko kirin ) , a human igg2 targeting the interleukin ( il)-17 receptor , is undergoing evaluation in three phase 3 studies ( amagine-1 , -2 , and -3 ) of patients with psoriasis . the amagine-1 study ( nct01708590 ) is evaluating the efficacy , safety , and effect of withdrawal and retreatment with brodalumab in patients with moderate - to - severe plaque psoriasis . in this study , either of two doses ( 140 mg or 210 mg ) or placebo is administered subcutaneously ( sc ) every two weeks until week 12 , when patients are rerandomized to placebo or continued treatment . the estimated primary completion date for the study is march 2014 . in the amagine-2 ( nct01708603 ) and amagine-3 ( nct01708603 ) studies , the efficacy and safety of induction and four maintenance regimens of brodalumab compared with placebo and ustekinumab in patients with moderate - to - severe plaque psoriasis is being evaluated . these studies have estimated primary completion dates of august and september 2014 , respectively . note : table compiled from information available as of april 25 , 2013 . abbreviations : cd , cluster of differentiation ; il , interleukin ; pcsk9 , proprotein convertase subtilisin / kexin type ; sle , systemic lupus erythematosus . note : table compiled from information available as of april 25 , 2013 . * in - licensed ; national cancer institute is sponsoring the phase 3 study in hairy cell leukemia patients . abbreviations : all , acute lymphoblastic leukemia ; cll , chronic lymphocytic leukemia ; hgf / sf , hepatocyte growth factor/ scatter factor ; nhl , non - hodgkin lymphoma ; nscl , non - small cell lung ; vegfr2 , vascular endothelial growth factor receptor 2 . a first phase 3 study ( nct01767857 ) of mabp1 ( xilonix , xbiotech , inc . ) , a human mab targeting il-1 , was recruiting patients as of march 2013 . the study will evaluate overall survival of metastatic colorectal cancer patients with cachexia who are administered the mab as a monotherapy . the effects of mabp1 administered intravenously ( iv ) every two weeks , plus best supportive care will be compared with those of megestrol acetate administered daily plus best supportive care . the difference between the study arms in median overall survival from baseline to 18 mo will be compared . the recombinant immunotoxin moxetumomab pasudotox ( cat-8015 , astrazeneca ) is undergoing evaluation in a phase 3 study ( nct01829711 ) of patients with relapsed / refractory hairy cell leukemia . the study is sponsored by the national cancer institute ( nci ) ; the mab is currently licensed to astrazeneca . in 2004 , nci licensed moxetumomab pasudotox to genencor , inc . the mab was then acquired in 2005 by cambridge antibody technology , which was itself acquired by astrazeneca in 2006 . moxetumomab pasudotox comprises a mouse disulfide stabilized variable fragment with the variable heavy domain fused with a 7-mer linker and the pseudomonas aeruginosa exotoxin a pe38 fragment . in the phase 3 study , patients will receive 40 g / kg of the immunotoxin administered iv over 30 min on days 1 , 3 , 5 of each 28 d cycle until complete response , progressive disease , initiation of alternate therapy or unacceptable toxicity is observed . initiated in march 2013 , the study s estimated primary completion date is december 2014 . two phase 3 studies of tildrakizumab ( sch900222/mk-3222 , merck ) , a humanized mab targeting the p19 subunit of il-23 , are recruiting patients . initiated in december 2012 , nct01722331 is a 64-week study to evaluate the efficacy and safety / tolerability of sc administration of tildrakizumab in patients with moderate - to - severe chronic plaque psoriasis . patients receive 100 mg or 200 mg tildrakizumab at week 0 and week 4 , and then every 12 weeks until study end or participant discontinuation . patients receive the same doses of tildrakizumab , but the study includes 50 mg etanercept as well as placebo as comparators . the two phase 3 studies have estimated primary completion dates of june and july 2015 , respectively . rilotumumab ( amgen ) is a human igg2 targeting human hepatocyte growth factor / scatter factor ( hgf / sf ) that blocks binding of hgf / sf to its receptor met , thereby inhibiting the met signaling pathways . the mab is undergoing evaluation in a phase 3 study ( nct01697072 ) of chemotherapy ( epirubicin , cisplatin and capecitabine ) with rilotumumab or placebo for untreated advanced met - positive gastric or gastresophageal junction adenocarcinoma . rilotumumab or placebo is administered iv at doses of 15 mg / kg every 21 d. the primary outcome measure is overall survival in a time - frame of three years . initiated in october 2012 , the estimated primary completion date for the study is december 2015 .", "recently announced results for three late - stage studies of two mabs for cancer ( farletuzumab , naptumomab estafenatox ) and tabalumab , a mab treatment for autoimmune disorders , indicate that clinical endpoints were not met . farletuzumab ( morab-003 , morphotek / eisai ) is a humanized igg1 mab that targets human folate receptor , which is overexpressed in most epithelial ovarian cancers , as well as some forms of endometrial , breast , renal , lung and colorectal cancers . the primary endpoint of progression - free survival ( pfs ) was not met in the phase 3 far-131 study ( nct00849667 ) of farletuzumab in combination with carboplatin and a taxane in patients with platinum - sensitive epithelial ovarian cancer in first relapse . patients received placebo or farletuzumab at either 1.25 mg / kg or 2.5 mg / kg weekly for ~six cycles . although the primary endpoint was not met , a trend toward improved pfs was observed in some patient subsets in a post - hoc analysis . the safety and efficacy of farletuzumab in combination with a platinum containing doublet in chemotherapy - nave subjects with stage iv adenocarcinoma of the lung are being evaluated in a phase 2 study ( nct01218516 ) . as announced in january 2013 by active biotech ab , initial results of a phase 2/3 study ( nct00420888 ) of naptumomab estafenatox ( abr-217620 , anyara ) in combination with interferon as a treatment for advanced renal cell carcinoma indicated that the study did not achieve its primary clinical endpoint of overall survival in the intention - to - treat cohort . a confounding factor was the presence of high levels of pre - formed anti - drug antibodies found in a majority of patients in this , but not previous , studies . a doubling of pfs and overall survival occurred in the 25% of patients who had low or normal levels of base - line il-6 and expected levels of anti - drug antibodies . naptumomab estafenatox is composed of an antigen - binding fragment ( fab ) that targets metastasis - associated 5t4 fused to a mutated form of staphylococcal enterotoxin a ( sea / e-120 ) . phase 3 studies of tabalumab ( ly2127399 , eli lilly and co. ) as a treatment for ra have been discontinued due to lack of efficacy . tabalumab , a human igg4 targeting b - lymphocyte stimulator , was being evaluated in five phase 3 studies of ra patients . tabalumab in combination with bortezomib and dexamethasone is also being evaluated in a phase 2/3 study ( nct01602224 ) of patients with previously treated multiple myeloma .", "historically , ~50% of mabs in the commercial clinical pipeline have been studied as cancer agents ; however , ~67% of the current cohort of mabs at phase 3 is in studies for non - cancer indications ( table 1 ) . these 20 mabs are human or humanized , with more than half ( 55% ) targeting an interleukin or an interleukin receptor . the mabs are being developed as treatments for immunological disorders such as ra , sle , ms and psoriasis , but also for hypercholesterolemia , cachexia and alzheimer disease . reflecting the overall greater challenge of demonstrating safety and efficacy in cancers , the molecular types of the 10 mabs in late - stage studies of cancer ( table 2 ) are more diverse than those for non - cancer indications . of particular note is the inclusion of a toxin in three molecules ( naptumomab estafenatox , moxetumomab pasudotox , inotuzumab ozogamicin ) and use of less than a full - length sequence for three molecules ( naptumomab estafenatox , moxetumomab pasudotox , onartuzumab ) . the majority ( 70% ) of the 10 product candidates are in studies of solid tumors , with the remainder undergoing evaluation as treatments for hematological cancers . the number of anti - cancer mabs at phase 3 is likely to increase in the next 35 y as more adcs move into and through the clinical pipeline . \n mabs will continue to track the progress of antibody therapeutics in clinical study throughout 2013 , and we look forward to reporting on results that may be released during the year in which are the antibodies to watch in 2014 ?" ]

[ "they may be seen in midline or para - axial location , from the brain to the sacral area . they display a particular tendency to locate in the sacrococcygeal and pre - sacral regions . reported incidence of tumors at these sites the oropharyngeal cavity is exceedingly rare for teratomas , 2% of all teratomas , present as large masses protruding from the oropharyngeal area . though oropharyngeal teratomas have a benign histopathology , they are potentially lethal as they may cause airway obstruction and respiratory compromise . so , principles of management in such patients comprise of the immediate postpartum establishment of a secure airway , if needed via tracheostomy and complete surgical resection of the mass .", "a 1980 g female infant born to 22-year - old mother after 38 weeks gestation presented with a growth in oral cavity associated with respiratory distress . the mother had undergone ultrasonography examination during pregnancy , but the report of the same was unavailable . on examination , cleft palate was present and a palatal tumor of size 4 cm 3 cm was found in the cleft area [ figure 1 ] . it was hampering feeding . computed tomography scan showed mass lesion in the palatal region ( attached to the palate , more on the right side ) . no intracranial extension [ figure 2 ] . computed tomography scan image after due preparation , the patient was operated , and the mass was excised in toto [ figure 3 ] . histopathologic examination revealed a teratoma composed of mature glial tissue , choroid plexus , glands lined by respiratory type , columnar mucin secreting , and melanin pigment bearing epithelium . pools of myxoid matrix bearing physaliferous cells are present ( notochord like / chondroid areas ) [ figures 4 and 5 ] . microscopic picture 1 microscopic picture 2 following excision , patient recovered faster and gained weight . now , the patient is under routine follow - up , and palatoplasty has been done .", "teratomas are the tumors which contain tissues derived from all three germ layers . these contain tissues foreign to the anatomical site of origin . most of the teratomas in the pediatric age group are benign , but reports of malignant teratoma do exist . basic histological classifications which are widely used ( arnold 's system ) are : \n dermoid tumors : these are composed of ectoderm plus mesoderm . this is the most common form of teratomateratoids : these are poorly differentiated tumors and contain all three germ layersteratomas : these also contain tissue from all three germ layers . they are histologically more identifiableepignathus : these are tumors which contain fully developed organs and appendages . the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios . this is the most common form of teratoma teratoids : these are poorly differentiated tumors and contain all three germ layers teratomas : these also contain tissue from all three germ layers . they are histologically more identifiable epignathus : these are tumors which contain fully developed organs and appendages . the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios . other imaging technique such as magnetic resonance imaging , is useful in finding out the anatomical relationship of these tumors . clinical differential diagnoses of oropharyngeal teratoma are cystic hygroma , lymphangioma , duplications and neuroblastoma . moreover , the histopathological differential diagnosis includes hamartoma , dermoid cyst , and a heterotopic gastrointestinal cyst . with growth , oral teratomas that is why , the respiratory distress may be less . the neonates with oral teratomas the basic principle of management of oropharyngeal teratomas is the establishment and maintenance of a secure airway . if an oropharyngeal teratoma is diagnosed antenatally , elective delivery or lower segment cesarean section is carried out . if orotracheal / nasotracheal intubation is failed / or difficult , tracheostomy must be done . surgical goal is to remove the complete mass in toto , as a residual tumor may cause recurrence . associated cleft palate can be repaired when the child grows up , that is , at the age of 1 years . during follow - up , afp should be monitored . in our case , all the afp levels were within normal range . early diagnosis , the establishment of the good airway , complete excision of tumor and timely follow - up should increase the survival of newborns with oral teratomas .", "", "" ]

[ "the rationale underlying treatment recommendations based on esthetic impairment come from the social science researches , which indicate that unacceptable dental appearance may stigmatize , impede career advancement and peer group acceptance , encourage negative stereotyping , and have a negative effect on self - concept . orthodontic problems are usually not associated with grave mortality or morbidity ; hence , they tend to be overlooked by most health professionals as less important . however , studies reveal that malocclusions have a significant impact on the psychosocial health of the affected individual . for many years , epidemiologic studies of malocclusion suffered from considerable disagreement among investigators , especially regarding how much deviation from the ideal should be accepted within the bounds of normal . the orthodontic treatment need in the study group was assessed by esthetic component ( ac ) of the index of orthodontic treatment need ( iotn ) . it is being widely used in dental epidemiology to prioritize orthodontic treatment and seeks to quantify the likely sociopsychological effects of each patient 's malocclusion . the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling .", "the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling .", "the sample comprised 753 patients ( 360 females and 393 males - table 1 ) irrespective of gender , caste , creed , and socioeconomic status essentially fulfilling the following criteria : \n 2025 years old [ table 2 ] \n no history of previous / current orthodontic treatmentpresence of all permanent teeth with / without third molarsno large restorationsinformed consent from patients for participation as part of the survey . \n 2025 years old [ table 2 ] \n no history of previous / current orthodontic treatment presence of all permanent teeth with / without third molars no large restorations informed consent from patients for participation as part of the survey . the division of sample according to gender division of the subpopulation according to the age patients were examined clinically using mouth mirror and explorer . ac was assessed by comparing and matching digitally clicked intraoral frontal view photograph in occlusion to the nearest resemblance on standardized photographs of iotn . number 1 is the most , and 10 , the least attractive arrangement of teeth . the anterior teeth were graded in their dental attractiveness as seen and no endeavor was made to predict the future appearance of the dentition . esthetic component of the index of orthodontic treatment need scale ( photographs 110 ) the ac of iotn has commonly been used to evaluate the treatment need on esthetic grounds by dentists ( operator - rated ) or patients ( self - rated ) . both patients and orthodontists can also assess the ac with the intent of removing the influence of parents and patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity . esthetic component grades all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment . this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment", "all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment . this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment", "malocclusions may range from the severe , rather uncommon conditions such as cleft lip and palate to the more common irregularities of teeth resulting from biological variations . management of malocclusion is contained by a number of disciplines in dentistry , primarily orthodontics and attempts to prioritize treatment have used the measure of iotn , which relates to treatment need rather than its complexity . a sample of 753 patients from among those reporting to the department for a routine dental check - up was considered . the age group of 2025 years old was chosen because by then , the patients would be in a permanent dentition stage . at this age , there are less individual variations in dental age and occlusal development . the ac scores were recorded by the orthodontist unlike other studies where the patients self - assessed with the intent of removing the influence of patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity . 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and 8.7% proved to have a definite need for orthodontic treatment [ figures 4 and 5 ] . esthetic component scores inference of esthetic component grading in evaluating the ac in a sample population in shiraz , 91.3% of the population was in no need for treatment , 2.44% in moderate need , and 6.21% in great need for treatment . on the contrary , 22.8% of the malaysian children were found to have a definite need according to ac in a study conducted by abdullah and rock in 2001 . analysis of registrations of the ac of the iotn should be interpreted with some caution . it has shortcomings , for example , the lack of photographs showing hypodontia , anterior spacing , and a class ii division 2 dentition . there is always some uncertainty in assessing the photographs of different types of malocclusion to identify one which corresponds to one 's own dental appearance . kok et al . , ( 2004 ) suggested that concern about a malocclusion is not closely related to the severity of the malocclusion in terms of esthetics as measured by the iotn - ac . 2002 suggested that the 10 photographs should be complemented with photographs of patients from other ethnic backgrounds . these studies not only provide a global perspective of high orthodontic treatment need , but also confirm a persistently high orthodontic treatment need over the past decade . in some european countries , general dentists and orthodontists have been using indices of treatment need to prioritize state - funded orthodontic treatment for children with major irregularities . in addition , the demand for orthodontic treatment has increased in contemporary setting due to increasing awareness and perceptions resulting in extensive waiting rolls . it is essential , therefore , that health authorities should carefully prioritize and plan the provision of orthodontic treatment . the introductions of easily comprehensible , reproducible , valid , and reliable indices of therapeutic need such as the ac of iotn have allowed improved focusing of services", "\n a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage . the data collected can help for worldwide comparisons with other populationsthe ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department . \n a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage . the data collected can help for worldwide comparisons with other populations the ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department .", "", "" ]

[ "in order to proliferate , cells must accurately transmit their chromosomes from one generation to the next . in eukaryotes , the chromosomes are confined to the nucleus , the perimeter of which is defined by the nuclear envelope . the nuclear envelope is made of a double membrane ; the outer nuclear membrane is continuous with the endoplasmic reticulum ( er ) and contains many of the same proteins . the inner nuclear membrane contains a distinct set of proteins , some of which interact with chromatin . the outer and inner nuclear membranes are fused at sites of nuclear pore complexes ( npcs ) , macromolecular structures that allow selective passage of proteins , rna and other molecules to and from the nucleus . in most eukaryotes , underlying the inner nuclear membrane is a network of proteins , called the nuclear lamina , made of lamins and lamin - associated proteins . this network provides rigidity to the nucleus and contributes to chromatin organization and other nuclear processes . interestingly , neither plants nor fungi have a lamin - based nuclear lamina , although other structures may serve a similar purpose . of note alterations in nuclear shape and size are characteristic of aging and certain disease states , such as various types of cancer . thus , understanding how nuclear shape is regulated and identifying genes that contribute to nuclear morphology are of interest from both a medical and a basic biology standpoint . the nuclear envelope not only serves as a diffusion barrier but also as a physical barrier separating the chromosomes from cytoplasmic structures . in most animal cells these cytoplasmic structures include the centrosomes , which function as microtubule organizing centers that nucleate spindle microtubules necessary for chromosome segregation . however , just before mitosis , the nuclear envelope stands between these microtubules and the chromosomes . different cell types have adopted different strategies to facilitate the access of chromosomes by microtubules : at one end of the spectrum are cells of most metazoans , in which nuclear envelope components , including the two nuclear membranes , the npcs and the nuclear lamina with their associated proteins , disperse during each and every mitosis ( fig . this type of mitosis is called open mitosis , and it necessitates the reassembly of the nuclear envelope around a full complement of chromosomes in each of the daughter cells once chromosome segregation is completed . parenthetically , the mechanism that ensures the formation of only a single nucleus at the end mitosis , as opposed to several nuclei each encompassing a subset of chromosomes , is not known . at the other end of the spectrum are certain fungi in which the centrosome equivalents , called spindle pole bodies , are either permanently embedded in the nuclear envelope ( such as in budding yeast ) or are embedded in the nuclear envelope prior to mitosis ( such as in fission yeast ) . these cells undergo what is called closed mitosis , which occurs without nuclear envelope breakdown , as spindle microtubules that assemble within the nucleus can readily access the chromosomes . however , during closed mitosis , the nucleus must elongate in a manner that is coordinated with chromosome movement as chromosome segregation takes place ( fig . the chromatin ( blue ) is contained within the nuclear envelope ( light green ) . as cells enter mitosis , the nuclear envelope disassembles , allowing spindle microtubules ( purple lines ) nucleated by centrosomes ( purple spheres ) to align the chromosomes on the metaphase plate . ( b ) closed mitosis . shown is mitosis as it occurs in s. cerevisiae . the spindle pole body ( purple ) is embedded in the nuclear envelope throughout the cell cycle . after spindle pole body duplication , an intra - nuclear spindle is formed ( s. cerevisiae chromosomes do not condense enough to visualize individual chromosomes or a metaphase plate ) . during anaphase , the nucleus elongates and the nuclear envelope expands as the sister chromatids move away from each other . ( c ) functionally open , structurally closed mitosis . a term defined by sazer to indicate a breakdown in the diffusion barrier while maintaining an intact nuclear envelope . shown is mitosis as it occurs in aspergillus nidulans . as in s. cerevisiae , during interphase the spindle pole body is embedded in the nuclear envelope . as cells enter mitosis the nuclear pores ( dark green ) partially disassemble , significantly reducing the diffusion barrier between the nucleus and cytoplasm . as in closed mitosis , the bulge in the center of the elongated nucleus during anaphase contains the nucleolus , which is left behind during chromosome segregation . schizosaccharomyces japonicus cells form an intra - nuclear spindle as in cells undergoing closed or partially open mitosis . however , during anaphase , the nuclear envelope ruptures as the nucleus elongates without nuclear envelope expansion . the historic definition of open vs. closed mitosis was based on cytology : during open mitosis the nuclear envelope disappears , opening the nucleus and exposing its content to the cytoplasm , while in closed mitosis the nuclear envelope remains intact . from a functional standpoint , the presence or absence of a nuclear envelope affects at least two processes : the free diffusion of molecules between the cytoplasm and the nucleus ( which occurs during open , but not closed , mitosis ) and the presence of a physical barrier between the chromosomes and cytoplasmic structures ( which exists in closed , but not open , mitosis ) . over the years , mitotic divisions that fall somewhere in between open and closed have been described , warranting a brief discussion on how these forms of mitosis should be defined . in aspergillus nidulans , which was originally defined as undergoing closed mitosis , npcs partially disassemble as cells enter mitosis , disrupting the diffusion barrier between the nucleus and the cytoplasm , while maintaining a physical barrier between the chromosomes and cytoplasmic structures ( fig . a breakdown in the diffusion barrier also occurs during a brief period in anaphase of meiosis ii of the fission yeast schisosaccharomyces pombe . unlike the situation in a. nidulans , however , the change in permeability in s. pombe meiosis ii occurs at only a short window of nuclear division and is not accompanied by changes in either npc composition or nuclear membrane integrity . this led sazer to define this type of division as functionally open but structurally closed , a term that can also be applied to a. nidulans mitosis . other types of mitosis involve a partial opening in the nuclear membrane itself . in schisosaccharomyces japonicas , the nuclear envelope does not expand during mitosis , nor does it disassemble . as a result , the nuclear envelope ruptures as the intra - nuclear spindle elongates ( fig . interestingly , rupturing is not a result of forces exerted by the spindle , but rather a programed cell cycle event , the regulation and mechanism of which remain to be discovered . in the early embryonic divisions of the nematode caenorhabditis elegans the nuclear envelope is breached in pro - metaphase , allowing diffusion of cytoplasmic proteins into the nuclear space . a similar phenomenon is seen during drosophila syncytial divisions , where the nuclear envelope displays a partial disassembly of nuclear pore complexes and fenestrations near the centrosomes , thereby allowing microtubules to access the chromosomes . live cell imaging suggests that just as in closed mitosis , the nuclear envelope during drosophila syncytial divisions expands as the spindle elongates . in these three cases , however , mitosis is not structurally closed , as nuclear envelope integrity is breached and cytoplasmic structures , such as microtubules , can enter the nucleus . consequently , the term functionally open but structurally closed may not fully apply when describing these types of mitosis , and several studies have previously referred to these mitoses as semi - open mitosis . we propose to use partially open mitosis as term that encompasses all forms of mitosis that are neither fully closed nor fully open , but that exhibit a change in the diffusion barrier at some point during mitosis that may or may not be accompanied by visible structural change to the nuclear envelope . among unicellular eukaryotes there are many variations of nuclear envelope behavior during mitosis ( for more examples see refs . 16 and 17 ) , and undoubtedly additional forms of mitosis still remain to be discovered . it is likely that the various types of mitosis have evolved to adapt to a particular environment , although it is currently not known what kinds of selective pressure shaped the different modes of mitosis . a possible driving force is the position of the centrosome , namely in the cytoplasm vs. embedded in the nuclear envelope , which likely coevolved with nuclear envelope behavior . in addition , the evolution of mitosis could have been affected by the need to eliminate rate - limiting diffusion barriers between the nucleus and the cytoplasm as occurs during open and partially open mitosis , but is either not a problem or is somehow overcome in closed mitosis . it is conceivable that , during syncytial divisions , partially open mitosis evolved to overcome the diffusion barrier and microtubule accessibility problems , while protecting chromosomes from aberrant attachments to microtubules from neighboring nuclei . the evolution of different forms of mitosis remains an unresolved , yet fascinating , question .", "closed mitosis appears at first glance to be an economical solution to the issue of microtubule - chromosome accessibility : nuclear envelope breakdown is circumvented , the chromosomes do not get entangled with cytoplasmic structures , and cells do not have to reassemble the nuclear envelope at the end of mitosis , risking leaving one or more chromosomes outside the nuclear perimeter . chief among them is how the nuclear envelope expands . in particular , how is the timing of nuclear expansion determined ? is it a consequence of spindle elongation or is it an independent process regulated by the cell cycle machinery ? and how are nuclear envelope components added ? finally , what happens to nuclear envelope expansion if cells are delayed in mitosis , for example due to checkpoint activation ? an insight into these questions came from a study by witkin et al . on the shape of the budding yeast nucleus . during normal growth , the budding yeast nucleus is round , except during mitosis when it elongates through the bud neck to accommodate chromosome segregation ( fig . witkin et al . screened for genes that when deleted lead to alterations in nuclear shape , aiming to identify genes that affect nuclear envelope dynamics . the authors used a collection of roughly 5000 budding yeast deletion mutants ( each deleted for a single non - essential gene ) , into which they introduced a nuclear gfp marker and a cytoplasmic rfp marker . this strategy then allowed them to screen visually for mutants that displayed abnormal nuclear morphology . one class of nuclear abnormalities was of nuclei that exhibited extensions , referred to as flares . further examination of these mutants revealed that this altered nuclear phenotype was not due to a direct involvement of the deleted genes in nuclear morphology , but rather due to the activation of a checkpoint pathway ( e.g. , the dna damage checkpoint or the spindle assembly checkpoint ) that induced a mitotic delay . in other words , the mutations isolated in the screen likely increased the formation of intracellular damage , which , in turn , led to a mitotic delay through checkpoint activation . indeed , other conditions that induced a mitotic delay ( e.g. , depolymerizing microtubules with nocodazole ; inhibiting the anaphase promoting complex ) also led to the formation of a nuclear flare . interestingly , the nuclear flare formed due to a mitotic delay was confined to the nuclear envelope region adjacent to the nucleolus ( fig . 2b , and see below ) , which normally forms a crescent - shaped structure at the nuclear periphery ( fig . are images taken 20 min apart of budding yeast cells expressing a gfp - tagged nucleoplasmic marker ( pus1 ) and an rfp - tagged nucleolar marker ( nsr1 ) . importantly , the change in nuclear morphology was specific to a cell cycle delay in mitosis ; witkin et al . found that nuclei of cells arrested in s phase or g2 were mostly round . moreover , the formation of the nuclear flare in cells treated with nocodazole began at the same time as nuclear elongation in untreated cells and was dependent on phospholipid synthesis . however , phospholipid synthesis alone was not sufficient to induce flares , as g2 arrested cells accumulated just as much phospholipid as mitotic arrested cells . taken together , these results suggest that nuclear expansion during closed mitosis of budding yeast is independent of spindle elongation , but rather occurs in response to a cell cycle cue that signals mitotic entry .", "first , what regulates nuclear envelope expansion ? as noted above , the process is independent of spindle elongation , much like nuclear envelope rupture in s. japonicus . thus , in these yeasts there are likely targets of the cell cycle machinery that affect nuclear envelope dynamics during mitosis the synthesis of phospholipids , the main components of the nuclear membrane , is required for nuclear envelope expansion , as shown previously for both budding and fission yeast . moreover , at least one enzyme that negatively regulates membrane expansion , pah1 , is regulated by cdk1 phosphorylation ( ref . the observation that a nuclear flare forms when checkpoint pathways are activated suggests that phospholipid synthesis is not subjected to checkpoint regulation . however , phospholipid synthesis is unlikely to be the only cell cycle target required for nuclear expansion , because g2-arrested cells accumulate just as much phospholipid as mitotic - arrested cells , yet they do n't form a flare ( ref . 18 ; note , however , that it is currently not known whether nuclear envelope of g2 cells also expands , but without forming a flare ) . it is tempting to speculate that the cell cycle target may be a protein or a process that is responsible for allocating membrane addition specifically to the nuclear envelope , either by localized phospholipid synthesis or by drawing membrane from the er preferentially during mitosis . spindle elongation may also play a role in nuclear envelope expansion : although the surface area of the nucleus increases during a mitotic arrest in the absence of a spindle ( i.e. , during an arrest induced by nocodazole ) , it is possible that spindle elongation contributes to the rate , and perhaps magnitude , of nuclear envelope expansion .", "observed that nuclear envelope expansion during a mitotic arrest occurs in a curious fashion : rather than expanding isometrically , thereby generating a larger sphere , the flare occurs at the nuclear envelope that is adjacent to the nucleolus ( fig . whether this occurs in other organisms that undergo closed mitosis is currently not known , as cell cycle analyses typically use dna staining , rather than a nuclear envelope marker , to follow cell cycle progression . nonetheless , the flare induced by a mitotic arrest is similar to the nuclear expansion observed when the pah1 pathway is misregulated . pah1 converts phosphatidic acid to diacylglycerol . in the absence of pah1 ( or in the absence of its activators , the nem1 and spo7 phosphatase complex ) the overall levels of certain phospholipids increase , the er loses its tubular shape in favor of membrane sheets , and the nucleus forms a flare at the nuclear envelope adjacent to the nucleolus . why , then , in both pah1 pathway mutants and in a mitotic arrest is the expansion of the nuclear envelope specific to the region adjacent to the nucleolus ? campbell et al . showed that in a spo7 mutant , if the nucleolus is reduced to a tiny sphere the flare still forms , with the diminished nucleolus often at the base of the flare . whether the nucleolus , or its remnant , affects the adjacent nuclear envelope remains unknown . alternatively , there may be a structure that prevents nuclear envelope expansion around the entire nuclear envelope except in the region adjacent to the nucleolus . there are indeed budding yeast nuclear membrane proteins that are excluded from the nucleolus ; none of the ones tested had properties consistent with an inhibitor of membrane expansion ( our lab , unpublished ) , but other such proteins may still exist . since the nuclear envelope is continuous with the er , it is quite possible that phospholipids are synthesized at the nuclear envelope itself . if this were the case , it is conceivable that the nuclear envelope adjacent to the nucleolus would have a higher capacity for phospholipid synthesis , or that membrane is added throughout the nuclear envelope , but , as mentioned above , there may be a mechanism that resists expansion everywhere except adjacent to the nucleolus . alternatively , the membrane could come from the er , and if this were the case then the cell would have a mechanism for allocating membrane from the er to the nuclear envelope specifically during mitosis . it is currently not known whether the er expands during a mitotic arrest , and if so , at what rate . thus , the mechanisms driving nuclear envelope dynamics , and in particular nuclear envelope expansion , remain to be uncovered .", "the study by witkin et al . highlights the dynamic nature of the nuclear envelope during the cell cycle . the observations in this study support a model whereby the nuclear envelope adjacent to the nucleolus is the preferential site of expansion during a mitotic delay . it is interesting that phospholipid synthesis is not under checkpoint control ; consequently , a cell delayed in mitosis needs to somehow deal with increased nuclear envelope surface area in the absence of chromosome segregation . we can envision at least three possibilities : first , sequestering the added nuclear envelope to the region adjacent to the nucleolus could preserve inter - chromosomal interactions that would otherwise be disrupted were the nucleus to expand isometrically . second , yeast cells are known to maintain a constant nuclear to cell volume ratio by a yet unknown mechanism . it is possible that by confining the extra nuclear envelope to a flare , rather than distributing it throughout the entire nuclear surface , the cell is better able to regulate its nuclear volume . finally , the nuclear envelope adjacent to the nucleolus may be the region of initial nuclear envelope expansion even under normal growth conditions , but in the absence of spindle elongation this expansion is not distributed throughout the entire nuclear surface . regardless of the mechanism , the appearance of a nuclear flare suggests that the budding yeast nuclear envelope has domains of distinct properties . how these domains differ in their ability to expand is not known . isolation of mutants that fail to produce a flare during a mitotic arrest will likely shed light on the mechanism that designates distinct nuclear envelope domains and on the function of flare formation . finally , can observations in closed mitosis illuminate processes related to the nuclear envelope in cells that undergo open mitosis ? as in closed mitosis , the nuclear envelope of metazoans must also expand , not during mitosis but during interphase , immediately following nuclear envelope reassembly . much like in yeast , the source of membrane , and both the temporal and spatial regulation of this process , are unknown . moreover , in certain cancer cells nuclei become larger and multi - lobed , and during aging the nuclear envelope appears if so , do changes in the nuclear envelope have consequences for cell function ? despite the different modes of mitosis in budding yeast and mammalian cells , cell cycle processes have often proved to be based on conserved proteins . thus , studies on nuclear envelope dynamics in budding yeast will likely shed light on processes related to nuclear envelope formation , tumorigenesis and aging in higher eukaryotes ." ]

[ "fibro - osseous lesions ( fol ) are a poorly defined group of lesions affecting the jaws and craniofacial bones . all are characterized by the replacement of bone by cellular fibrous tissue containing foci of mineralization that vary in amount and appearance . recent world health organization classification ( 2005 ) for fibro - osseous lesions was considered while sub - dividing these into various groups [ table 1 ] . so proper categorization requires good correlation of the history , clinical findings , radiographic characteristics , operative findings , and histologic appearance . the aim of this study was to analyze various clinico - pathological and radiological features in the benign fol reported and to compare the features between fibrous dysplasia ( fd ) and cemento - ossifying fibroma ( cof ) . these patients were treated for fol , reported to the hospital between 1989 and 2009 .", "the clinical parameters included were age , sex , location , duration , family history , associated symptoms , and behavior of the lesion . the radiographic appearance , histologic features , treatment , and follow - up data were also recorded . basic clinical parameters such as age , sex , duration of lesion , family history , history of trauma , associated symptoms , palpatory findings , status of associated / involved teeth , associated pigmentation , site of the lesion , treatment , and recurrence details were retrospectively analyzed . regarding site distribution , the maxilla was divided into two anatomic regions as anterior ( midline to distal surface of canine ) and posterior ( mesial surface of first premolar distally ) . the mandible was divided into four anatomic regions such as anterior ( midline to distal surface of canine ) , posterior ( mesial surface of first premolar distally ) , angle ( from distal of third molar to the inferior portion of ramus ) , and ramus ( upper portion of the ramus beyond the occlusal plane ) . radiological features were assessed for radiolucency , radiopacity , margin of the lesion , cortical - plate expansion , involvement of antrum , displacement , and resorption of teeth . histopathologically , parameters such as type of bone ( mature / immature ) , cellularity , presence of cementum - like material , and nature of stroma were assessed .", "a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol , the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) . the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) . however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] . the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd , a biopsy was done because of a non - healing extraction socket . in case of cof , the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis . none of family history and past medical history data collected were contributory to our study except few which are listed below . there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident . the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) . moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone . there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible . the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ] . extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) . when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] . the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof , the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth . radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these , 55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline . a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds , most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum . only one case showed resorption of the associated teeth . in case of cof , on 34 ( 56% ) occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof . other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd . the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue . the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts . basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof psammamatoid type . other features evident with regular histopathology were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) . fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a comparison of histopathological features between cemento - ossifying fibroma and fibrous dysplasia the treatment procedures rendered in these cases has been summarized in the table 3 . eight patients of cof showed recurrences within a period ranging from 2 months to 4 years . one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time .", "a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol , the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) . the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) . however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] . the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd , a biopsy was done because of a non - healing extraction socket . in case of cof , the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis . none of family history and past medical history data collected were contributory to our study except few which are listed below . there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident . the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) . moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone . there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible . the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ] . extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) . when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] . the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof , the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth .", "radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these , 55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline . a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds , most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum .", "in case of cof , on 34 ( 56% ) occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof . other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd .", "the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue . the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts . basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof psammamatoid type . other features evident with regular histopathology were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) . fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a chinese letter ", "eight patients of cof showed recurrences within a period ranging from 2 months to 4 years . one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time .", "the fol of the jaws comprise a diverse , interesting , and challenging group of conditions that pose difficulties in classification and treatment . common to all is the replacement of normal bone by a tissue composed of collagen fibers and fibroblasts that contain varying amounts of mineralized substance , which may be bony or cementum - like in appearance . langdon et al . , suggested that certain fols of the jaw may represent different stages in the evolution of a single disease process . although the first case report of fibro - osseous lesion was reported 60 years ago , there are only few retrospective studies regarding their clinic - pathological correlation . among them , the four important studies were carried out by alsharif et al . , on chinese population , ogunsalu et al . , on jamican population , langdon et al . comparison of clinical parameters with previous studies however , although fol are one of the common lesions occurring in india , such a retrospective study is not reported in the english language literature . we also tried to compare the features between cofs and fds , which are the only two groups of entities reported in this region . aggressive variants seen in young adults are called as jof ( who 1992 ) . according to recent who classification of fols , of is a well - demarcated lesion composed of fibrocellular tissue and mineralized material of varying appearances . juvenile trabecular of and juvenile psammamatoid of are two histologic variants of of . due to the presence of cementum - like material of have been called as cementifying fibroma ( cf ) or cof if they have both cementum and bone - like material . previous studies considered cementoid lesions as separate groups which included cemento - osseous dysplasia , gigantiform cementoma , and cementoblastoma . however , alsharif et al . , has considered cof as cementoid lesions containing cementum - like material , since he has considered of as a separate entity . however , in 1992 the who grouped such lesions under the common denomination of cof on the grounds that they represented histologic variants of the same type of lesion . in this study , of and cof are combined and no cementoid lesions were reported till today to our hospital . in our data , 24 cases were diagnosed as of , 21 as cof , 11 as cf , and 4 as jof . however , we considered all 60 cases as a single entity ( cof ) in contrast to the study on chinese population where cof and of are assessed separately . none of these cases showed any familial history as we know there are two case reports of familial cof . the term fd was given by lichtenstein in 1938 , which was earlier described as osteitis fibrosa disseminate . it is a genetically based sporadic disease of bone that may affect single or multiple bones ( monostotic or polyostotic ) or if it is occurring in multiple adjacent craniofacial bones , it is regarded as craniofacial fd . fd may be part of jaffe - lichenstein 's , macunae albright 's , or mazabraud 's syndrome . in over 80% of cases it is monostotic , whereas all 20 cases in our data showed a solitary lesion in the jaws , although few of maxillary lesions extending up to infra - orbital margin affect vision . , was 27 years and 34 years , contrasting to what we got as 21 years for the present study . early lesions may be radiolucent , but they become increasingly radiopaque and typically show a diffuse radiopacity or ground glass appearance . in the present study , 75% of the lesions showed mixed opacity and lucency , whereas two cases were completely radiolucent and three cases with complete radiopaque picture . the key histologic features of fd are delicate trabeculae of immature bone with no osteoblastic rimming , enmeshed within a fibrous stroma giving a mature bone was seen in one of our case and osteoblastic rimming was evident in few areas of five cases and stroma was mostly fibrocellular , although few showed a completely fibrous or vascular background . compared with previous studies,[468 ] both cof and fd were predominantly seen in younger population ( 25.5 years and 21 years respectively ) . however , in our data , there was a definite male predilection ( 1.8:1 ) for fd which was contrasting to the previous studies.[468 ] when compared with fd , many of cof presented with associated symptoms like pus discharge , egg shell crackling , tenderness , ulceration of the overlying mucosa , numbness of lip , and proptosis which is unusual for these groups of lesions ( this involvement was seen in four patients diagnosed as jof ) . other than jof , there are no previous reports of conventional cof showing highly aggressive behavior . only few fds presented with aggressive symptoms . the involvement of other facial bones ( frontal , ethmoidal , antrum ) was a frequent finding in cof when compared with fd .", "both cof and fd showed multiple recurrences . since few of our cof cases showed history of trauma and association with osteosarcoma , a careful detailing of these in the history is emphasized although its correlation with the occurrence of the lesion can not be established through this study . as aggressive behavior and recurrence was a frequent finding in few patients with cof , a long follow - up was advised . juvenile ossifying fibroma was of predominantly psammamatoid type and finally , not a single cementoid lesion was reported for last 20 years ." ]

[ "green tea , unfermented and made from the leaves of the camellia sinensis plant , is one of the most popular beverages consumed across the world [ 13 ] . the property and chemical components of green teas are influenced by many factors , such as tea species , harvest season , climate , geographical locations , and processing . in china , among various factors , the geographical origin is recognized as an important aspect of tea . because of the similar tea species , cultivation and processing conditions in a specific tea - producing area , many teas are named after their geographical origins . longjing tea is a green tea produced in xihu and its surrounding areas ( hangzhou , china ) . as a famous green tea with protected designation of origin ( pdo ) , longjing is recognized as one of the top green teas for its special appearance ( flat and straight leaves ) , flavor , and taste . however , little information has been available on the feasibility of discriminating longjing from its three specific subproducing areas , namely , xihu , qiantang , and yuezhou . as the quality and prices of longjing tea from the above three producing areas are different , it is necessary to develop effective analysis methods for discrimination of longjing from different subproducing areas . because of the similarity ( processing , appearance , and taste ) among different subproducing areas , the specific geographical origins of longjing are usually distinguished by sensory analysis . however , because it is very expensive and may take years to train a tea taster , it would be more efficient to use some nonhuman techniques . recent years have witnessed increased applications of electronic tongue technology to analysis of wines , milk , tea , beer , juice , and so on [ 710 ] . in these applications , a very good time stability and sensitivity , a good correlation between human and electronic tongue judgment has been observed , which makes it a promising alternative to human sensory analysis of teas . this paper was focused on developing a rapid analysis method for discriminating specific subproducing areas of longjing tea by electronic tongue and chemometrics . robust principal component analysis ( robpca ) [ 11 , 12 ] was used to detect outliers in each class . partial least squares discriminant analysis ( plsda ) was used to develop the classification model .", "155 longjing samples were collected from the local tea plantations in xihu ( 32 samples , class a ) , qiantang ( 59 samples , class b ) , and yuezhou ( 64 samples , class c ) . all the samples were preserved in a cool ( about 4c ) , dark , and dry place with integral packaging before preparation of tea extract . six gram of each sample was added with 250 ml boiling deionized water and infused for 10 min . the infusion was then filtered into a beaker and cooled to the room temperature ( 25c ) by water bath for electronic tongue analysis . the detection system consists of one reference electrode ( ag / agcl ) and 7 liquid cross selective sensors ( zz , ba , bb , ca , ga , ha , and jb ) . the cross - sensitivity and selectivity of the sensor array are listed in table 1 . the sensors array analyzed the solutions of tea samples with sampling interval of 1 s. each sample was measured for 150 s , which can ensure a stable response . all the data analysis was performed on matlab 7.0.1 ( mathworks , sherborn , ma , usa ) . the responses of the 7 sensors reported at 150 s were used for the subsequent data analysis . outliers in the data would degrade the classification models and prediction performance , so robust principal component analysis ( robpca ) was used to detect outliers in each class of samples . robpca can obtain robust projections of the original data points and avoid the masking effects caused by multiple outliers . with the computed robust score distance ( sd ) and orthogonal distance ( od ) , robpca can classify an object into one of the four groups : regular points ( with small sd and small od ) , good pca - leverage points ( with large sd and small od ) , orthogonal outliers ( with small sd and large od ) , and bad pca - leverage points ( with large sd and large od ) . with outliers deleted , the duplex algorithm was used to divide the measured data into a training set and test set . duplex algorithm can obtain a test set of samples distributed uniformly in the range of training samples . for multiclass classification , plsda can be performed by regressing each column of a dummy response matrix y on the measured data x by pls . for the ith ( i = 1 , 2 , and 3 ) column in y , an element is set a value of 1 if the corresponding object is from class i ; otherwise , it is assigned a value of 0 . for prediction , a new sample is classified into class i when the ith element of its predicted response vector is nearest to 1 . monte carlo cross validation ( mccv ) was used to estimate the number of components in plsda by minimizing the mean percentage error of mccv ( mpemccv ) : \n ( 1)mpemccv=i=1bnii=1bmi , \n where b is the numbers of mccv data splitting , mi is the number of prediction objects , and ni is the number of wrongly predicted for the ith mccv data splitting . model sensitivity and specificity of prediction for each class were used to evaluate the performance of classification models : \n ( 2)sensitivity = tptp+fn , specificity = tntn+fp , \n where tp , fn , tn , and fp represent the numbers of true positives , false negatives , true negatives , and false positives , respectively . for classification , objects in each class were denoted as positives and the other two classes were denoted as negatives .", "the average electronic tongue features for each class of longjing tea are shown in figure 1 . seen from figure 1 , the features of the three classes have very similar response patterns . the features of class b and c are very similar and different from those of class a , especially in the responses of ga and ha . robpca was used for outlier detection with a significance level of 0.05 . because each class of tea has a different probability distribution , robpca models were performed separately on each class . figure 2 demonstrates the robpca plots for the three classes . for each class , a robpca model with three principal components ( pcs ) three pcs explained 89.7 , 91.5 and 92.1 percents of the total variances of each class , respectively . for outlier diagnosis , bad pca - leverage points , good pca - leverage points and orthogonal outliers were excluded to obtain a representative data set distributed in the entire range of measured samples . the numbers of objects with large sd and od were denoted . as a result , for class a , two orthogonal outliers ( objects 1 and 26 ) and two good pca - leverage points ( objects 15 and 31 ) were deleted ; for class b , four orthogonal outliers ( objects 1 , 19 , 24 , and 43 ) were deleted ; for class c , three orthogonal outliers ( objects 12 , 17 , and 29 ) and one bad pca - leverage point ( object 55 ) were deleted . the duplex algorithm was then performed to divide the remaining 143 tea samples into training and test objects . finally , 95 samples ( class a , 18 ; class b , 37 ; and class c , 40 ) were used for training and 48 samples ( class a , 10 ; class b , 18 ; and class c , 20 ) for prediction . plsda model was developed and mccv was used to select the number of latent variables ( lvs ) . for mccv , the original 95 training samples were randomly split into training ( 50% ) and test objects ( 50% ) for 100 times . the training and prediction results of a three - component plsda are demonstrated in figure 3 . the sensitivity / specificity of plsda for classes a , b , and c was 1.000(10/10)/1.000(38/38 ) , 1.000(18/18)/0.967(29/30 ) , and 0.950(19/20)/1.000(28/28 ) , respectively . the training accuracy was 1 and for prediction only one object from class c was wrongly assigned to class b , indicating the effectiveness of electronic tongue for classification of longjing tea samples .", "rapid and effective discrimination of longjing green tea from different subproducing areas was performed using electronic tongue and chemometrics . the sensitivity / specificity of plsda for classes a , b , and c was 1.000/1.000 , 1.000/0.967 , and 0.950/1.000 , respectively . electronic tongue and chemometrics can provide a rapid and reliable tool for discriminating the specific producing areas of longjing . compared with human sensory analysis , this method is easier to perform and the more attractive economically . in the future studies , the comparison of chemical methods , for example , lc / uv / ms , to the electronic tongue analysis will be performed to investigate the statistical correlation between the chemistry and the tastes of longjing tea ." ]

[ "aging is associated with changes in various body systems including in the neuromuscular structures , which lead to a reduction in important components of physical fitness.1 the preservation of fitness - related components such as muscular strength , endurance , and flexibility are essential to the performance of daily activities , which thus helps to maintain autonomy and quality of life in the elderly population.25 of these fitness - related components , flexibility is often less appreciated with respect to its contribution to optimal health and functional status as well as independent living for the elderly.3,6 flexibility reductions may increase the risks of injury , falling , back pain , and physical dependence in older adults.7 several mechanisms have been proposed for reductions in flexibility , among them alterations in soft - tissue structures and physical inactivity.7 when a joint is relatively inactive due to sedentary behavior , the muscles that cross it shorten , thereby reducing its range of motion . moreover , independent of activity levels , the aging process plays a role in flexibility decreases.8 joint structures such cartilage , ligaments , and tendons change mechanically and biochemically with aging , increasing muscular and tendon stiffness and thus impeding mobility.911 on the other hand , maintaining a physically active lifestyle may result in improvement of functional performance with advancing age , thus enabling the execution of activities of daily living with more vigor and less fatigue.12,13 while resistance training ( rt ) is recommended for older people for the development of muscular strength,13,14 some studies have indicated that regular participation in rt programs may also contribute to increased flexibility.11,1519 in fact , there is evidence that regular rt serves as an active form of flexibility training and can improve range of motion to a similar extent as typical static stretching protocols.20 regular performance of rt may improve flexibility by reducing passive tension and stiffness of the tissues surrounding a joint.11 thus , from a time - saving standpoint , rt is a good way to develop both strength and flexibility as well as achieve improvements in body composition within a single session of training . the fitness - related benefits associated with rt are dependent on the manipulation of variables such as intensity , volume , exercise order , and rest intervals.12 with respect to training volume , manipulation can involve varying the number of repetitions , sets , and/or frequency defined here as the number of sessions performed per week . the american college of sports medicine recommends that older adults engage in two to three rt sessions a week for improving neuromuscular fitness.12 however , there is a current paucity of research regarding the effects of different rt weekly frequencies on flexibility adaptive responses . previous studies are limited to analyzing the impact of rt on flexibility without regard for manipulation of the variables that make up the training program . considering the importance of flexibility in the health and wellness of the elderly therefore , the main purpose of this investigation was to analyze the effect of rt performed at different weekly frequencies on flexibility in untrained older women . we hypothesized that higher rt frequencies would result in greater flexibility increases than lower frequencies . the rational for our hypothesis response relationship between training volume and muscle strength and hypertrophy,21,22 whereby greater training volumes are associated with enhanced muscular adaptations . a secondary aim of the study was to assess rt frequency - related changes in skeletal muscle mass . in accordance with the aforementioned dose response relationship , it was hypothesized that muscle protein accretion would be greater with the higher frequency program .", "recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil . a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires . the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions . the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group g2x ) or a group that performed rt three times per week ( n=32 ; group g3x ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis . participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study . this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee . the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing . anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 . subjects were instructed not to perform any other type of physical exercise during the entire study period . body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes . height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes . body mass index was calculated as body mass in kilograms divided by the square root of height in meters . the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal . scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis . feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region . the software generated standard lines that set apart the limbs from the trunk and head . these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer . analyses during the intervention were performed by the same technician who was blinded to intervention time point . retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass . to evaluate a subject s flexibility , five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion . these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale . all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf , the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground . for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure at this point the participants were instructed to remain in the final position until the reading was completed . the highest score obtained from the three measurements at each joint motion was adopted as a reference standard . the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results . it is worth emphasizing that the evaluator had over 2 years experience , and based on the test retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees . during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects . afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week . supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities . the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance . g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays . the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise . participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) . instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique . progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively . two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test , the analyses were adjusted using a greenhouse geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences . baseline differences between groups were explored with an independent t - test . the effect size ( es ) was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 . the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) .", "recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil . a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires . the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions . the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group g2x ) or a group that performed rt three times per week ( n=32 ; group g3x ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis . participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study . this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee .", "the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing . anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 . subjects were instructed not to perform any other type of physical exercise during the entire study period .", "body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes . height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes . body mass index was calculated as body mass in kilograms divided by the square root of height in meters .", "the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal . scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis . feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region . the software generated standard lines that set apart the limbs from the trunk and head . these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer . analyses during the intervention were performed by the same technician who was blinded to intervention time point . retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass .", "to evaluate a subject s flexibility , five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion . these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale . all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf , the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground . for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure at this point the participants were instructed to remain in the final position until the reading was completed . the highest score obtained from the three measurements at each joint motion the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results . it is worth emphasizing that the evaluator had over 2 years experience , and based on the test retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees .", "during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects . afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week .", "supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities . the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance . g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays . the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise . participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) . instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique . progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively .", "two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test , the analyses were adjusted using a greenhouse geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences . baseline differences between groups were explored with an independent t - test . the effect size ( es ) was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 . the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) .", "a significant main effect for group - by - time interaction ( f=2.53 , p<0.05 ) was observed , in which g3x showed a higher increase than g2x ( g2x=+87.8% , es=7.31 ; g3x=+92.2% , es=10.39 ) . the information regarding flexibility indicators pre - and post - training according to group are presented in table 2 . a main effect of time ( p<0.01 ) was observed for ce ( g2x=+19.1% , g3x=+20.0% ) , right hip flexion ( g2x=+14.6% , g3x=+15.9% ) , and left hip flexion ( g2x=+25.7% , g3x=+19.2% ) . significant group - by - time interaction ( p<0.01 ) was observed only for fhf , in which g3x showed a higher increase than g2x ( + 12.8% , and + 3.0% , respectively ) . figure 3 shows the absolute ( a ) and relative ( b ) variations from pre- to post - training on skeletal muscle mass indicators according to group . only the main effect of time reached statistical significance ( f=22.05 , p<0.001 ) , with both groups having a similar increase after the 12 weeks of the rt program . the ess for skeletal muscle mass changes were 0.21 and 0.43 for twice and thrice per week sessions , respectively .", "the main finding of the present study is that 12 weeks of rt was sufficient to increase or at least maintain flexibility in elderly women . the increases in flexibility after an rt program in older individuals have been previously reported,11,1619 and our findings are consistent with these studies . for example , fatouros et al18 recruited eight older men to perform 16 weeks of rt three times per week , and observed significant range - of - motion increases in knee flexion , elbow flexion , shoulder flexion , hip flexion , shoulder extension , and hip extension at study end . gonalves et al19 investigated the effect of 8 weeks of rt three times per week on the flexibility of elderly men and women , and noted an increase in shoulder extension , hip flexion , and hip extension after the intervention . barbosa et al16 investigated the effect of 10 weeks of rt three times per week in elderly women and observed an increase in flexibility as measured by the sit - and - reach test after 10 weeks . the exact mechanisms responsible for increased flexibility after an rt program have not yet been established in the literature and the outcomes drawn from our study do not provide mechanistic insight . joint movement is related to morphological elements such as muscle , bone , and connective tissues . in particular , muscle and fascia are responsible for ~41% of a joint s resistance to movement,27 suggesting that an rt - mediated reduction in passive tension and stiffness of these tissues translates into a greater range of motion.11 this hypothesis requires further study . the novel and important feature of our study was the comparison of two versus three rt sessions per week with respect to flexibility outcomes . results show that only fhf benefited from an increased rt frequency , while all other flexibility outcomes were similar regardless of the number of weekly sessions . the fact that hip - flexion range of motion showed greater improvement when training was undertaken three times per week rather than twice per week has implications for exercise prescription . hip flexion measures the flexibility of muscles in both the lumbar region and on the posterior thigh . these structures are highly relevant to the mobility of the torso and lower back , which has particular implications for functional capacity in the elderly.28 moreover , hip flexibility is a predictor of low back pain , and is strongly associated with aging.29 the muscles of the low back and the hamstrings are considered to be particularly relevant to hip mobility.24 given that es analysis showed the group with higher volume had a greater positive effect on skeletal muscle mass compared with the lower volume group , it is conceivable that the associated increased strength of muscles surrounding the hip may have led to a greater improvements in flexibility . an analysis of the weekly training load , which refers to the load used to perform a given exercise , was adopted in this study as the muscular strength indicator . analysis of the training load is an alternative and potentially more practical method for monitoring muscular strength changes compared with one repetition maximum test.30 our results indicate that a greater rt frequency is associated with higher increments of specific muscle strength in the sum of exercises used ( 88% and 92% , for g2x and g3x , respectively ) . to date , as far as we are aware , only a few studies have investigated the effects of different training frequencies in older people,3133 with conflicting results . for example , both difrancisco - donoghue et al32 and taafee et al33 showed similar increases in strength between programs with high ( twice ) and low ( once ) weekly training frequencies . alternatively , farinatti et al31 investigated the effects of rt once , twice , and three times per week , and observed that higher frequencies promoted greater increases in strength for certain exercises ( seated dumbbell curl and knee extension ) but not for others ( bench press and standing calf raise ) . no statistically significant differences in changes in skeletal muscle mass were noted between training two versus three times a week in the present study . however , thrice - weekly training showed greater absolute increases compared with the twice - weekly condition ( 7.4% vs 4.4% , respectively ) and the ess were considerably larger when training three times a week as well ( 0.43 versus 0.21 , respectively ) . , g3x performed three sets per muscle per week while the g2x group performed two sets per muscle per week . thus , our findings suggest a potentially meaningful benefit to increasing the volume of training for a given muscle from two to three sets per week with respect to skeletal muscle mass . considering the relatively short duration of our study , future research is recommended to determine the extent to which such differences in skeletal muscle mass would continue or perhaps narrow over time . the data found in our study are limited to the joint movements analyzed and time of training applied . it is possible that the range of motion of these joints might continue to increase if training were to exceed 12 weeks , and the results should not be extrapolated to populations other than older women . moreover , the absence of monitoring the physical activity and dietary intake habits is a limitation as well . on the other hand , to our knowledge , the present study is the first to have investigated the effect of different rt frequencies on flexibility in older women . our findings indicate that rt performed at a minimum of twice per week can result in positive effects on flexibility in the elderly , reversing or slowing the aging - induced losses .", "the results of our study suggest that 12 weeks of rt improves or at least preserves the flexibility of different joint movements in older women . in addition" ]

[ "bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death . the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed . the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution . we divided the zonular fibers into two groups : the equatorial zonule ( blue fibers in fig . other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens . because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues . once the equatorial fibers had been removed , the hyaloid zonule was exposed . the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous . they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face , it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor . samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor , a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor . human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample . thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) . thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) . human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax . sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence . deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) . sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody . the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3 10 minutes ) , sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) . sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) . bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each . samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c . five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard . five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) . the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al . briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate . a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c . peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate . the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation . bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine . samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark . an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin . digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction . peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration . samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed . the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m 2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid . after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m 25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c . peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min . survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2 10 . the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% . top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans . dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 . , is provided as supplementary materials s1 . the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line . an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files . ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 . databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain . common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method . the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences . the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 . searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev . 2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively . other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring . the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates . comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet . target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% . protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed . protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles these results were used for quantitative analyses using ms2 fragment ion intensity weighted spectral counting . to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded . it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins . comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot . a fasta database file of all human ecm proteins was constructed . for both human and bovine samples , the lists of identified proteins were used to extract separate fasta database files for identified proteins . an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins . analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins . common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis . extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample . the total abundances of putative zonule proteins ( using ms2 intensity weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined . average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes . this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples , the ms2 intensity weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together . full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 , s5 ) for each experiment are included .", "bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death . the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed . the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution . we divided the zonular fibers into two groups : the equatorial zonule ( blue fibers in fig . other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens . because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues . once the equatorial fibers had been removed , the hyaloid zonule was exposed . the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous . they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face , it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor . samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor , a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor . human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample . thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) . thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .", "human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax . sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence . deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) . sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody . the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3 10 minutes ) , sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) . sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .", "bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each . samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c . five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard . five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) . the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al . briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate . a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c . peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate . the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .", "bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine . samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark . an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin . digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction . peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .", "samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed . the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m 2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid . after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m 25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c . peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min . survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2 10 . the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% . top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans . dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .", "the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line . an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files . ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 . databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain . common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method . the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences . the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 . searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev . 2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively . other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring . the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates . comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet . target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% . protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed . protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles these results were used for quantitative analyses using ms2 fragment ion intensity weighted spectral counting .", "the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line . an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files . ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain . common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method . the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences . the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 . searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev . 2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively . other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates . comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet . target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% . protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed . protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles these results were used for quantitative analyses using ms2 fragment ion intensity weighted spectral counting .", "to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded . it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins . comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot . a fasta database file of all human ecm proteins was constructed . for both human and bovine samples , the lists of identified proteins were used to extract separate fasta database files for identified proteins . an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins . analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins . once matrisome proteins were identified common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis . extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample . the total abundances of putative zonule proteins ( using ms2 intensity weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined . average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes . this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples , the ms2 intensity weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together . relative abundances within each sample by gene were computed as described above . full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 ,", "we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) . for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) . our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins . liquid chromatography ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) . 3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species . two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins . the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans . the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors . of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs . however , when the data were weighted according to protein abundance , based on ms2 intensity weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) . the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 . the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) . overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome . however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) . shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule . we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig . , the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig . the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs . fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) . emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) . fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) . all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig . the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome . fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively . all family members are present ( with the exception of ltbp4 in the human zonule ) . the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins . mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients . mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms . together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule . ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm . both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) . curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved . another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family . adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae . adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 . unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) . we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig . adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig . in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm . immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig . a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig . the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells . work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds . we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils . several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig . transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule . the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome . exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma . given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule . one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 . the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils . expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule . the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) . only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 . col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium . megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant . immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule . this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals . similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes . cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions . proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels . unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 . fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample . recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous . comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs . proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor . protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) . some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous . note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule . the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous . if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values . although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule . such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample . the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig . histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .", "we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) . for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) . our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins . liquid chromatography ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) . 3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species . two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins . the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans . the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors . of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs . however , when the data were weighted according to protein abundance , based on ms2 intensity weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) . the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 . the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) . overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome . however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) . shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule . we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig . , the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig . the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs . fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) . emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) . fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) . all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig . the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome . fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively . all family members are present ( with the exception of ltbp4 in the human zonule ) . the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins . mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients . mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms . together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule . ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm . both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) . curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved . another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family . adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae . adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 . unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) . we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig . adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig . in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm . immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig . a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig . the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells . work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds . we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils . several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig . transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule . the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome . exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma . given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule . one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 . the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils . expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule . the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) . only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 . col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium . megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant . immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule . this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals . similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes . cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions . proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .", "unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 . fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample . recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous . comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs . proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor . protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) . some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous . three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous . note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule . the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous . if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values . although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule . such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample . the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig . histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .", "the ciliary zonule plays a key role in ocular function , centering the lens on the optical axis and transmitting the forces that mold its shape during accommodation . the zonule is adversely affected in several pathologic conditions , including exfoliation syndrome , homocystinuria , and marfan syndrome . to understand how and why the zonule is compromised in these diseases , a detailed analysis of its composition microfibrils ( the main structural elements of the zonule ) are ubiquitous components of the ecm but questions remain regarding their organization , synthesis , and role as signaling hubs . the ciliary zonule , a readily accessible , cell - free system composed of bundled microfibrils , represents a useful model system in which to gain insights into microfibril organization . the composition of the equatorial zonule was broadly similar in humans and cows , and the set of 52 proteins identified in both samples accounted for > 95% of the mass in either case . the fact that the human samples were from middle - aged individuals , whereas the bovine samples were from young animals , might in part explain why more proteins were identified in the bovine zonulome ( 176 vs. 144 ) . the bovine samples were also obtained shortly after death , whereas the human samples were obtained 2448 hours postmortem . it is possible that labile proteins were lost from the human samples during the postmortem period . in both humans and cows , the zonule consisted of a microfibril backbone comprised primarily of fibrillin and a relatively restricted set of glycoproteins . ltbp2 , a fibrillin - like protein implicated in autosomal recessive weill - marchesani syndrome type 3 ( wms3 : mim#614819 ) , was a surprisingly abundant component , comprising 10% of the total zonular protein . there are three fibrillin genes in humans and the presence of all three proteins was confirmed in the zonulome , consistent with earlier observations . fbn2 , the second most abundant fibrillin in the zonule , is synthesized early in development and is essential for normal zonule formation in mice . immunolocalization studies have previously failed to detect fbn2 in the adult zonule , but its presence in the human and bovine zonulome suggests that fbn2 epitopes may become inaccessible to antibody staining during postnatal development , as reported in other systems . several proteins implicated in tgf signaling were detected in the zonule . despite its name , latent tgf binding protein 2 ( ltbp2 ) is not a bona fide tgf-binding protein , but two other family members , ltbp1 and ltbp3 , were detected in the zonulome . in other systems , ltbp1 and ltbp3 facilitate the sequestration ( and release ) of tgf in the ecm through interaction with fbn1 and other components . of note another protein with a potential role in modulating tgf signaling was emilin-1 , a prominent component of the human and bovine zonulome . emilin-1 has been shown to inhibit tgf signaling by binding to pro - tgf precursor and preventing its furin - mediated maturation . emilins were originally identified as elastic fiber components located at the interface between microfibrils and the underlying elastin core . more recent studies have suggested that they associate directly with microfibrils . because zonular fibers insert into the lens capsule directly above the lens germinative zone ( the region of the lens epithelium containing the mitotically active cell population ) , tgf or other growth factors sequestered in the zonule are well placed to influence the growth of the lens directly . it is also possible that the cyclical stretching of the zonule during accommodation might serve to release bound factors from the surface of zonular fibers . one of the novel ( and abundant ) proteins detected in our study was adamtsl-6 ( thsd4 ) . adamts proteins and adamtsl proteins have well - documented roles in ocular biology and pathology . adamtsl-6 has not been identified previously in the zonule but , in other systems , is known to bind to the n terminus of fbn1 , where it may promote microfibril formation . among the proteins that we expected to detect in the zonulome , but did not , was adamts10 ; adamts10 mutations underlie weill - marchesani syndrome type 1 ( wms1 ) . ectopia lentis is commonly observed in wms1 , implying a mechanical defect in the ciliary zonule . clinically , wms1 is indistinguishable from wms2 and wms3 , syndromes caused by mutations in fbn1 and ltbp2 respectively , genes encoding two of the most abundant zonular proteins . immunoelectron microscopy studies have previously suggested that adamts10 is abundant in the human ciliary zonule so its absence from the proteome is perplexing . a striking feature of the zonulome in human and cows was the presence of a diverse set of protease inhibitors , the most abundant in humans being timp3 . the zonule is known to be exquisitely sensitive to proteolysis . before the introduction of modern cataract surgery , ophthalmologists removed the cataractous lens in a single piece , a procedure facilitated by brief exposure to dilute solutions of -chymotrypsin , which served to lyse the zonule and release the lens from the eye . the presence of timp3 and other protease inhibitors may help explain the evident durability of the zonule , despite lifelong exposure to matrix metalloproteinases and other proteases present in the ocular humors . the hyaloid zonule is intimately associated with the anterior face of the vitreous . during ophthalmic surgery , the vitreous face behaves , physically , as if it were a distinct membrane ( the hyaloid membrane ) . ophthalmologists take great care not to damage the hyaloid membrane during surgical procedures , lest the contents of the vitreous leak . the current study may provide some clues as to the nature of the hyaloid membrane . first , it is apparent that the hyaloid membrane is reinforced by the presence of a meshwork of fibrillin - rich microfibrils , the hyaloid zonule . during dissection of the hyaloid zonule it is likely that proteins from the anterior - most region of the vitreous were coisolated . the hyaloid zonule sample was characterized by the presence of high concentrations of opticin , consistent with previous immunolocalization studies of opticin in human eyes . our analysis also suggested that the hyaloid sample was enriched in fibrillar collagens ( especially col2a1 , col5a2 , col5a3 , and col11a1 ) , which were detected elsewhere in the vitreous but at much lower concentrations . interestingly , mutations in col2a1 and col11a1 result in stickler syndrome , a condition characterized by vitreous pathology . in stickler syndrome type 1 ( caused by col2a1 mutations ) , a membranous vitreous is often observed ; whereas in stickler syndrome type 2 ( col11a1 ) a beaded vitreous is observed . in each case the anterior face of the vitreous appears to be compromised . thus , the phenotypes in stickler syndrome are consistent with a model in which fibrillar collagens form part of a meshwork ( along with fibrillin - rich microfibrils and opticin ) that defines and supports the vitreous face . one question arising from the current study is why such an unassuming structure as the ciliary zonule should require a proteome with fifty or more components ? in other settings , it is evident that microfibril scaffolds , decorated with latent growth factors , serve as both mechanosensors and signaling hubs . the pleitropic ocular effects of mutations in zonular genes ( which include congenital glaucoma , cataract , ectopia lentis , and microspherophakia ) are consistent with the notion that the role of the ciliary zonule is not solely to secure the lens in place .", "" ]

[ "identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types . the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay . the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized . normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information . new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin .", "identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types .", "the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay .", "the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized .", "normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information . new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin .", "", "this study was in part financially supported by the netherlands genomic initiative / netherlands organisation for scientific research .", "" ]

[ "zoonotic cutaneous leishmaniasis ( zcl ) is polymorphic disease with various clinical manifestations ( 1 , 2 ) . leishmaniasis , a group of parasitic infections , has a worldwide distribution ( 3 ) ; the world health organization estimates a prevalence of approximately 12 million cases , with an annual mortality rate of 60,000 and an at - risk population of approximately 350 million ( 4 ) . approximately 90% of cl cases occur in just seven countries , including iran ( 5 ) . several molecular targets for diagnostic pcr testing have been evaluated in leishmania , including minicircle kinetoplast dna ( kdna ) ( 5 , 6 ) , the mini - exon ( spliced leader rna ) gene ( 7 ) , gp63 polymerase chain reaction analysis of restriction fragment length polymorphism ( pcr - rflp ) ( 8) , and the internal transcribed spacer ( its ) ( 1 , 3 , 9 , 10 ) . pcr is a new alternative to existing diagnostic procedures , such as direct smear of parasites from clinical specimens or by cultivation , with microscopic examination .", "in the present study , as described previously ( 1 , 11 , 12 ) , we used the rflp analysis of amplified its1 in ribosomal operons to investigate the parasite that causes zcl , and the genetic variations among l. major isolates from chabahar , a port city in southeast iran ( situated at the iran - pakistan border ) . in this region , the manifestation of zcl as a new focus of disease was in doubt , but our findings showed that the species is l. major with variable genes . the disease was correlated with the clinical manifestations of zcl in chabahar , so this is a new major public health problem .", "a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) . the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) . the study was approved by the institutional ethics committee , and all patients signed informed consent forms . we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients . these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases . the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages . each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation , two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25 1c . the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin . the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations , three skin samples were collected from the edges of the lesions using sterile surgical blades . two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25 1c . parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700 g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) . for species identification , the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions . the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) . the reaction was carried out with the pcr - ready supreme mix in 47 l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix . amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide . major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes . the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) . the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions .", "a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) . the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) . the study was approved by the institutional ethics committee , and all patients signed informed consent forms . we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients . these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases . the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages . each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation , two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25 1c . the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .", "the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations , three skin samples were collected from the edges of the lesions using sterile surgical blades . two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25 1c . parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700 g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) .", "for species identification , the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions . the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) . the reaction was carried out with the pcr - ready supreme mix in 47 l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix . amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide . major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls . the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .", "the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) . the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions .", "we used the pcr - rflp assay of its1 genes for the direct identification of leishmania species in 24 out of 33 patients suspected to have cl . these 24 patients , who were positive on smears and cultures for l. donovani bodies demonstrated microscopically , were selected for the study . the patient characteristics are shown in table 1 , including gender , age , number of lesions , ulcer duration , and distribution of ulcers on the body . with the use of litsr / l5.8s primer for pcr amplification , single products of the expected size of 350 bp of l. major were detected in 21 patients ( figure 1 ) . in three isolates , a nonspecific 450 bp band was seen . rflp was performed on the its1 region in the ribosomal operons of 24 isolates of l. major . after using the restriction enzyme haeiii , banding patterns , including fragments of 140 bp and 210 bp bands , were observed in 19 cases ( lma ) . in three cases with this enzyme , 300 bp and 150 bp bands ( figure 2 ) m , molecular size marker ( 50 bp ) ; lane 1 , standard l. major ( mrho / ir/75/er ) ; lane 2 , standard l. tropica ( mhom / ir/99/yaz1 ) ; lane 3 , pcr products ( 350 bp ) as a standard ; lane 4 , pcr products ( 450 bp ; different from standard ) ; lanes 5 - 10 , pcr products ; lane 11 , negative control . m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , standard l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control . digestion of its1 from ribosomal dna with taq1 enzymes enabled us to identify two different patterns . using the taq1 restriction enzyme , banding patterns including 150 bp and 200 bp were observed in 19 cases , same as the standard of l. major ( genotype pattern lma ) . in three isolates , digestion with this enzyme showed 300 bp and 150 bp banding patterns , dissimilar to the l. major reference strain . in two cases , m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control . m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lane 3 , pcr products ; lane 4 , pcr products ; lane 5 , pcr products ; lane 6 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 7 , pcr products ; lanes 8 - 10 , pcr products ; lane 11 , negative control .", "leishmaniasis is a major societal health problem that mostly affects populations where essential health services are not easily accessible and the detection of cl is based on clinical characteristics ( 14 ) . the present study shows that cl is caused by l. major in chabahar , iran . parasitological methods are the gold standard for the diagnosis of cl , with a high dependence on the number of parasites in the samples and a requirement of technical skill for sampling . the sensitivity of these methods varies from 27% to 85% for the diagnosis of leishmaniasis , which is their main disadvantage ( 15 ) . in addition , contamination of the cultured medium is an occasional problem ; such material must be discarded . molecular techniques have proved to be sensitive and powerful tools for detecting leishmania directly in clinical samples , as well as for parasite characterization using pcr ( 1 ) . some studies on viannia isolates from different hosts and geographic areas have found high levels of intra- and interspecific variation . schonian et al . established a diagnostic its1 pcr - rflp method using the restriction enzyme haeiii for leishmaniasis , combining high sensitivity for detecting leishmania directly in clinical materials and the ability to identify all medically relevant species groups ( 12 ) . in our study , using the haeiii restriction enzyme , the restriction analysis of the amplified its1 ribosomal dna revealed two different schizodeme patterns ( lma and lmb ) in l. major . on the other hand , some researchers have found that haeiii was not sufficient to distinguish all species of viannia ( 16 , 17 ) . we found that haeiii , rsa , and hinf1 can produce two profile bandings ; alu1 produces three ; dde1 and scrf1 produce four ; and taq1 produces five ( 1 ) . in this study , ( 20 ) , two profile bandings ( 220 bp and 140 bp ) in haeiii digestion were detected , similar to the standard . taq1 digestion showed two patterns with 150 bp and 200 bp bands . in contrary , in a study in yazd ( 20 ) on molecular identification using taq1 restriction enzymes , four patterns were produced . it should be emphasized that leishmaniasis in chabahar is new , and genetic variation is limited . the results of the present study showed that cl infections were more common in males ( 48.4% ) than in females ( 24.2% ) ( table 2 ) , probably because males spend more time outdoors than females do in this region . some studies have shown that pcr methods achieved positive results in more than 90% of cl cases ( 13 ) . the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub - families of leishmania , and move to different hosts along with them . some researchers believe that the only way to isolate crithidia from leishmania is through kdna and/or ribosomal rna genes . it has been suggested that by extracting kdna and/or rdna , then amplifying them with pcr using the pcr - rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia , these two parasites can be isolated from each other ( 21 ) . however , its1-rflp pcr showed good sensitivity , and two its1 analyses showed the same results , as in a recent study by doudi et al . thus , the results of the present study confirm that in the border area of sistan va baluchestan , on the peninsula of chabahar , l. major is the causative agent of cl , with genotypic variation in this area ." ]

[ "cystic fibrosis ( cf ; omim 219700 ) is the most common lethal autosomal recessive disorder ( 1 , 2 ) , with an incidence rate of 1 in 2000 - 3000 live births in europe and 1 in 10,000 births for asians living in the united kingdom ( 1 ) . it is a multisystem disorder affecting the gastrointestinal , respiratory , hepatobiliary , and reproductive systems , as well as the sweat glands ( 1 - 6 ) . cf is caused by a mutation in the cystictransmembrane conductance regulator ( cftr ) gene , leading to defective ion transport of the cftr protein ( 7 - 10 ) . although numerous mutations in the cftr gene have been reported as a primary cause in the clinical manifestations and outcome of cf ( 9 ) , recent research has focused on the role of other factors such as gender ( 11 , 12 ) , race ( 1 ) , age ( 11 ) , geographic area ( 2 ) , infection with pseudomonas aeruginosa ( 11 , 13 ) , religion ( 8) , culture ( 1 , 14 ) , and nutritional status ( 13 - 15 ) . recently , improvement and progress in outcomes have been observed through better understanding of the disease , finding the effective factors in the start and progression of the disease , and ultimately in better management of the disease ( 16 ) . these factors have led to earlier diagnosis and more timely treatment , which could affect the clinical course and change the outcome for a patient . therefore , the link among researchers and clinicians will be stronger due to extensive research on the disease and improved introduction of the disease ( 17 , 18 ) . in addition , the research results could have an effective role in changing of the culture and laws of a society . retrospective research , in spite of some disadvantages , plays an important role in depicting the disease ( 16 , 19 ) . this research has been conducted after searching the electronic databases and performing a literature review , to ensure that no similar study had been undertaken in the region .", "this study aimed to assess the association between outcomes and demographic status in azeri turkish patients with cystic fibrosis .", "this was a cross sectional study conducted at the educational and treatment children s hospital of the university of medical sciences and the medical genetic laboratory , tabriz , iran , from march 2001 to september 2014 . the educational and treatment children s hospital is a governmental , specialized , and referral hospital with 150 beds and six wards in northwestern iran and dr . m. rafeey is the top , and the most professional , physician in this field and region . this study was performed on azeri turks , who are members of one of the largest ethnic groups in iran ( 20 ) . all the medical records of the patients were reviewed using the census method , and further information was gathered from patients , or their parents , through telephone interviews . the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) . the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors . patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement . the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end , 331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 . data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth . ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group . a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) . a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree , first cousins were classified as the first group and the others as the second group . demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables . the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test . logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses , odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance . ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) . participants were assured of confidentiality , and consent was obtained from the patients or their parents .", "the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) . the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors . patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement . the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end , 331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 .", "data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth . ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group . a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) . a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree , first cousins were classified as the first group and the others as the second group . demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables . the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test . logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses , odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance .", "ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) . participants were assured of confidentiality , and consent was obtained from the patients or their parents .", "demographic data of this study is shown in table 1.there was a significant difference between frequencies of deceased and living patients ( p < 0.001 ) . in the application of the kolmogorov - smirnov test , age was assumed as non - normal distribution that uses the sample median and the iqr ( minimum - maximum ) , consequently , median age for living patients was 0.4 years ( 7 days30 years ) and median age at death was 4.8 years ( 3 months 43 years ) . abbreviations : d , days ; iqr , minimum - maximum ; m , months ; y , years . all data were mentioned as frequency ( % ) . for deceased cf - patients , median age at death and living cf - patients , median age at time of the study have been considered , because of non - normal of age and weight at birth , median and iqr were applied . eighty - five patients expired during the 13 years of the study with a mean of six deaths per year and at a ratio of 1:3 . during the time of the study , 77 ( 90.6% ) of the 85 deceased cf patients had died under the age of four years . among the 246 living cf patients , 131 ( 53.3% ) were less than four years of age . ( % ) . at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females . the test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step , there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) . there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) . there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) . the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages . finally , weight at birth was computed and compared in cf patients , both deceased and living . there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) . there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) . associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced . results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 .", "at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females . the test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step , there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) . there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) . there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) . the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages . finally , weight at birth was computed and compared in cf patients , both deceased and living . there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) .", "there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) . associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced . results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 .", "the association between non - genetic variables and mortality in individuals with cf has been established ( 2 , 6 ) . despite improved management and increased awareness , outcome remains an important issue for cf patients , because non - genetic parameters , especially demographic factors , have been ignored by researchers and clinicians . the current study was performed to highlight the associations between demographic parameters and outcomes in cf patients . in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference . eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) . therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf . in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18 12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients , the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study , also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care were risk factors for a younger median age at death in non - european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries . we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) . in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females . a number of studies have indicated that males have a better survival rate than females , while lannefors et al . in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males . expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) . reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study , there was a significant difference between outcome and location of residence . the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) . the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival . growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) . continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) . the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) . preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) . in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born . management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand , high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families , patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history . there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study . ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease . patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research . considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) . social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently . the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) . most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) . ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found . the mean of weight at birth in the living cf population was slightly higher than the deceased cf population . cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) . patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) . an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) . it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival . the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients . the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research . another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results . data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers . the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time . data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes . this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies . this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes . clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas . it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity . couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy . other studies were performed for evaluating the association , if any , between outcome and other demographic factors .", "in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference . eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) . therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf . in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18 12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients , the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study , also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries . we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) .", "in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females . a number of studies have indicated that males have a better survival rate than females , while lannefors et al . 15 ) showed 45% and 49% of males with cf expired , respectively . in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) may be reasons for the differences . for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males . expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) . reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) . the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival . growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) . continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) . the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) . preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) . in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born . management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand , high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families , patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history . there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study . ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease . patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research . considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) . social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently . the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) . most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) . ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found . the mean of weight at birth in the living cf population was slightly higher than the deceased cf population . cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) . patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) . an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) . it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival . the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients .", "the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research . another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results . data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers . the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time . data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes . this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies .", "this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes . clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas . it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity . couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy . other studies were performed for evaluating the association , if any , between outcome and other demographic factors ." ]

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