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acute generalized exanthematous pustulosis (agep) is a pustular eruption, mainly drug induced, often accompanied by fever and neutrophilic leukocytosis appearing as scarlatiniform erythema over the flexures evolving into numerous tiny non - follicular pustules. we report a case of agep to the epidermal growth factor receptor (egfr) inhibitor, lapatinib. a 56-year old woman presented with erythema, scaling and oozing from the flexures, and erythematous scaly papules and plaques over the back, trunk, thighs and forearms of 4-month duration [figure 1 ]. the skin lesions had started gradually and increased in extent and intensity in the last 1 month. the periphery of the papules and plaques were studded with pustules [figure 2 ]. painful lesions resembling pyogenic granuloma were present over the pulp of the right toe and over the proximal nail folds of both thumbs [figures 3 and 4 ]. she complained of breathlessness, weakness and feverishness inspite of the temperature being normal. in march 2008, she underwent left mastectomy for ductal carcinoma breast (her-2 receptor 3 + - strongly positive) with supraclavicular metastasis detected in may 2009. she developed the lesions described above two months after initiating treatment with the two drugs. upon developing skin lesions, discrete and confluent scaly, erythematous papules over the back scarlatiniform erythema of the flexures, with the periphery of the papules and plaques showing pustules painful pyogenic granuloma - like lesions over the right toe painful pyogenic granuloma - like lesions over the proximal nail folds of the thumbs clinically, pustular psoriasis and agep were considered in the differential diagnoses. lapatinib was withheld for a week and she was treated with topical corticosteroids and antihistamines (amitryptiline) for the burning pain over the finger and toe pulps. investigations revealed anemia (hb-9.6 g %) and increased polymorphs (80%), total count- 10.310/l. biopsy of the pustule and plaque revealed subcorneal and intraspinous collection of neutrophils with spongiosis, upper dermal edema, perivascular inflammatory cell infiltrate of neutrophils, lymphocytes and eosinophils, neutrophilic vasculitis and extravasation of rbc confirming agep [figures 5 and 6 ]. subcorneal and intraspinous collection of neutrophils with spongiosis, upper dermal edema, perivascular inflammatory cell infiltrate of neutrophils, lymphocytes and eosinophils, neutrophilic vasculitis and extravasation of rbc (h&e, 10) dermis with vasculitis and extravasation of rbcs (h&e, 40) after the withdrawal of lapatinib, lesions showed clearing, but she was advised by the oncologist to restart the medication at a lower dose (from 1250 to 750 mg). as lapatinib was reintroduced while lesions were clearing, they recurred on restarting lapatinib but were less severe. systemic prednisolone 30 mg / day, tapered to 10 mg / day over a month, was administered to control the reaction and she was reasonably controlled with this maintenance dose 1 month later, but for erythema and few lesions over the flexures [figure 7 ]. her breathlessness and feverishness subsided and the pyogenic granuloma - like lesions over the proximal nail folds and over the pulp of the toe showed signs of resolution [figures 8 and 9 ]. she subsequently stopped the medication on her own and achieved complete clearance of the erythema. the pyogenic - granuloma - like lesions resolved completely. skin lesions resolving after withdrawal of lapatinib and institution of systemic corticosteroids lesion over the pulp of right toe showing resolution resolution of lesions over the proximal nail folds lapatinib is an oral dual kinase inhibitor belonging to the egfr antagonist group of drugs targeting both the erbb1 and erbb2 receptors. epidermal growth factor receptor or erbb1 is a member of the erbb family is a 170-kd transmembrane protein with an extracellular ligand - binding domain and an intracellular protein tyrosine kinase. upon ligand binding, the phosphorylated protein kinase activates a signal transduction pathway regulating proliferation, differentiation, migration and apoptosis of cells. erbb1 (or her-1) is overexpressed in 27 - 30% of breast tumors and erbb2 (or her-2) is overexpressed in 20 - 25% of breast cancers. lapatinib, a dual tyrosine kinase inhibitor has shown efficacy in breast cancer and as a result its use has been approved, in combination with capecitabine, for the treatment of disease positive for the human egfr. the most frequent adverse effects of lapatinib include diarrhea, rash, nausea and fatigue. the skin rash is characterized by papules and pustules over the face, chest and back resembling folliculitis or an acneiform drug eruption. this appears within 1 and 3 weeks of treatment and peaks at about the fifth week, subsiding within 4 weeks of withdrawal of treatment. dermatologic adverse reactions to egfr inhibitors include follicular or pustular lesions, painful periungual granulation - type or friable pyogenic granuloma - like changes, fissures of the lateral nail folds and/or distal finger tufts, hair changes, dry skin, hypersensitivity reactions and mucositis. our patient had non - follicular pustules and scarlatiniform erythema and scaling with marked accentuation over the flexures, with sparing of the face and seborrheic areas clinically suggestive of agep which was confirmed by histopathology. the skin rash associated with the skin toxicity of egfr inhibitors is follicular and involves the face and seborrheic areas. the painful pyogenic granuloma - like lesions of the proximal nail folds and finger pulp are possibly related to the skin toxicity of lapatinib also described with other egfr inhibitors. pustular psoriasis was considered in the differential diagnosis, but the presence of eosinophils in addition to intraspinous collections of neutrophils and the rapid clearance of lesions after withdrawal of lapatinib, and recurrence after reintroduction favored the diagnosis of agep. also after lapatinib was stopped by the patient on her own leucocytoclastic vasculitis is also reported in a few cases of agep and is not seen in pustular psoriasis. serious adverse skin reactions like stevens - johnson syndrome and agep have been reported with imatinib, a multikinase inhibitor which is a first - line therapy for chronic myeloid leukemia. to date, four cases of imatinib - induced agep have been described and in one patient this been referred to as " scarlatiniform erythema with non - follicular pustules confined to flexural areas. " another patient had " fever, exfoliative dermatitis, and non - follicular pustules. " all these patients developed the eruption within 1 - 3 months after the start of imatinib therapy. imatinib induces agep via inhibition of tyrosine kinases by a mechanism similar to that seen in mercury - induced agep. various adverse reactions have been reported with multikinase inhibitors, whereas reported reactions to egfr inhibitors have been comparatively milder. although agep has been reported with imatinib (a multikinase inhibitor), there have been no reports of serious reactions with lapatinib, an egfr inhibitor. this case could represent the first case report of agep to the egfr inhibitor, lapatinib.	acute generalized exanthematous pustulosis (agep) is a pustular eruption, mainly drug induced often accompanied by fever and neutrophilic leukocytosis presenting as scarlatiniform erythema over the flexures evolving into numerous tiny non follicular pustules. we present a case report of a 56-year old woman, who had undergone mastectomy, treated with lapatinib for metastatic disease, and who presented with multiple erythematous papules and plaques with peripheral pustules. she also developed painful pyogenic granuloma - like lesions over the pulp of toe and over the proximal nail folds.all the lesions subsided following withdrawal of lapatinib. although agep has been reported with imatinib (a multikinase inhibitor), there have been no reports of serious reactions with lapatinib, an egfr inhibitor. this case could represent the first case report of agep to the egfr inhibitor, lapatinib.
post - stroke depression (psd) is one of the symptoms observed in stroke patients. the prevalence of psd is high, as it is diagnosed in 53% of patients at 3 months and in 42% of patients at 12 months after stroke1. additionally, psd has been reported to have a high incidence even 1 month from the onset of stroke2,3,4. psd is the most important reason for impaired quality of life in stroke patients15,16,17. furthermore, several studies have shown that psd is associated with decreased activities of daily living (adl)1,18,19,20,21,22,23,24 and impaired recovery of adl25,26,27,28,29. bhogal.4 and hackett.30 suggested that depressive symptoms differ depending on the stage of stroke recovery (e.g., acute, subacute, or chronic) and the living environment (e.g., in a hospital or in the community). however, little is known about the association between psd and the improvement in adl among patients in a rehabilitation ward setting. sinyor.18 reported that adl were worse in depressed patients than in non - depressed patients on admission and at discharge ; however, both groups showed similar improvement over the course of rehabilitation in a subacute hospital setting, suggesting that the effect of psd on improvement in adl may be minimal. patients in that previous study, however, did not show an equal level of adl between groups on admission, and the study did not distinguish between patients with and without psd on admission. the level of adl on admission has been reported to be related to improvement in adl during the hospital stay31, 32. therefore, to clarify the effect of psd on improvement in adl, there needs to be an equivalent level independence in adl on admission in the psd group and the non - psd group. in another study, chemerinski.27 reported that psd patients showed significantly greater recovery in adl than non - psd patients in the subacute hospital setting. in that study, the level of independence in adl on admission for the psd and non - psd groups was equal ; however, the study had a small sample size. finally, there are no reports on the effects of psd on improvement in various adl domains (i.e., the motor domain and the cognitive domain). therefore, the purpose of this study was to investigate improvement of adl between admission to and discharge from a convalescent rehabilitation ward in patients with or without psd who had equal levels of adl on admission. there were 396 stroke patients who were admitted to and discharged from a convalescent rehabilitation hospital ward from april 4, 2011 to december 26, 2012. on the basis of clinical evaluations, 206 patients with dysphasia, low cognitive function (1 point on the japanese coma scale), or a history of depression were excluded. another 82 patients were missing data on one or more of following items : the brunnstrom recovery stage (brs), geriatric depression scale, short version (gds), and functional independence measure (fim). therefore, 108 patients fulfilled the inclusion criteria for enrollment into the study (fig. this study was approved by the institutional research board of aida memorial rehabilitation hospital (approval no., 014). because of the study s design, there were no risks, disadvantages, or infringements to individual and/or family rights. flow chart of participant selection information obtained from the medical records included sociodemographic variables (i.e., age and gender), laterality of the lesion, primary disease, whether discharge was to home or to a geriatric facility, medication information, illness duration, length of stay, total units of therapy, units of therapy per day, and brs. the brs was assessed for upper limbs, fingers, and lower limbs at the time of admission and at discharge. brs is classified in 6 stages, as follows : (1) flaccidity, (2) synergy development, (3) voluntary synergistic movement, (4) some movements deviating from synergy, (5) independence from basic synergies, and (6) isolated joint movements. the gds has scores ranging from 0 to 15, with a score of > 6 points indicating a likelihood of depression. the short version of the test used in this study is more convenient and less of a burden on the patient. the gds does not examine stroke symptoms (e.g., fatigability, constipation, and decreased appetite)33, 34. as a screening test, the gds is predictive for psd35 and has a high sensitivity for psd as diagnosed by a psychiatrist36. the fim contains 18 items composed of 13 motor tasks and 5 cognitive tasks. the dimensions assessed by the fim include eating, grooming, bathing, upper body dressing, lower body dressing, toileting, bladder management, bowel management, bed - to - chair transfer, toilet transfer, shower transfer, locomotion (ambulatory or wheelchair - bound), stairs, comprehension, expression, social interaction, problem solving, and memory. tasks are rated on a 7-point ordinal scale that ranges from total assistance to complete independence. scores range from 18 (lowest) to 126 (highest), indicating the level of function. scores were rated on admission and at discharge. the reliability and validity of the fim have already been confirmed in stroke patients37. based on standard a cut - off score of 6 for the gds at the time of admission, patients were divided into the non - psd group or the psd group, and the two groups were compared. the degree of improvement in the fim (fim gain) was defined as the difference between the fim score on admission and at discharge. fim scores were compared in total (fim total), by the subscale of motor activities (fim motor), and by the subscale of cognition (fim cognition). tests were used to compare differences between groups for gender, laterality of the lesion, primary disease, whether discharge was to home or to a geriatric facility, and medication. an independent t - test was used to compare age, illness duration, length of stay, total units of therapy, and units of therapy per day between groups. the mann - whitney u - test was used to compare fim total, fim motor, fim cognition, fim gain scores, and brs between groups. analysis of variance (anova) was used to examine the effect of the time period on psd. fim total, fim motor, and fim cognition scores were analyzed with a repeated - measures two - way anova, with psd (non - psd / psd) and the time period (admission / discharge) being within - subject variables. factors that affected fim scores at the time of discharge were examined using multiple regression analysis. explanatory variables were gds, age, length of stay, illness duration, units of therapy per day, and brs of the lower limbs on admission. explanatory variables (length of stay, total units of therapy, brs of upper limbs and fingers on admission and at discharge, and brs of lower limbs at discharge) that caused multicollinearity were eliminated. data were analyzed using the japanese version of spss statistics for windows version 20.0 (ibm corporation, new york, ny, usa). at the time of admission, 45 patients (42%) were classified into the non - psd group, and 63 patients (58%) were classified into the psd group. with respect to the sociodemographic variables, none (age, gender, laterality of the lesion, primary disease, discharge to home or to a geriatric facility, medication information, illness duration, length of stay, total units of therapy, units of therapy per day, and brs) was significantly different between the two groups (table 1table 1.demographic datanon - psd group (n=45)psd group (n=63)age, years66.3 15.267.2 14.7gender, nmale : 31 female : 14male : 39 female : 24laterality of lesion, nright : 17right : 20left : 24left : 39bilateral : 4bilateral : 4primary disease, nch : 14ch : 23ci : 28ci : 39sah : 3sah : 1discharge destination, nfacility : 2facility : 10home : 43home : 53medication information(antidepressant / ataractic), nnothing : 41nothing : 55antidepressant : 1antidepressant : 4ataractic : 3ataractic : 4illness duration, day39.8 39.843.9 13.7length of stay, day94.6 44.299.6 43.4total units of therapy, units608.4 259.9616.8 235.7units of therapy per day, units6.6 1.26.4 1.0gds, score3 (05)9 (615)brs (admission)upper limb, score5 (16)5 (16)hand, score4 (16)5 (16)lower limb, score5 (26)5 (16)brs (discharge)upper limb, score5 (16)5 (16)hand, score5 (16)5 (16)lower limb, score6 (26)5 (26)scores are presented as mean sd, median (minimum to maximum). one unit is defined as 20 minutes of rehabilitation. tests were used to compare differences between groups for gender, laterality of the lesion, primary disease, discharge destination, and medication information. independent t - tests were used to compare differences between groups for age, illness duration, length of stay, total units of therapy, and units of therapy per day. brs : brunnstrom recovery stage ; ch : cerebral hemorrhage ; ci : cerebral infarction ; gds : geriatric depression scale short version ; psd : post - stroke depression ; sah : subarachnoid hemorrhage ; sd : standard deviation). tests were used to compare differences between groups for gender, laterality of the lesion, primary disease, discharge destination, and medication information. independent t - tests were used to compare differences between groups for age, illness duration, length of stay, total units of therapy, and units of therapy per day. brs : brunnstrom recovery stage ; ch : cerebral hemorrhage ; ci : cerebral infarction ; gds : geriatric depression scale short version ; psd : post - stroke depression ; sah : subarachnoid hemorrhage ; sd : standard deviation when the fim scores on admission were compared, there was no significant difference between the two groups. when the scores for fim total (p=0.003), fim motor (p=0.002), and fim cognition (p=0.011) at the time of discharge were compared, there were significant differences between the two groups. the psd group had worse scores than the non - psd group did. when the fim gain score was compared between groups, there were significant differences in fim total (p=0.022) and fim motor (p=0.035) between the two groups. however, there was no significant difference between the two groups in the fim gain score for cognition (p=0.317) (table 2table 2.comparison of fim scores in the psd group and the non - psd groupnon - psd group(n=45)psd group(n=63)admissionfim total, score76.7 16.772.4 21.7fim motor, score50.6 14.047.5 18.4fim cognition, score26.1 6.324.9 5.8dischargefim total, score110.1 6.791.2 25.2fim motor, score80.3 5.465.4 22.1fim cognition, score29.7 3.827.5 4.7gainfim total, score33.3 12.926.8 16.1fim motor, score29.7 11.024.5 13.4fim cognition, score3.6 4.72.3 5.5p<0.05, p<0.01scores are presented as means sd.fim scores were analyzed between groups with the mann - whitney u test.fim : functional independence measure ; psd : post - stroke depression). fim : functional independence measure ; psd : post - stroke depression the anova revealed significant interactions between the time period and psd factors for fim total (f [1, 106]=5.07, p=0.030) and fim motor (f [1, 106]=4.61, p=0.030) (table 3table 3.results of a two - way anova in the psd group (n=63) and the non - psd group (n=45)time periodinteractionadmissiondischargemean sdmean sdfim total, scorenon - psd76.7 16.6110.1 14.7psd72.3 21.799.1 22.2fim cognition, scorenon - psd26.1 6.229.7 5.1psd24.9 5.827.2 5.6fim motor, scorenon - psd50.6 13.980.3 11.7psd47.4 18.371.9 18.5p<0.05. anova : analysis of variance ; fim : functional independence measure ; psd : post - stroke depression ; sd : standard deviation). p<0.05. anova : analysis of variance ; fim : functional independence measure ; psd : post - stroke depression ; sd : standard deviation the multiple regression analyses for fim total and fim motor were significant for gds, age, and brs (p<0.05). the multiple regression analysis for fim cognition was significant for gds, age, and illness duration (p<0.05). the multiple regression analysis for gds was significant for fim total (r=0.28, =0.22, p=0.011), fim motor (r=0.30, =0.19, p=0.025), and fim cognition (r=0.19, =0.22, p=0.014) (table 4table 4.results of a multivariate regression analysis to identify factors related to fim score in the patients (n=108)dependent variableexplanatory variablefim totalfim motorfim cognitiongds0.220.190.22age0.400.370.34illness duration0.130.100.19units of therapy of per day0.060.040.10brs (lower limb, admission)0.290.380.08adjusted r0.280.300.19p<0.05, p<0.01, : standardized coefficient ; brs : brunnstrom recovery stage ; gds : geriatric depression scale, short version ; fim : functional independence measure). p<0.05, p<0.01, : standardized coefficient ; brs : brunnstrom recovery stage ; gds : geriatric depression scale, short version ; fim : functional independence measure adl improvement was investigated in patients who were categorized into the non - psd or psd groups on admission in a convalescent rehabilitation ward setting. in general, the results indicated that psd decreased both the level of adl independence at discharge and adl improvement in stroke patients. in this study, there were no significant differences in adl between patients with and without psd on admission, but there were significant differences in adl at discharge and in the degree of improvement in adl. these results indicate that there is less of a restorative effect on adl in psd patients. in addition, there was a statistically significant interaction between the time period and psd in fim scores. this finding supports the idea that psd is related to adl recovery and that it adversely affects a patient s prognosis for adl improvement in a convalescent rehabilitation ward setting. generally, the effects of the gain in adl score are associated with age and the intensity of rehabilitation38, 39. therefore, differences in age and intensity of rehabilitation were controlled between psd patients and non - psd patients. however, there was a statistically significant difference in the improvement of adl between the two groups, which also indicated that psd affects the improvement in adl. several studies have also shown that there is impaired recovery of adl in psd patients compared with that in non - psd patients25,26,27, similar to the results shown here in the setting of a convalescent rehabilitation ward. on the other hand, in the multiple regression analysis, gds, age, and brs influenced the adl at the time of discharge. other studies have also reported that the adl of stroke patients were influenced by age and the degree of paralysis of the lower limbs39,40,41. the results of the present study indicate that the level of adl independence at discharge was related not only to these factors but also to the severity of psd at admission. feigin.42 investigated the course of stroke - related function and concluded that depression was associated with adl independence at 5 years after stroke. accordingly, this study concluded that psd had a negative impact on recovery of adl and led to negative outcomes with respect to rehabilitation in a convalescent rehabilitation ward. in addition, the present results revealed that psd particularly inhibited fim motor adl, which may be due to psychological rather than to physiological mechanisms. for example, depressed patients may be hopeless about the future and thus may be psychologically less motivated to put any effort into rehabilitation or recovery27. in addition, because training may cause mental stress in psd patients, it is important to train by avoiding mental load and to listen to the complaints of patients43. high - intensity training is needed to promote recovery of adl44, and such training is often difficult for psd patients. in addition, depression leads to reduced attention and to an increased risk of falling45, 46. therefore, the falling risk induced by psd may be associated with low independence levels in adl. however, this study indicated a poor outcome with respect to rehabilitation in patients who had psd on admission to a convalescent rehabilitation ward. previous studies have shown that alleviation of depressive symptoms is associated with better functional recovery27, 49. therefore, early detection of psd by screening and early treatment may lead to better functional recovery in patients in convalescent rehabilitation wards. the study sample was only from one convalescent ward, and it excluded patients with aphasia and mild paralysis. furthermore, this study did not assess the effects of higher brain dysfunction and balance, which are related to adl and gait ability50,51,52,53,54,55,56,57,58,59,60,61.	[purpose ] there have been no investigations into the improvement of activities of daily living among patients suffering from post - stroke depression on admission to convalescent rehabilitation wards in japan. this study aimed to assess the improvement of activities in daily living in patients with or without post - stroke depression at the time of admission to a convalescent rehabilitation ward. [subjects and methods ] this retrospective study included 108 stroke patients divided into two groups according to their geriatric depression scale 15-item short form scores. activities of daily living were assessed using the functional independence measure. the degree of improvement on the functional independence measure was defined as the difference between scores on admission and at discharge. [results ] the functional independence measure gain score was significantly different from the functional independence measure total score. there was a significant interaction between time period and post - stroke depression factors for the functional independence measure total score. a multiple regression analysis revealed a significant association between geriatric depression scale score and functional independence measure total score. [conclusion ] the present study suggests that post - stroke depression has a negative impact on recovery of activities of daily living and on rehabilitation outcomes in a convalescent rehabilitation ward setting.
aging is an important risk factor for metabolic disorders, including obesity, impaired glucose tolerance, and type 2 diabetes (t2d). it has been reported that the prevalence of t2d increases with age and peaks at 6074 [24 ]. almost one third of the elderly have diabetes and three quarters have diabetes or prediabetes. the higher incidence of diabetes is especially alarming considering that diabetes in itself increases the risk for multiple other age - related diseases such as cardiovascular disease (cvd), atherosclerosis, stroke, alzheimer disease (ad), parkinson 's disease, nonalcoholic fatty liver disease (nafld), and cancer. the pathogenesis of t2d in aging is characterized by two major features : peripheral insulin resistance and impaired insulin secretion from cells. here, we review how aging predisposes to diabetes and impaired glucose tolerance through effects on insulin secretion and insulin action. age - related defects in insulin secretion have been demonstrated in rodents as well as humans. glucose and amino acid are major stimuli for insulin release from the pancreatic cell. with aging, there is a decrease in insulin secretion following stimulation with glucose as well as amino acid arginine. decrease in glucose - stimulated insulin secretion (gsis) in vivo has been shown in rodents using the state - of - the - art hyperglycemic clamps. in humans, disorderly insulin release, a decrease in insulin pulse amplitudes, and decreased response to glucose oscillations as well as alterations in insulin clearance have all been demonstrated. when the insulin secretory defects are superimposed over an increased need for insulin as in old age, impaired glucose homeostasis, glucose intolerance, and diabetes can result. we have demonstrated in rodent models that older rodents are unable to increase insulin secretion in proportion to the increased demands imposed by insulin resistance, thus contributing to impaired glucose tolerance. similarly, studies in humans have demonstrated a secretory defect that is consistently observed even after controlling for insulin action. many factors contribute to the decrease in insulin secretion in aging, including the age - associated loss of sirt1-mediated gsis, decreased -cell sensitivity to circulating incretins, age - associated decrease in mitochondrial function, as well as increased oxidative stress. in this section of the review, we will specifically address age - related decline in various aspects of -cell function and mass that could contribute to the observed defects in insulin secretion. insulin, secreted from pancreatic cells, is the major hormone in regulating glucose homeostasis. the secretion of insulin from cells is a complex process involving the integration of multiple stimuli, such as nutrients, hormones, neurotransmitters, and drugs, but the primary stimulus for insulin secretion is circulating glucose. aging is associated with a marked decline in gsis in both humans and rodents [13, 14 ] and the impairment of gsis is one of the hallmarks of t2d. regulatable steps involved in glucose - induced insulin secretion (as shown in figure 1) : (1) glucose is transported into the cells through the translocation of the glucose transporters (gluts), especially glut2 ; (2) generation of atp through the oxidation of glucose ; (3) elevation the ratio of atp / adp induces closure of cell - surface atp - sensitive k (katp) channels, leading to cell membrane depolarization ; (4) extracellular ca influx into the cell ; (5) a rise in cytosolic ca triggering the exocytosis of insulin granules as shown in figure 1, initiation of the glucose transport is the first step that links glucose metabolism to insulin release in the cell. glut2 is the major glucose transporter expressed in pancreatic cells and ensures large bidirectional fluxes of glucose and other dietary sugars, such as fructose and galactose, in and out the cell due to its low affinity and high capacity. loss of pancreatic -cell glut2 expression in humans is associated with hyperglycemia and impaired gsis, and the loss of gsis directly correlates with decreased expression of the cell glut2 in several rodent t2d models. hou. observed that high extracellular glucose concentrations enhance glut2 endocytosis, which leads to the insulin secretion in glut2 overexpressed -cell line. however, the internalized glut2 protein undergoes rapid degradation induced by chronic high - glucose stimulation, which indicates that hyperglycemia directly affects cells function. moreover, mice lacking glut2 in pancreatic cell display an almost complete absence of first phase glucose stimulated insulin secretion. taken together, these studies suggest that glut2 is essential for gsis and lack of glut2 causes hyperglycemia. glut2 expression was diminished in very old animals compared with juvenile and adult rhesus monkeys, implying the potential connection between age and glut2 expression level. age - associated decrease in expression of glut2 has been demonstrated in aged rodent models along with other cell specific genes. the study by ohneda. shows that glut2 is underexpressed with increased age ; however, neither the magnitude of the underexpression of glut2 nor of the reduction in glut2 transport function in islets of goto - kakizaki (gk) rats is sufficient by itself to explain the profound reduction in gsis. compared to rodents, recent evidence demonstrates that human cells express three glucose transporters, glut1, 2, and 3. the higher levels of glut1 and glut3 may introduce differences in the regulation of glucose sensing in humans versus rodent islets. after uptake into cells, glucose undergoes oxidation and eventually generates atp in cytosol and mainly mitochondria via the citric acid cycle also known as the tricarboxylic acid (tca) cycle, or the krebs cycle (figure 1). in the pancreatic cells, glucose oxidation results in the increase of atp production which is required for insulin secretion. islets from t2d patients exhibit lower atp content and blunted gsis, implicating the mitochondria in the pathogenesis of cell dysfunction. the metabolism of glucose is initiated by its phosphorylation by glucokinase (gck), a member of a family of evolutionary and structurally related hexokinases. the reaction catalyzed by gck is the first reaction in glycolysis as well as the first rate - limiting reaction in the metabolism of glucose. the gene expression level of gck was significantly increased with age in healthy rats, suggesting a potential mechanism by which cells attempt to overcome age - associated glucose intolerance and insulin resistance. interestingly, there is evidence to show that glucose oxidation rates are lower in older animals. by using [1-c ] and [6-c ] glucose - incubated islets isolated from pancreases of 2-month and 12-month - old rats, g. m. reaven and p. d. reaven demonstrated that the amount of glucose converted to co2 by islets from 12-month - old rats was only half that of 2-month - old rats. in humans, there is a lower glucose oxidation but higher lipid oxidation rates in elderly than in the young, suggesting that an enhanced randle cycle may play a major role in producing a reduction in insulin - mediated glucose oxidation. macdonald has pointed out that pancreatic islets contain 4070 times the activity of mitochondrial glycerophosphate dehydrogenase (gpdh) compared to other tissues, implicating the potential importance of gpdh in islets. since gpdh plays a crucial role in transporting and reducing equivalents from cytosol to mitochondria, a decrease in its activity leads to an accumulation of cytosolic nadh and consequently, an increase in the cytosolic nad / nad+ ratio, a decrease in glycolysis, and a reduction in gsis. an approximate 50% reduction in the activity of gpdh in islets of 12-month - old compared with 2-month - old rats, suggesting a role for gpdh in diminished gsis in aging. increased atp / adp ratio from glucose oxidation reduces the whole cell k permeability, leading to cell membrane depolarization and extracellular ca influx into the cell. these changes are thought to be mediated through the atp - sensitive k (katp) channels and voltage - dependent ca channels (figure 1). studies from ammon and colleagues have shown that raising the glucose concentration from 3 to 5.6 and 16.7 mm had no effect on k efflux from islets of 24-month - old male rats whereas that from 24-month - old female rats were decreased. also, net uptake of ca was significantly diminished in islets of 24-month - old compared to islets of 3-month - old rats. in the presence of 16.7 mm of glucose, islets of 24-month - old rats exhibited only 6070% of the insulin release obtained with islets from 3-month - old rats. these data suggest that the decreased insulin secretory response to glucose during old age is due, at least in part, to inadequate inhibition of k efflux and diminished net uptake of ca. however, it is important to consider that the differences in ion fluxes could relate to age - related deficits in the steps leading up to ion flux such as decreased glucose transport and oxidation. the final step of insulin secretion is the exocytosis of insulin granules (figure 1). insulin is stored in large dense core vesicles (ldcvs), also called insulin granule, and released by exocytosis, a multistage process involving vesicle trafficking, docking, and eventually fusion with the plasma membrane. ca regulates several steps in exocytosis, such as the size of vesicle pools, the fusion event, and the size of the fusion pore, and may act on distinct protein targets [31, 32 ]. since the net uptake of ca is decreased with age, it is reasonable to speculate that insulin granule exocytosis is also inhibited by age. to date, insulin secretion is known to involve the same soluble n - ethylmaleimide, sensitive factor attachment protein receptor (snare) isoforms as those utilized in synaptic vesicle exocytosis and neurotransmitter release, namely, syntaxin 1, vesicle - associated membrane protein (vamp) 2, and synaptosomal - associated protein of 25 kda (snap-25) [3337 ]. the exocytosis is induced by the pairing of snare proteins on the vesicle membrane, termed v - snare (such as vamp2), with cognate proteins on the target membrane, the t - snares (syntaxin and snap-25). in addition, other proteins such as munc18/sec1 and munc13 have also been reported to be involved in the regulation of insulin granule exocytosis [38, 39 ]. vanguilder and colleagues showed that neurotransmission - regulating proteins such as vamp2, syntaxin1, and snap-25 decline with age in hippocampal synaptosomes in rats. altered synaptic protein expression may decrease stimulus - induced neurotransmission and vesicle replenishment during prolonged or intense stimulation, two processes that are necessary for learning and the formation and perseverance of memory. however, it is still unclear whether these proteins also decline with age in pancreatic cells. the proliferation and apoptosis of cells and islet neogenesis are three major factors that tightly regulate -cell mass. as shown in figure 2, in this section, we will review the effects of aging on these factors. it has been shown that age correlates with decreased proliferative activity and enhanced sensitivity to glucose - induced -cell apoptosis (figure 2). in cultured islets from 2 to 3-month - old rats, increasing glucose from 5.5 to 11.1 mm decreased -cell apoptosis and increased -cell proliferation, which was further augmented when glucose was increased to 33.3 mm. in contrast, in islets from 7 to 8-month - old rats, increasing glucose concentrations from 5.5 to 33.3 mm induced a linear increase in -cell death and a decrease in proliferation. this has also been observed in cultivated human islets where age correlated positively with the sensitivity to glucose - induced cell apoptosis and negatively to baseline proliferation. it is reported that there is a three - to - ten - fold increase in -cell apoptosis in diabetic patients compared to nondiabetic individuals as detected by tunel staining. maedler. demonstrated that high glucose induces apoptosis in human pancreatic cells through inducing the expression of fas and activation of caspase 8 and 3. report that relative -fcell volume in human pancreatic islets remains constant with aging and -cell replication decreases age dependently, while -cell apoptosis does not change significantly. however, the most recent study performed on 4-month, 14-month and, 24-month - old wistar rats has shown that there is a significant reduction in -cell proliferation and increase in apoptosis in cells in the islets. the study also showed that age associated decreases in activities of many antioxidant enzymes and suggests that an increase in oxidative stress in the cells contributes to the increased apoptosis. islet amyloid polypeptide (iapp) or amylin is cosecreted with insulin from cells. amylin suppresses glucagon secretion and helps to regulate glucose homeostasis. in insulin - resistant states, careful analyses show that the amylin could aggregate into amyloid plaques that increase -cell apoptosis leading to reduced islet volume and -cell mass, and subsequent diabetes. accumulating evidence implicates toxic iapp oligomers in the mediation of cell apoptosis in t2d. in support, freshly dissolved human iapp (hiapp, but not rodent iapp) induces apoptosis when added to cells in culture. although research has shown that with age there is an increased deposition of amylin in the islets of diabetic individuals but not nondiabetic individuals, it is still too early to rule out the potential influence of aging on amylin aggregation. -cell mass normally grows well into adulthood to provide increased insulin secretion capacity to match the greater insulin requirements of maturity. -cell mass can slowly expand in adult rodents in response to increased insulin requirements or during pregnancy. several mechanisms have been invoked to explain adult -cell mass expansion, including neogenesis from pancreatic ducts or hematopoietic tissues, replication of specialized -cell progenitors, and self - renewal by cell [54, 55 ]. -cell proliferation and the capacity of cell to regenerate declines with age in mice. the rate of -cell proliferation gradually declines with aging in rats to a steady state by 7 months of age. young mice respond to high - fat diet by increasing -cell mass and proliferation and maintaining normal blood glucose levels. old mice, in contrast, do not display any increases in -cell mass or -cell proliferation in response to high - fat diet and become diabetic. there is a four - fold increase in cell proliferation in young mice after the administration of streptozotocin (stz), a chemical that is toxic to the cells in mammals and normally is used for inducing insulin secretion deficiency models in rodents, whereas no changes are observed in older mice. similarly, stz stimulated -cell replication in young mice but had little effect in old mice. partial pancreatectomy greatly stimulated -cell proliferation in young mice but failed to increase -cell replication in old mice. p16 is an effector of senescence and a potent inhibitor of proliferative kinase cyclin - dependent kinase 4 (cdk4), which is essential for pancreatic cell proliferation in adult mammals [60, 61 ]. another cyclin - dependent kinase, cdk6, is not expressed in mouse islets but is very effective in driving cell replication in human islets. lack of cdk4 expression in mice leads to insulin - deficient diabetes. to perform their kinase activity, cdks bind some kinds of regulatory proteins called cyclins. without cyclins, cdks have little kinase activity. mouse cells express 3 d - cyclins, termed cyclin d1, d2, and d3 ; d2 is the most abundant one whereas d3 is nearly undetectable. however, -cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin d2 mice, causing glucose intolerance that progressed to diabetes by 12 months of age. although cyclin d1 mice never developed diabetes, life - threatening diabetes developed in 3-month - old cyclin d1 d2 mice as -cell mass decreased after birth. thus, cyclins d2 and d1 were essential for cell expansion in adult mice. in contrast, in the human cell, cyclin d3 is highly expressed, whereas cyclin d1 and d2 levels are much lower. overexpression of cyclin d3 in isolated human islets, especially in combination with cdk6, induced the greatest increase in -cell proliferation when compared with over - expression of other cyclins. although cell cycle proteins play crucial roles in the proliferation of pancreatic cells, less is known about the effects of aging on the expression or / and activity of cyclins and cdks ; therefore, studies in this area may contribute to a better understanding of the relationship between aging and loss of -cell mass. cell proliferation is self - replication of the cell, whereas islet neogenesis is the differentiation of progenitor cell or transdifferentiation of pancreatic non- cells to cells. neogenesis of islets occurring during normal embryonic development and in very early postnatal life can lead to -cell mass expansion. in addition to fetal and neonatal periods, cell neogenesis has been shown to be important in increasing -cell mass in the adult during periods of increased insulin demand such as obesity and pregnancy. rosenberg. developed a model for islet neogenesis in adult mammalian pancreas in the 1980s, which showed that pancreatic ductal cells can be induced to differentiate into normal functioning adult endocrine cells. -cell neogenesis may occur through two pathways, stem / progenitor cell activation and transdifferentiation of adult pancreatic cells. islet neogenesis - associated protein (ingap), found in pancreatic exocrine secretions, appears to stimulate the growth and proliferation of duct cells and their differentiation into endocrine cells [7274 ]. ingap and a bioactive 15 amino acid synthetic peptide (ingap peptide) are inducers of islet neogenesis in a human islet system. other stimuli have been demonstrated to exert neogenic effects on the endocrine pancreas. the combination of epidermal growth factor (egf) and gastrin has been shown to stimulate islet neogenesis in both animal and human studies. glucagon - like peptide 1 (glp-1), an incretin from enteroendocrine l cells of the intestine, has been shown to induce islet neogenesis in rodents. although the potential for -cell replication appears to decline substantially with age as evidenced by decreased pdx expression, the rate of islet neogenesis (expressed as percentage of insulin positive duct cells) is not affected by aging in humans. in summary, various aspects of -cell mass and function decline as a feature of age, thus contributing to the age - associated defects in insulin secretion. this defect when superimposed for an increased need for insulin, could contribute to impaired glucose homeostasis, glucose intolerance and diabetes. in the subsequent section, we will discuss and review literature on age - associated deterioration of insulin action. insulin action is the ability of insulin to bind to its receptors located on tissues including muscle, liver, and adipose tissue and initiate signaling effects. the net physiological metabolic effects that result from insulin signaling include (a) regulation of glucose homeostasis through a decrease in hepatic glucose output (via decreased gluconeogenesis and glycogenolysis) and increase in glucose uptake, primarily into striated muscle and adipose tissue as well as (b) increase in lipid synthesis in fat cells, and attenuation of release of free fatty acid from triglycerides in fat. insulin resistance results when normal circulating concentrations of the hormone are insufficient to regulate these processes appropriately. it is well documented that aging is associated with a decline of insulin action [7779 ]. the decline in insulin action with age is thought to contribute to the high prevalence of impaired glucose tolerance and type 2 diabetes among the elderly [80, 81 ]. notably, some studies support the hypothesis that the decline in insulin action in the elderly persons is related to increased abdominal fat rather than to aging per se. however, other studies suggested that age - associated insulin resistance may not be explained solely by concomitant abdominal obesity. abdominal fat (also called visceral fat, vf), which is located in and around the viscera, has been demonstrated to be strongly related to many health conditions, including cvd, insulin resistance, and t2d. several cross - sectional studies suggest that visceral fat increases throughout the lifespan in men and women of all ages and race, independent of increases in body weight [8588 ]. many factors contribute to the increased vf seen with aging such as physiologic decline in gh / igf-1 axis, decrease in sex steroids as well as sedentary life style. the mechanisms how vf links to the metabolic syndrome are still not entirely clear, but it has been suggested to involve its anatomical location, leading to a portal. showed that extraction of vf from 20-month - old fischer 344 brown norway (fbn) rats was sufficient to restore peripheral and hepatic insulin action to the levels of young rats. moreover, removal of vf in zucker diabetic fatty (zdf) rats prevented the progressive decrease in insulin action and delayed the onset of diabetes, but vf extraction did not alter plasma free fatty acid levels. borst. reported that vf removal in sprague dawley (sd) rats tended to improve glucose tolerance and lowered some pro - inflammatory adipokines in serum ; these animals displayed increased insulin - stimulated glucose transport in excised soleus and digitorum longus muscles as compared to control group. these studies provide verification that vf is a potent modulator of both hepatic and peripheral insulin action. calorie restriction (cr) extends life span and retards age - related chronic diseases in a variety of species, including rats, mice, fish, flies, worms, and yeast. our studies have shown that a reduction in fat mass, specifically vf, may be one of the possible underlying mechanisms of the antiaging effect of caloric restriction. nowadays, adipose tissue is recognized as an active metabolic - endocrine organ, and obesity is considered as a low - grade inflammatory condition strongly linked to adverse metabolic outcomes. a putative key link between increasing fat mass and obesity - related complications, including insulin resistance, is a chronic low - grade inflammatory state within adipose tissue, related to infiltration by macrophages. we and others have shown that vf depots display a unique profile of inflammatory mediators compared to subcutaneous adipose tissue, including the clearly higher expression levels of macrophage migration inhibitory factor (mif) and chemokine receptor 2 in vf [96, 97 ]. other studies also suggested that vf is a stronger risk factor for metabolic disorders and mortality than subcutaneous fat [82, 98 ]. mif, a proinflammatory cytokine, can activate nuclear factor (nf)-b signaling but directly inhibits the function of p53 [99, 100 ]. the possible explanation is aging is associated with inflammation and inflammation could accelerate aging process, whereas lack of mif could downregulate nf-b, mediated inflammatory signaling, which will subsequently mitigate the process of aging. macrophages are considered to be a significant source for many fat - derived proinflammatory cytokines, and the percentage of macrophages in fat has been shown to increase in obesity. interestingly, our study suggested that the percentage of macrophages in the stromal vascular cell fraction from both visceral and subcutaneous fat increased with age regardless of obesity status. taken together, increase of vf is a hallmark of aging and a source of increased chronic inflammation. inflammation could accelerate the aging process and eventually lead to the metabolic dysfunction. breaking this vicious cycle by decreasing the vf will be a potential therapeutic method for treating metabolic and related diseases (figure 3). lack of or resistance to insulin leads to two metabolic crises : a marked increase in the rate of lipolysis in adipose tissue and activation of hepatic gluconeogenesis in spite of high plasma glucose levels. the increased rate of lipolysis increases circulating ffa levels, which, in turn, exacerbates insulin resistance in the whole body. it has been very well documented that the acute elevation of plasma ffa produces insulin resistance in both diabetic and nondiabetic individuals [106108 ]. it is also shown that chronically elevated plasma ffa levels cause insulin resistance, and lowering elevated plasma ffa levels overnight normalizes insulin sensitivity in obese nondiabetic subjects and significantly improves insulin sensitivity in obese diabetic patients. the mechanisms by which elevated levels of ffa produce insulin resistance have not been fully understood. however, studies have shown that increasing plasma ffas acutely decreases insulin - stimulated glucose uptake and glycogen synthesis in human. it is also reported that increase of ffa level in human inhibits pi3 kinase activity in skeletal muscle, suggesting the impairment of insulin signaling by ffa. pointed out that, with the infusion of ffa, increased accumulation of diacylglycerol (dag) and protein kinase c (pkc) activity in muscle contribute to the impairment of insulin signaling, potentially through activation of nf-b. the steatotic liver is also resistant to insulin in terms of inhibition of hepatic glucose production and stimulation of glycogen synthesis. the high ffa levels may be the unifying mechanism that accounts for the insulin resistance in obesity, type 2 diabetes, lipodystrophy, and aging. we and others have shown that the circulating ffa levels are significant higher in 9- to 20-month - old sd rats compared to 3-month old, demonstrating that circulating ffa increases with age. although peripheral insulin resistance is a hallmark of the development of t2d, more recent evidence has shown that insulin resistance also exists in central nervous system (cns), and that central insulin action plays an important role in regulating whole body glucose metabolism. like peripheral tissues, molecules in insulin signaling such as insulin receptor (ir), insulin receptor substrates (irs), and phosphatidylinositol 3-kinase (pi3k) are universally expressed in the brain, indicating a potential role of insulin signaling in the brain. koch. showed that mice lacking ir in cns exhibit significantly more severe impairment of peripheral glucose homeostasis than mice lacking ir in the peripheral tissues. has shown that central infusion of pi3k inhibitor attenuated insulin - induced glucose lowering by 35%40% in both acute and chronic insulin treatment paradigms, while hypothalamic overexpression of either irs-2, a upstream kinase of pi3k, or protein kinase b (pkb / akt), a key downstream mediator of pi3k action, enhanced the glycemic response to insulin by 2 folds in stz - induced diabetic rats, suggesting that hypothalamic insulin signaling is an important determinant of the response to insulin in the management of uncontrolled diabetes. interestingly, an increasing body of evidence shows a link between diabetes and ad, a neurodegenerative disorder and the most common form of dementia. it has been reported that patients with t2d increase the prevalence of ad by two - to - three folds, and insulin levels and insulin activity in the central nervous system are reduced in ad. studies in human subjects show that both peripheral and central administration of insulin improves memory in ad patients [118121 ], suggesting impairment of insulin signaling in the brain as a risk factor of neurodegenerative disorders, and restoration of insulin signaling could be a potential therapy for ad. fernandes. demonstrated that the protein levels of elements in the insulin signaling pathway such as irs and src homology adaptor protein (shc) did not change significantly in the forebrain cortex and cerebellum of rats aged 1 d to 60 wk. however, insulin induced tyrosine phosphorylation of ir and shc, and the association of shc / growth factor receptor binding protein-2 (grb2) decreased significantly in both types of tissues. other studies showed that intracerebroventricular administration of insulin was more efficient at reducing food intake and body weight in 3-month - old rats than in 8- and 24-month - old rats, indicating the development of hypothalamic insulin resistance with age in wistar rats. furthermore, the tyrosine phosphorylation of ir and irs-2 and the phosphorylation of downstream target genes such as foxo1 and p70s6k declined, whereas serine phosphorylation of ir and irs-2 increased with age in rat hypothalamus. aging - associated increase in central and peripheral insulin resistance could contribute to both diabetes and ad. the field of central insulin resistance and its role in the development of neurodegenerative disorders and the control of whole body glucose homeostasis is complicated and further studies are needed to fully understand the underlying mechanisms. for a detailed review of the insulin signaling in the brain, it is clearly established that the risk for impaired glucose tolerance and diabetes increase with age in rodents and humans. the specific factors such as increased vf and circulating ffa that contribute to impaired insulin action and various defects in -cell mass / function have been highlighted in the previous section. however, the integrated whole body glucose homeostasis is complex with various age - related parameters playing a crucial role on both aspects of glucose homeostasis, namely, insulin action and insulin secretion. leptin, a hormone secreted from adipose tissue, plays a key role in energy intake and expenditure. deficiency of leptin and its receptor leads to severe obesity, insulin resistance, and diabetes in rodents and humans. resistant to the effects of leptin, termed leptin resistance, is seen in obesity and aging. leptin resistance associated with aging [127129 ] and decline in growth hormone (gh)/insulin - like growth factor (igf)-1 axis could play a key role in the alterations of glucose homeostasis in aging. accumulating evidence suggests that endoplasmic reticulum (er) stress plays a role in the pathogenesis of diabetes, contributing to both pancreatic -cell function and peripheral insulin resistance. it has been reported that aging is related to increase in proapoptotic markers with er stress in multiple tissues, including lung, liver, kidney, and brain [131, 132 ]. in the past decade, a family of nicotinamide adenine dinucleotide- (nad-) dependent protein deacetylases, termed sirtuins, have been shown to contribute to longevity. interestingly, overexpression of sirtuins or treat with activators of sirtuins, such as resveratrol protect against metabolic decline in aging, increases insulin sensitivity, increases insulin secretion, improves life quality, and extends lifespan [11, 117, 133 ]. in addition to the above - mentioned variables, aging - associated sedentary life style and diminished physical activity may be important factors for age - related changes of glucose homeostasis. research has shown that healthy elderly with greater degrees of physical fitness have better glucose tolerance and lower level of insulin resistance than less active old people. in addition, aging is associated with defects in the balance of insulin secretion and insulin action (demands). in the young, a hyperbolic relationship exists between insulin secretion and insulin sensitivity, whereby pancreatic cell compensates for changes in whole - body insulin sensitivity through a proportionate increase in insulin secretion. our data shows that compared to younger animals, when challenged with a prolonged hyperglycemic stimuli older animals are unable to maintain the insulin secretion proportional to the degree of resistance. whole body glucose homeostasis is a complex balance of glucose production and utilization by different tissues. food intake and hepatic glucose production are the two sources of glucose production, while skeletal muscle contributes to the majority of the glucose uptake and utilization. utilizing tracer technology, it is possible to differentiate between the effects on glucose production (liver) and glucose utilization (primarily the muscle). hepatic glucose production (hgp) plays crucial roles in glucose homeostasis, both in the fasting and postprandial states. in contrast to rodents, where there is an increase in hgp in age, there are no differences in either the basal hepatic glucose production or the dose - response curve of its suppression by insulin between young and old individuals. the european group for the study of insulin resistance reported that hepatic glucose production does not increase with age, when adjusted for lean body mass. furthermore, hepatic glucose output has not been shown to be increased in elderly patients with t2d. thus, hepatic insulin resistance does not seem play a significant role in decreased glucose tolerance of elderly people. as mentioned earlier, glut4 is the major glucose transporter in skeletal muscle responsible for insulin - stimulated glucose uptake. muscle glut4 protein level is not altered in obesity and t2d, however, its expression levels decline with age, and are related to insulin sensitivity in normal controls. european group for the study of insulin resistance demonstrated that glucose uptake is not altered as a function of aging per se but is secondary to increased body fat accumulation. moreover, the decrease of lean body mass and contractile strength with age are other factors that contribute to the reduction in insulin stimulated glucose uptake. the above factors, along with changes in body composition, accumulation of vf, and increase in circulating ffa levels, contributes to the decreased glucose uptake with age. glucose intolerance, insulin resistance, and t2d associated with aging are leading causes of morbidity and mortality through its multiple complications as well as increases in the risk for multiple other age - related diseases such as cancer, stroke, cardiovascular diseases, parkinson 's disease, and ad. though various factors that contribute to the changes in glucose homeostasis are relatively well characterized, there are still areas that are not yet fully elucidated such as the roles of aging on -cell mass and function, and the crosstalk between central and peripheral insulin action. a comprehensive understanding of all the defects that impair glucose homeostasis in the elderly will lead to development of appropriate, novel treatments that will substantially improve quality of life and over all life span.	aging is a risk factor for impaired glucose tolerance and diabetes. of the reported 25.8 million americans estimated to have diabetes, 26.9% are over the age of 65. in certain ethnic groups, the proportion is even higher ; almost 1 in 3 older hispanics and african americans and 3 out of 4 pima indian elders have diabetes. as per the nhanes iii (third national health and nutrition examination) survey, the percentage of physician - diagnosed diabetes increased from 3.9% in middle - aged adults (4049 years) to 13.2% in elderly adults (75 years). the higher incidence of diabetes is especially alarming considering that diabetes in itself increases the risk for multiple other age - related diseases such as cancer, stroke, cardiovascular diseases, parkinson 's disease, and alzheimer 's disease (ad). in this review, we summarize the current evidence on how aging affects pancreatic cell function, cell mass, insulin secretion and insulin sensitivity. we also review the effects of aging on the relationship between insulin sensitivity and insulin secretion. understanding the mechanisms that lead to impaired glucose homeostasis and t2d in the elderly will lead to development of novel treatments that will prevent or delay diabetes, substantially improve quality of life and ultimately increase overall life span.
head injury is a trauma to the head that may or may not include injury to the brain. road traffic accidents (rtas) constitute a considerable concern to the general public and may be a major cause of traumatic head injury in nigeria. mortality rate from rtas in nigeria is higher than those of both the industrialized and other developing countries probably due to the poor road network. head injury is a major cause of morbidity in survivors and a major cause of traumatic brain injury. traumatic brain injury (tbi) is the leading cause of death and severe disability in people under 45 years of age in western industrialized countries affecting the young and adults in the most productive years of their lives. although tbi is a problem of major medical and socioeconomic significance, its pathogenesis is not well understood and is often difficult to manage that may lead to primary and secondary lesions of varying severity. the immune system response to damage done by the closed head injury as a result of free radicals produced neutrophils to remove the damaged tissue. the cells of the immune system such as t - cells, b - cells, and macrophages have membranes that are particularly rich in long - chain unsaturated fatty acids, thus making them more vulnerable to free radicals oxidation initiated by the closed head injury than other cells. the brain itself is rich in unsaturated fatty acids and consumes large amounts of oxygen that may generate excess free radicals which could overwhelm the antioxidant defense systems that can exacerbate the brain injury. the antioxidants vitamin e and vitamin c protect the brain from oxidative stress by directly scavenging the free radicals. increased intake of vitamins c and e may positively influence the healing processes by maintaining the immune system, lymphocytes proliferation and decrease production of immunosuppressive prostaglandin e2, and suppressing neuroinflammatory processes. decreased levels of antioxidants or inhibition of the antioxidant enzymes the current work was designed to study the effect of -tocopherol and ascorbic acid on the severity and management of traumatized brain injury in albino rats. ascorbic acid (vitamin c) was obtained from pal pharmaceutical industries ltd., kano, nigeria, while - tocopherol (vitamin e) was obtained from pharco pharmaceuticals, bolkely alexandria, egypt. the experimental protocol was approved by the ethical and animal care and usage committee of the usmanu danfodiyo university, sokoto nigeria. seventy - two (72) apparently healthy albino rats of wistar strain, weighing between 170 and 200 g, were randomly divided into eight groups of nine rats per group. group a : non - tbi - untreated group, group b : tbi untreated, group c : tbi treated with 45 mg / kg of vitamin c, group d : tbi treated with 45 mg / kg of vitamin e, group e : tbi treated with 45 mg / kg each of vitamins c and e, group f : tbi treated with 60 mg / kg of vitamin c, group g : tbi treated with 60 mg / kg of vitamin e, group h : tbi treated with 60 mg / kg each of vitamins c and e. the vitamins were suspended in 0.9% nacl and administered to the rats for 2 weeks before induction of the trauma. the animals were then anesthetized with 7.5 mg / kg propofol intraperitoneally and were incubated and ventilated on a harvard rodent ventilator. the body temperature of the animals was maintained throughout with a thermostatically controlled heating pad set at 37 c. the skull was exposed by midline incision and a stainless steel disk measuring 10 mm in diameter and 3 mm in depth was cemented centrally along the control suture between the lambda and the bregma with a polyacrylamide adhesive. the animals were secured in the prone position on a 10 cm deep foam bed. injury was induced by dropping 80 g brass weight from a distance of 1 m. the stainless steel disk was immediately removed from the skull and the animals were weaned off the ventilator and recover in the cages. the treatment continued for additional period of 2 weeks during which the rats were followed up for recovery and survival from the trauma. the animals were then killed by decapitation and blood was collected in labeled centrifuge tubes, then centrifuged, and serum collected was used for estimation of oxidative stress indices. brain tissues were taken from frontal cortex, hippocampus, and cerebellum and perfused with normal saline followed by 4% para - formaldehyde. the brain tissue samples were homogenized in four volumes of 0.2 m phosphate buffer, ph 7.8 and centrifuged for 2 minutes at 1,000 rpm. the samples were frozen immediately on dry ice and stored at 20 c until required. the tocopherols in the sample were oxidized to tocopheryl quinine by fecl3 and fe + and the resultant fecl2 formed a red - colored complex with,-dipyridyl which was estimated at 520 nm using a spectrophotometer. the ascorbic acid in the samples was oxidized to dehydroascorbic acid which in the acid solution reacts with 2,4-dinitrophenylhydrazine to form a corresponding hydrazine and after treated with sulfuric acid developed an orange red - colored complex which was measured spectrophotometrically at 520 nm. copper sulfate was then added to enhance the ascorbic acid and to prevent the effect of interfering compound in 2,4-dinitrophenyl hydrazine. trichloroacetic acid was added to serum and brain tissue homogenate to precipitate the protein content to avoid its interference with the process. potassium trioxochlorate iv and manganese iv oxide reaction was used to generate oxygen and flavin adenine dinucleotide accepts an electron from oxygen produced to form superoxide anion. in the absence of superoxide dismutase, the radical reacts with the detector nitroblue tetrazolium salt. in the event fad reduces oxygen to generate superoxide radicals and nitroblue tetrazolium salt scavenges the free radicals. creatine kinase activity (ck - bb) was estimated by the enzymatic method using a standard enzyme kit supplied by agappe diagnostics. the procedure involves measurement of ck - activity in the presence of an antibody to the ck - b monomer. this antibody completely inhibits the activity of ck - bb and half of the activity of ck - mb without affecting the m subunit activity of ck - mb and ck - mm. the ck - bb activity is obtained by multiplying the ck - b by 2. lipid peroxidation generates peroxide intermediates which upon cleavage releases mda, a product that reacts with thiobarbituric acid (tba) forming a colored complex, which is measured at 532 nm. dunnett 's test was used for multiple comparisons and values were considered statistically significant at p 0.05) difference. effects of -tocopherol and ascorbic acid on serum levels of nonenzymatic antioxidants of traumatized rats grp a = non - tbi - untreated group ; grp b = tbi untreated ; grp c = tbi treated with 45 mg / kg vitamin c ; grp d = tbi treated with 45 mg / kg vitamin e ; grp e = tbi treated with 45 mg / kg each of combined vitamins c and e ; grp f = tbi treated with 60 mg / kg vitamin c ; grp g = tbi treated with 60 mg / kg vitamin e ; grp h = tbi treated with 60 mg / kg each of combined vitamins c and e the effect of -tocopherol and ascorbic acid on the brain tissue of traumatized rats is presented in figure 3. the results indicated that supplementation with combined 45 mg / kg of vitamins c and e significantly (p 0.05) decrease in the activity of the group treated with 60 mg / kg of vitamin c. effects of -tocopherol and ascorbic acid on serum sod, mda and ck - bb of traumatized rats the level of mda of the tbi - untreated group increased significantly (p 0.05) effect on the level of serum mda compared with the tbi - untreated control but treatment with 45 mg / kg (p 0.05) increase in the activity of sod in the remaining treated groups. effect of -tocopherol and ascorbic acid on brain tissue homogenate sod and mda level of traumatized rats there was a significant (p < 0.05) decrease in the tissue mda of the treatment groups compared with the tbi - untreated group. traumatized brain injury is an oxidative related injury associated with biological and metabolic alteration which generates free radicals from various cellular activities. widespread and bilateral damage of the neurons, axons, dendrites, and microvasculature have been shown to be associated with tbi. according to ref. tbi induces brain edema that begins 20 minutes after injury and peaks at 24 hours after trauma. the current study indicated high mortality (55%) rate in the untreated traumatized group compared with the treatment group while treatment with combined vitamins performed better in the prevention of mortality. report has shown that primary injury was not adequate to explain the degeneration, rather the deterioration was caused by a complex set of biochemical cascade that occurred in hours to days following trauma. pretreatment of animals with -tocopherol has been reported by hall. to lessen secondary damage in several models of ischemic or traumatic injury to the central nervous system. impaired antioxidant defense and increased lipid peroxidation have been reported in traumatic brain injury rats and chronic schizophrenic patients. free radicals play an important role in the pathogenesis of structural and functional changes of neuronal membrane that could be responsible for the beginning or aggravation of the schizophrenic disease. the significant increase in the levels of serum and brain tissue homogenate of ascorbic acid and vitamin e in the treated rats when compared with tbi - untreated rats indicated that oxidative stress has been implicated in the traumatized brain injury and supplementation with antioxidant vitamins can ameliorate the oxidative stress insult. vitamin e and ascorbic acid are important chain - breaking antioxidants, responsible for scavenging the free radicals and suppression of peroxidation in the aqueous and lipid region of the cell. studies have indicated a decrease in lipid peroxidation and an increase in the activity of antioxidant enzymes after the administration of -tocopherol and selenium during the treatment of brain impairment. intensive pretreatment of animals with endogenous lipid peroxyl radical scavenger, -tocopherol, with 21- aminosteroids has been shown to decrease posttraumatic spinal ischemia and enhance chronic neurological recovery. low levels of serum and brain tissue homogenate of vitamin e in the traumatized untreated control as shown in the current study may facilitate oxidative damage in the traumatized brain injury of rats. the marked decrease in serum ascorbic acid level after tbi first tbi might disrupt the blood brain barrier and decrease the concentration gradient for ascorbate accumulation. second movement of ascorbate from brain to blood could occur and ascorbate may have been consumed by the free radicals associated with tbi. this is the most likely scenario and one that provides a unifying explanation for the results in the current study. lipid peroxidation product, mda, levels were significantly reduced in serum and brain tissue homogenate of treated rats compared with the tbi - untreated group. reduction in mda could be due to decreased generation of reactive oxygen species (ros), due to its destruction by the antioxidants treatment. the raised serum and brain tissue homogenate of mda level in tbi - untreated control reflects the oxidative injury which is attributed to free - radical formation that abstracts hydrogen atoms from lipoproteins causing lipid peroxidation. increased levels of thiobarbituric acid reaction products have been found in the serum of patients experiencing craniotomy and also in plasma of schizophrenic patients, with or without tardive dyskinesia. the results also indicated that vitamin e and vitamin c supplementation was found to increased sod levels in both serum and brain tissue homogenate compared with the tbi - untreated group. the role of sod is to protect cells from the toxic effect of superoxide radicals as it catalyzes the dismutation reaction of the radicals. the levels of ck - bb were found to be significantly elevated immediately after the head injury and that the greater the degree of external cranial injury inflicted the higher isoenzyme activity. this seems to provide evidence that ckbb activity is an early indicator of brain damage and that its level may reflect the extent of cerebral damage involved. in the present study perhaps the amount of ck - bb released from the brain damage caused an appreciable rise in the serum level of ck - bb of tbi rats. this indicated that the model was good enough to cause the released of ck - bb into peripheral blood and so this may possibly offer new criteria for early assessment of the severity of brain damage. the current study provides evidence of the involvement of oxidative stress in the pathogenesis of tbi as manifested in the elevation of ck - bb activity and mda, and reduction in the serum and brain tissue homogenate of vitamin c and vitamin e. treatment also significantly improved the serum and brain tissue antioxidant parameters an indication of the effectiveness of these vitamins in reversing the complications of tbi. treatment does not only reduce oxidative stress in tbi but also effectively reduce the mortality rate and healing time. thus combined therapy is more effective in protecting and reversing the effects against trauma - induced damage.	background : traumatic brain injury (tbi) is accompanied by substantial accumulation of biomarkers of oxidative stress and depletion of antioxidants reserve which initiate chain reactions that damage brain cells. the present study investigated the role of ascorbic acid and -tocopherol on the severity and management of tbi in rats.materials and methods : wistar rats were subjected to closed head injury using an accelerated impact device. rats were administered 45 mg / kg and 60 mg / kg body weight of ascorbic acid, -tocopherol or a combination of the two vitamins for 2 weeks pre- and post injury. blood and brain tissue homogenates were analyzed for vitamin c, vitamin e, malondialdehyde, superoxide dismutase, and creatine kinase activities.results:the results indicated that tbi caused significant (p < 0.05) decreased in vitamins c and e levels in the blood and brain tissue of tbi - untreated rats. the activities of superoxide dismutase in tbi rats were markedly reduced when compared with non traumatized control and showed a tendency to increased following supplementation with vitamins c and e. supplementation of the vitamins significantly (p < 0.05) reduced malondialdehyde in the treatment groups compared with the tbi - untreated group.conclusion:the study indicated that pre and post treatment with ascorbic acid and -tocopherol reduced oxidative stress induced by brain injury and effectively reduced mortality rate in rats.
common variable immunodeficiency (cvid) is a heterogeneous group of diseases characterized by hypogammaglobulinemia and repeated, predominantly, bacterial infections. b lymphocytes, however, show defects of differentiation and proliferation after antigen stimulation. the number of plasma and memory cells is reduced. as a result of this, the values of serum igg and iga are reduced and also irregularly the values of igm (di renzo., 2004). the antibody response to protein and polysaccharide antigens is insufficient or completely missing in some cases. the attempts to classify cvid based on in vitro immunoglobulin (ig) production and b lymphocyte subsets have confirmed marked heterogeneity of this antibody defects (weiler & bankers - fulbright, 2005). differentiation of precursor cells into mature b lymphocytes and then into plasma cells is a process regulated by products of a number of genes. mutation of genes for inducible costimulator (icos), the first single gene defect, has been described in patients with cvid (grimbacher., subsequently, the mutations of genes encoding transmembrane activator and calcium - modulator and cytophilin ligand interactor (taci) and cd19 have been found. it ranks in the family of costimulation molecules similarly as cd28 and ctla-4 (cd152) molecules. it is encoded by three genes that are mapped to chromosome 2q33. a great number of single nucleotide variants that participate in the icos polymorphisms have been reported. the comparison of the frequency of single nucleotide polymorphisms between patients and healthy controls showed no significant differences (haaning andersen., 2003 ; guzman., 2005 ; ohm - laursen., the icos and cd28 molecules share up to 20% amino acid homology and both are type i transmembrane receptors. icos occurs as a homodimer, with an extracellular (ig) v like domain. unlike the cd28 molecule, which is expressed constitutively, the icos molecule is expressed only on activated t lymphocytes. its membrane expression persists on recently activated as well as on memory th1 and th2 cd4 lymphocytes (van berkel & oosterwegel, 2006 ; warnatz., 2006). the icos expression appears to be higher on th2 cells compared to th1 cd4 t cells (coyle., 2000). the th2-mediated response is more dependent on the icos function than th1 response (lhning., 2003 ; grunig., the density of icos membrane expression correlates with cytokine production. in case of icos gene mutation, especially the synthesis of cytokine associated with high - level icos expression (il-10, il-2, il-4, il-5, il-13) within the course of viral or bacterial infections in knock - out icos/ mice, large secondary germ centres fail to develop, and specific antibody response is missing. both the reduced proliferation th2 activity and the failure of t and b lymphocyte co - operation participate in the developed antibody defect (van berkel & oosterwegel, 2006 ; warnatz., 2006) (figure 1). the cascade of co - stimulation signals between t and b lymphocytes. the expression of cd154 (cd40l) and icos is up - regulated through constitutively surface expressed cd28 molecule and its cd80/cd86 ligands. another support of co - stimulation ligands between t and b lymphocytes occurs through icos - icos - l which up - regulates cd154 expression. the intracellular tail of the icos molecule contains two tyrosine residues, one of which lies within the ytyr180mem motive, which is associated with adapter molecule grow factor receptor - bound protein-2 (grb-2). one amino acid replacement at the position in this motive includes explaining of the lack of capability to recruit grb-2. this molecule plays a key role during the initiation of il-2 synthesis (harada., 2003). this finding explains why activating signals coming through defective icos molecules do not lead to synthesis of il-2 (itoh., 2002). activated t lymphocytes amplify the icos - l expression on b lymphocytes through il-4, il-5, il-6, granulocyte macrophage colony - stimulating factor (gm - csf), ifn and tumour necrosis factor (tnf) cytokines (bayry., 2005). icos - l expression on b lymphocytes is also up - regulated through cd40 and cd154 ligands or bacterial superantigens (salzer., 2004 ; janke., 2006). the icos : icos - l interaction is necessary for il-10, il-17 synthesis (chtanova., 2004 ; warnatz., the icos - l molecule is encoded by one gene, which is found on the long arm of chromosome 21 (mapped 21q22.3). the b7h2 variant is encoded by exon 17 and appears on both lymphoid and non - lymphoid cells. the hgl-50 variant is encoded by exon 16 and 8, and is found only on lymphoid cells (ohm - laursen., 2005 ; warnatz., interaction of icos icos - l molecules is important for differentiation of naive b lymphocytes into memory and plasma cells. icos - l ligand shares up to 20% homology with cd80 and cd86 molecules (receptors for cd28 and ctla-4 molecules) (salzer., 2004 ; van berkel & oosterwegel, 2006). the clinical phenotype includes recurrent and serious bacterial infections, autoimmune phenomena, splenomegaly, lymphadenopathy and malignancy. patients with cvid, based on icos defect, have mild b lymphopaenia and significantly reduced number of memory cd19/cd27 b lymphocytes (warnatz., 2002 ; piqueras., this laboratory finding is not, however, characteristic for icos mutations because up to 75% of patients with cvid have a reduced number of memory isotype - switched cd19/cd27/igm b lymphocytes (schejbel., 2005). however, patients with icos deficiency belong to cvid type i group (freigburg classification) (warnatz., 2002 ; carsetti., 2005). icos deficiency, based on one point mutation, was the first example of an autosomal recessive disorder that occurs only in a small amount of patients with cvid (approximately 2%) (warnatz., 2006). in order to ensure physiological antibody response after antigen stimulation, other activation signals mediated by both intracellular interactions and soluble factors are important (kroczek., 2004). class switch recombination (csr) of ig synthesis requires four signals. the first one is mediated by il-3, il-4, il-5, il-6, il-13 cytokines, which participate in ch genes transcription regulation. in case of a t - cell dependent antibody response, the interaction of cd40l (cd154) molecule expressed on activated t lymphocytes with cd40 molecule on b lymphocytes is necessary (mcheyzer - williams, 2003). costimulatory icos icos - l interaction constitutes the third necessary stimulus for sufficient antibody production. the fourth signal is mediated by other interactions of molecules, which belong to the superfamily of tnf molecules (b - cell acting factor of the tnf family (baff), a proliferation - inducing ligand (april)), and their receptors (b - cell maturation antigen (bcma), baff receptor (baff - r), taci). the baff and april are among these molecules (castigli., 2005a). baff is present predominantly on monocytes / macrophages, dendric cells and t lymphocytes (mackay & ambrose, 2003). on the contrary, it is not expressed on b lymphocytes although this molecule has been detected on b - cell derived chronic lymphatic leukaemia (novak., 2002). the april molecule is expressed on a large variety of cells, monocytes / macrophages, dendritic cells and activated t lymphocytes. baff and april molecules create both homotrimers and heteromers capable of stimulating b lymphocyte differentiation (wu., 2000 ; it has been proved that april and baff molecules interfere with csr of t - independent antibody response (litinskiy., 2002 ; martin & vishva, 2005). both april and baff molecules bind two type iii membrane receptors, bcma, taci (schneider., 1999). bcma is expressed exclusively on b lymphocytes while taci is expressed both on b lymphocytes and activated t lymphocytes. the third receptor belonging to the tnf - r family is the baff - r, which is the unique receptor for baff (bodmer., 2002 ; losi., this membrane molecule is predominantly expressed on b lymphocytes and only on part of activated t lymphocytes (schneider & tschopp, 2003 ; martin & vishva, 2005). interactions of april and baff, expressed on monocytes and macrophages, with their receptors on b lymphocytes are evidence of the significance of natural immunity for the correct antibody response (mcheyzer - williams, 2003 ; de durana., 2006). the absence of any of the previously mentioned signals is the cause for defect of terminal differentiation of a mature b lymphocyte into plasmatic and memory cells, which are manifested by antibody immunodeficiency (salzer & grimbacher, 2005) (figure 2). tnf receptor family members, baff - r, bcma and taci, and their ligands baff and april check terminal differentiation of b lymphocytes and/or csr of ig synthesis. tnf receptors and their ligands regulate both the survival and the apoptosis during immune system development and immune responses. receptor taci molecules are encoded by the tnfrsf13b gene, which is situated at the short arm of chromosome 17 (17p11.2). the described mutations affect taci molecule in its extracellular (c104r, s144x), transmembrane (a181e) and intracellular parts (s194x, r202h, ins204) (castigli. tnfrsf13b mutations are associated with antibody deficiencies (salzer., 2005). this suggests that taci deficiency may manifest in autosomal dominant as well as in recessive traits in familial and in sporadic cvid. in the case of heterozygous mutations, the clear evidence that they are the cause of humoral deficiencies has not been shown. it is not possible to exclude that heterogeneous mutations may be the only modifying factor that leads to the manifestation of other defects. the taci mutations show a range of clinical symptoms from no infection to very severe infections, suggesting that other genetic and environmental factors contribute to the variable disease spectrum. in addition, taci deficiency may also represent a common genetic defect for cvid and selective iga deficiency (sigad), which has been long proposed to be based on the clinical observations in cvid / sigad families (salzer & grimbacher, 2006). the numbers of b cells in homozygous taci deficiency are normal or tend to be slightly reduced. patients with homozygous mutations in taci showed selective impairment of april and baff - induced b - cell proliferation and csr. they have low numbers of cd19/cd27/igm b lymphocytes and suffer from severe infections caused by encapsulated microbes. the vaccination with non - conjugated vaccines is not effective. in the case of homozygous taci mutations, polysaccharide antigens are followed by a disturbance in the proliferation of b lymphocytes (castigli., 2005b). unlike knock - out taci/ mice, in which spontaneous development of lymphomas occur, only one case of malignant lymphoma has been described in patients with taci mutation (seshasayee., 2003). the increased incidence of lupus - like disease seen in taci/ mice was not observed in cvid patients with the same defect. in case of patients with cvid, the incidence of autoimmune manifestations in patients with and without taci defect is the same. the incidence of tnfrsf13b mutations is estimated to be in 510% of patients with cvid (salzer & grimbacher, 2006). the cd19 molecule is detectable in the cytoplasm of b lymphocyte precursors, and from the stage of immature b lymphocytes it is imbedded in the cell membrane. it participates in feedback regulation of signals which come through bcr. on the other hand, it facilitates the recognition of antigen - antibody complexes that link to bcr and the cd21 molecule. together with the cd21 molecule, it ensures another communication between natural immunity and b lymphocytes. van zelm described the homozygous mutation in the cd19 gene in four members of a family with antibody deficiency that complies with classification criteria for cvid. the encoding gene is situated on the short arm of chromosome 16 (mapped 16p11.2) (van zelm., 2006). its real frequency is unknown in patients with cvid because the findings discovered up to now are rare. the numbers of peripheral b lymphocytes are normal or only slightly reduced in affected individuals. cd19 expression on b lymphocytes is missing and as a result of this, the expression of cd21 surface molecules is reduced. the hypersomatic mutation process proceeds in a normal way ; however, cd27 b lymphocytes are missing. autoimmune and lymphoproliferative symptoms were not observed (van zelm., 2006). in 1953 janeway described a case of a 39-year - old woman with hypogammaglobulinaemia, which is considered the first mention of cvid. more than 50 years have passed during which separate pathological units, as for example hyper igm syndrome linked and unlinked to x - chromosome and autosomal recessive agammaglobulinaemia, have been excluded. in the last 3 years, mutations of genes encoding icos, taci and cd19 have been described. these defects are found in less than 15% of the patients and for this reason the etiology of most of the patients with cvid remains unknown. the described mutations and findings of other expected gene defects will probably cause disintegration of this heterogeneous antibody deficiency syndrome.	common variable immunodeficiency (cvid) is the most frequent clinically manifested primary immunodeficiency. according to clinical and laboratory findings, cvid is a heterogeneous group of diseases. recently, the defects of molecules regulating activation and terminal differentiation of b lymphocytes have been described in some patients with cvid. in this study, we show the overview of deficiencies of inducible costimulator, transmembrane activator and calcium - modulator and cytophilin ligand interactor, cd19 molecules, their genetic basis, pathogenesis and clinical manifestations.
prolonged pregnancy occurs in approximately 10% of all singleton pregnancies and is associated with an increased risk of fetal macrosomia, intrapartum fetal heart abnormalities, meconium staining, perinatal death, and cesarean delivery (1 - 3). an early prediction of this condition is important because several methods to decrease the rate of prolonged pregnancy, such as membrane stripping and outpatient prostaglandin therapy, have been already proposed (4, 5). therefore there has been considerable interest in the development of tests for the prediction of prolonged pregnancy. these tests include fetal fibronectin, cytokine, or nitric oxide concentrations in cervicovaginal secretions and cervical length as determined by ultrasonography (6 - 10). cervical length measured by transvaginal ultrasound in the second trimester has been well known to be effective in identifying pregnancies at high - risk of spontaneous preterm delivery (11 - 16). recent studies have reported that sonographic measurement of cervical length at term is a useful predictor of the likelihood of successful induction of labor and the spontaneous onset of labor in a 7-day period (17 - 21). moreover, romero. have proposed that cervical ripening is part of the common terminal pathway of human parturition, which includes uterine contractility and membranedecidua activation (22). these observations suggest that a passively shortened or dilated cervix may play a role in the mechanisms responsible for the initiation of parturition (preterm and term). therefore if the shorter the cervix, the greater the risk of preterm delivery, the likelihood of successful induction of labor and the spontaneous onset of labor, it is possible that a longer cervix may predict a higher risk of prolonged pregnancy. the purpose of this study was to determine whether the transvaginal ultrasonographic measurement of cervical length at 20 to 24 weeks and 37 weeks can predict prolonged pregnancy in nulliparous women. this was a prospective observational study conducted at seoul national university bundang hospital (seongnam, korea) between june 2004 and may 2006. women attending the antenatal clinic were enrolled at 37 weeks. in our institution ultrasound examination is carried out routinely at 11 - 14 weeks and at 20 - 24 weeks of gestation. the scan at 20 - 24 weeks included fetal examination and the option of having a transvaginal scan to measure cervical length as a screening test for spontaneous preterm delivery. the inclusion criteria were : 1) nulliparous women ; 2) singleton pregnancy ; 3) live fetus with vertex presentation ; 4) intact amniotic membranes ; 5) known gestational age confirmed by ultrasound measurement of crown - rump length (crl) (gestational age was corrected on the basis of crl measurement if a discrepancy of 7 days or more exists) ; 6) with sonographically measured cervical length between 20 and 24 weeks ; 7) no pregnancy complications (i.e., fetal growth restriction, preeclampsia, and major fetal abnormalities) ; 8) absence of labor ; 9) no history of previous uterine surgery. prolonged pregnancy was defined as a pregnancy that extended beyond 41 weeks and 2 days (289 days) (this definition was developed because we usually induced labor in patients who did not go into spontaneous onset of labor by that date). week of gestation was defined as completed week ; i.e., week 37 refers to 37.0 weeks to 37.6 weeks menstrual age. this study was approved by the institutional review board of our institution seoul national university bundang hospital. transvaginal ultrasonographic assessment of the cervical length was performed by one of the authors at 20 - 24 weeks using an aloka ssd 5500 (aloka co. ltd, tokyo, japan) ultrasound machine with a 6.0 mhz transducer and at 37 weeks using an envisor (philips medical system, netherlands) ultrasound machine with a 6.0 mhz transducer. women were asked to empty their bladder and were placed in the dorsal lithotomy position. the probe was gently placed at the anterior fornix of the vagina to obtain a sagittal view of the complete cervix, including the internal os, external os, and endocervical canal. the probe was slowly withdrawn until the image blurred and then the insertion pressure was increased only enough to restore a clear cervical image. this cervical image was magnified to at least 75% of the screen and the electronic markers were placed at the furthest points between the internal os and external os, then cervical length was measured as a straight line. univariate analysis was conducted using the mann - whitney u test or the test. receiver operating characteristic (roc) curves were constructed to describe the relationship between the sensitivity (true - positive rate) and the false - positive rate for the cervical length in predicting prolonged pregnancy. pearson correlation analysis was used to determine the relationship between sonographically measured cervical length and gestational age at delivery. this was a prospective observational study conducted at seoul national university bundang hospital (seongnam, korea) between june 2004 and may 2006. women attending the antenatal clinic were enrolled at 37 weeks. in our institution ultrasound examination is carried out routinely at 11 - 14 weeks and at 20 - 24 weeks of gestation. the scan at 20 - 24 weeks included fetal examination and the option of having a transvaginal scan to measure cervical length as a screening test for spontaneous preterm delivery. the inclusion criteria were : 1) nulliparous women ; 2) singleton pregnancy ; 3) live fetus with vertex presentation ; 4) intact amniotic membranes ; 5) known gestational age confirmed by ultrasound measurement of crown - rump length (crl) (gestational age was corrected on the basis of crl measurement if a discrepancy of 7 days or more exists) ; 6) with sonographically measured cervical length between 20 and 24 weeks ; 7) no pregnancy complications (i.e., fetal growth restriction, preeclampsia, and major fetal abnormalities) ; 8) absence of labor ; 9) no history of previous uterine surgery. prolonged pregnancy was defined as a pregnancy that extended beyond 41 weeks and 2 days (289 days) (this definition was developed because we usually induced labor in patients who did not go into spontaneous onset of labor by that date). week of gestation was defined as completed week ; i.e., week 37 refers to 37.0 weeks to 37.6 weeks menstrual age. this study was approved by the institutional review board of our institution seoul national university bundang hospital. transvaginal ultrasonographic assessment of the cervical length was performed by one of the authors at 20 - 24 weeks using an aloka ssd 5500 (aloka co. ltd, tokyo, japan) ultrasound machine with a 6.0 mhz transducer and at 37 weeks using an envisor (philips medical system, netherlands) ultrasound machine with a 6.0 mhz transducer. women were asked to empty their bladder and were placed in the dorsal lithotomy position. the probe was gently placed at the anterior fornix of the vagina to obtain a sagittal view of the complete cervix, including the internal os, external os, and endocervical canal. the probe was slowly withdrawn until the image blurred and then the insertion pressure was increased only enough to restore a clear cervical image. this cervical image was magnified to at least 75% of the screen and the electronic markers were placed at the furthest points between the internal os and external os, then cervical length was measured as a straight line. the shortest of three measurement obtained was taken as the cervical length. univariate analysis was conducted using the mann - whitney u test or the test. receiver operating characteristic (roc) curves were constructed to describe the relationship between the sensitivity (true - positive rate) and the false - positive rate for the cervical length in predicting prolonged pregnancy. pearson correlation analysis was used to determine the relationship between sonographically measured cervical length and gestational age at delivery. during the study period, 184 consecutive women met the inclusion criteria in this study. of these, 35 women had labor induced before 41 weeks and 2 days because of oligohydramnios (n=28), non - reassuring non - stress test (n=3), and request for social reason (n=4). thus, these women were excluded from the study, leaving a total of 149 women qualified for inclusion. spontaneous onset of labor and delivery at or before 41 weeks and 2 days occurred in 126 (85%) women. in 23 women (15%) that remained undelivered beyond 41 weeks and 2 days, cervical length was successfully measured in all cases and the mean (standard deviation) cervical lengths at 20 to 24 weeks and 37 weeks were 396 mm and 286 mm, respectively. table 1 presents the clinical characteristics and obstetric outcomes of patients according to the study group. no significant differences were found in terms of median maternal age and median gestational age at ultrasound examination of cervical length measured between 20 and 24 weeks between the study groups. however, women going beyond term had a significantly higher rate of cesarean delivery and greater mean birth weight than did those with spontaneous term delivery. moreover, the median cervical length at 37 weeks was significantly longer in women going beyond term than in those with spontaneous term delivery, but the median cervical length at 20 - 24 weeks were not significantly different between the two study groups. 1 displays the roc curve for sonographically measured cervical length at 37 weeks in predicting prolonged pregnancy. the curve constructed for sonographically measured cervical length was above the 45 line, indicating that there was a significant relationship between this variable and prolonged pregnancy (area under the curve 0.702 ; se 0.060 ; p<0.005). the best cut - off value for the prediction of prolonged pregnancy was 30 mm with a sensitivity of 78% and a specificity of 62%. a significant positive correlation between sonographically measured cervical length at 37 weeks and gestational age at delivery however, there was no correlation between cervical length at 20 - 24 weeks and gestational age at delivery. the results of our study clearly demonstrate that cervical length assessed by transvaginal ultrasonography at 37 weeks can predict the likelihood of prolonged pregnancy and is associated with the gestation at spontaneous onset of labor in low - risk nulliparous women. furthermore, our data indicate that these associations did not exist as early as 20 to 24 weeks. similar observations have been made in other gestational ages (i.e., at 11 to 14 weeks and 22 to 24 weeks of gestation) in the setting of spontaneous preterm delivery (12, 13). several investigators have reported that measurement of cervical length in the second trimester provides accurate prediction of risk for spontaneous preterm delivery and this risk is inversely correlated with cervical length (11 - 16). however, there are few reports on cervical length in the second trimester of pregnancy in relation to prolonged pregnancy. some studies have begun cervical assessment at 37 weeks and demonstrated that cervical length at 37 weeks is associated with the incidence of prolonged pregnancy and the spontaneous onset of labor in a 7-day period (9, 10, 21). to our knowledge, this is the first report to examine the relationship between cervical length in the second trimester and the occurrence of prolonged pregnancy. the results are unexpected, in that relationship found in spontaneous preterm delivery does not exit (i.e., the longer the cervical length in the second trimester, the higher the rate of prolonged delivery). these findings imply that cervical assessment provide sensitive prediction of spontaneous delivery (preterm or term) within the next some weeks, not the next some months, regardless of whether this assessment is performed in the second trimester or at term. a major finding in this study is that cervical length at 37 weeks can predict the likelihood of prolonged pregnancy. this finding is in keeping with the observation made by ramanathan. who demonstrated that measurement of cervical length at 37 weeks can be used to determine the likelihood of prolonged pregnancy and the risk of cesarean section in those requiring induction for prolonged pregnancy (9). they demonstrated that the measurement of cervical length at 39 weeks and 40 weeks was significantly shorter in a group who had spontaneous onset of labor before 41 completed weeks of gestation than in that who did not, but the measurement of cervical length was similar in both group at 37 weeks and 38 weeks. the disparity among these studies is probably attributable to a combination of factors, including the definition of prolonged pregnancy, the study population to participate (i.e., nulliparity or primiparity), and sample size. nulliparity was chosen as the enrollment criterion of our study because primiparity and prior prolonged pregnancy are the most common identifiable risk factor for prolongation of pregnancy (9, 23, 24). it is noteworthy that a significant positive correlation between cervical length at 37 weeks and gestational age at delivery was noted. this finding is consistent with the observations of ramanathan. who documented a high association between cervical length at 37 weeks and gestation at spontaneous onset of labor (9). moreover, in the current study, cervical length at 37 weeks 30 mm had a sensitivity of 78% and a specificity of 62% in the prediction of prolonged pregnancy. these data have several clinical implications for the management of patients at term. first, if routine measurement of cervical length at 37 weeks can identify patients at high risk of prolonged pregnancy, the incidence of prolonged pregnancy or the risks associated with prolonged pregnancy will be reduced because simple method to promote spontaneous onset of labor (i.e., membranes stripping and outpatient prostaglandin therapy) was already proposed (4, 5). second, these data may be utilized in individualizing the timing of elective cesarean section rather than performance of this operation at 38 weeks. third, from the patients ' point of view these data may give patients information to arrange their social activities and to deal with their anxiety. our study has demonstrated that the mean cervical lengths at 20 to 24 weeks and 37 weeks are, respectively, 39 mm and 28 mm in singleton low - risk pregnancies delivered at or beyond term. these findings are similar to those of previous reports (9, 12, 25). moreover, our data indicated that the cervical length decreased from the second trimester to term and this spontaneous shortening was, however, more pronounced in women delivered at term than in those going beyond term. the mean cervical length in women delivered at term decreased from 39.1 mm to 27.7 mm. on the other hand, in women going beyond term, the shortening of the cervix was from 40.9 mm to 31.9 mm between the 20 - 24-week and the 37-week evaluations. these findings indicate that the relatively slow rate of spontaneous cervical shortening between the second trimester and term pregnancy might be related to the occurrence of prolonged pregnancy. in a similar study, vimercati. also reported that women who remained undelivered beyond 41 weeks has a delayed effacement process and the measurement of cervical length at 41 weeks in these women would possibly show the same pattern as that at 39 weeks in women who had spontaneous onset of labor before 41 weeks (10). on the other hand, in the context of spontaneous preterm delivery, carvalho. have found that there is a spontaneous shortening in the pregnant cervix from the first to the second trimester of pregnancy and this shortening is more rapid in pregnant women who deliver prematurely (12). a limitation of the current study is that cervical length was measured as a straight line because from the practical point of view, it is reasonable to measure the linear distance between the internal and external os. although in cases with a curved cervix the longer the measurement taken along the endocervical canal, the longer inevitably the measurement of cervical length taken as a straight line, cervical length in our study population, especially measured at 20 - 24 weeks, may not represent true cervical length. in conclusion, cervical length at 37 weeks can predict the likelihood of prolonged pregnancy and is associated with the gestation at spontaneous onset of labor in low - risk nulliparous women. however, cervical length at 20 to 24 weeks is associated with neither prolonged delivery nor the gestation at spontaneous onset of labor.	this study was done to evaluate transvaginal ultrasonographic measurement of cervical length at 20 to 24 weeks and 37 weeks as a predictor of prolonged pregnancy (defined as a pregnancy that extended beyond 41 + 2 weeks of gestation [289 days ]) in nulliparous women. this prospective observational study enrolled 149 consecutive nulliparous women with singleton gestation at 37 weeks. cervical length was measured by transvaginal ultrasonography at 20 to 24 weeks and 37 weeks. cervical length at 37 weeks, but not at 20 to 24 weeks, was significantly longer in women delivered at > 41 + 2 weeks than in those delivered at 41 + 2 weeks (p<0.005). there was a significant correlation between cervical length at 37 weeks and gestational age at delivery (pearson correlation coefficient, r=0.387, p<0.0001). in the receiver operating curve, the best cut - off value of cervical length at 37 weeks for the prediction of prolonged pregnancy was 30 mm, with a sensitivity of 78% and a specificity of 62%. cervical length assessed by transvaginal ultrasonography at 37 weeks can predict the likelihood of prolonged pregnancy in nulliparous women. however, there is no association between cervical length at 20 to 24 weeks and the occurrence of prolonged pregnancy.
nurr1 also known as nr4a2, belongs to the nuclear receptor superfamily of transcription factors, is highly expressed in midbrain dopaminergic (da) neurons, and plays an essential role in the development, survival and function maintenance of midbrain da neurons. nurr1 is a potential target for parkinson 's disease (pd), which is the second common neurodegenerative disease and both genetic and environmental neurotoxin are important causes. nurr1 can activate the transcription of tyrosine hydroxylase and enhance the expression of da transporter. it was reported that homozygous nurr1 knockout mice failed to develop da neurons, and heterozygotes reduced their brain da and developed age - dependent locomotor deficits including impaired horizontal and vertical movement, difficultly performance on rotarod test compared to age - matched wild - type mice. reduction of nurr1 in the adult brain may increase the vulnerability of da neurons to stress and participate in the pathogenesis of pd. several polymorphisms of nurr1 gene including 291tdel and 245t-->g have been reported to result in a marked decrease of nurr1 mrna levels in transfected cell lines and lymphocytes of affected individuals. furthermore, le. reported a decreased nurr1 gene expression in peripheral blood mononuclear cells (pbmcs) of pd patients, which anticipated nurr1 as a potential biomarker for pd diagnosis. but to date, little has been done to investigate the association among nurr1 expression in pbmc, disease progression of pd, and da agents. this study was to measure the nurr1 mrna level in pbmc and evaluate the effect of nurr1 expression by da agents in vivo and in vitro. the 362 pd patients at department of neurology, the first affiliated hospital of sun yet - sen university between january 2012 and august 2014 were recruited. the diagnosis was made in accordance with the united kingdom pd society brain bank criteria. besides recent - onset pd patients without any anti - pd treatments (de novo), each patient reported to have taken anti - pd medicines at least 2 years and all patients were assessed according to the hoehn and yahr (h and y) scale. meantime, 193 healthy controls (hcs) were enrolled either from physical examination center of sun yat - sen university or the spouses of the patients. the study was approved by the ethics committee of sun yat - sen university. peripheral blood samples (5 ml) of pd patients and hcs pbmc from venous blood of pd patients and hcs were prepared by ficoll - hypaque density gradient centrifugation. after centrifugation at 2200 r / min for 20 min, buffy coats were collected and washed three times with phosphate - buffered saline (pbs), then centrifuged at 1800 r / min for 10 min to collect pbmc cells. then pbmc were lysed by adding 1 ml trizol reagent (invitrogen, carlsbad, ca, usa) and stored at 80c until used. in order to further explore the pattern of nurr1 expression, an in vitro analysis on cultured human pbmc was conducted. pbmc from venous blood of 10 hcs was prepared by ficoll - hypaque density gradient centrifugation through centrifuging at 2200 r / min for 20 min. the buffy coats were collected and washed twice with pbs, then washed once with complete rpmi 1640 medium (gibco, german) containing 2 mmol / l l - glutamine, 100 u / ml penicillin, 100 g / ml streptomycin, and 10% fetal bovine serum and suspended at a concentration of 2 10 cells / ml with complete rpmi 1640 medium. pbmc was delivered in 3.5 cm diameter dishes with 2 10 cells separately with complete rpmi 1640 medium. after 12 h cultured, the cells were treated with 10 mol / l pramipexole (sigma, st. louis, mo, usa) for 2, 4, 8, 12 and 24 h and cultured at 37c under the humidified 5% co2 atmosphere. selected time, the pbmc cells were collected for rna extraction. then total rna was isolated using trizol (invitrogen) according to the manufacturer 's protocol. the 1 g of total rna was reverse transcribed into first - strand complementary dna (cdna) using a reverse transcription (rt) kit (takara, japan) according to the manufacturer 's protocol in a final volume of 20 l. for analysis, on nurr1 expression with quantitative real - time rt - polymerase chain reaction (rt - pcr), human glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as internal control, and the sequences of primers as follows : nurr1 forward : 5-tccaacgaggggctgtgcg-3 ; nurr1 reverse : 5-cactgtgcgcttaaagaagc-3 ; and gapdh forward : 5-gaaggtgaagg tcggagtc-3 ; gapdh reverse : 5-gaagatggtgatgggatttc-3. the 2 l of the synthesized cdna were used in all real - time pcr together with the sybr green i master mix (sybr premix ex taq, takara, japan) on mj research opticon2 real - time thermocycler (bio - rad, hercules, ca, usa). fluorescent reading from real - time pcr reaction was quantitatively analyzed by determining the difference of ct (delta ct) between ct of nurr1 and its internal gapdh, and the nurr1 gene expression was determined by the formation of 2. in addition, for all the analyzed samples, only triplicates with a standard deviation of the ct 0.05), which was in accord with the half - life of 812 h. nurr1 mrna relative levels in vitro measured by real - time polymerase chain reaction treated with pramipexole. all experiments were performed at least three independent times (p 0.05), which was in accord with the half - life of 812 h. nurr1 mrna relative levels in vitro measured by real - time polymerase chain reaction treated with pramipexole. peripheral blood mononuclear cells from health volunteers were treated with 10 mol / l pramipexole for 024 h. glyceraldehyde-3-phosphate dehydrogenase was monitored as internal control. all experiments were performed at least three independent times (p < 0.001). nurr1 is highly expressed in the developing and adult ventral midbrain and required for the acquisition and maintenance of the da phenotype in nigrostriatal neurons. it has been reported that besides central nervous system, nurr1 is also an expression in many tissues including bone, endothelial cells, and pbmc. the key point for clinical evacuation of nurr1 is whether the alteration of nurr1expression is secondary to anti - pd drug effects. using rt - pcr, we analyzed the nurr1 mrna level in pbmc from 362 sporadic pd patients and 193 hcs from southern china, and found that the nurr1 expression level in de novo subgroup with a tendency of nonsignificant decrease compared to hcs, but a statistically significant increase in both da agonist or da agonist and l - dopa subgroups, which indicated that da agonists may exert an up - regulation effect on the expression of nurr1 in pbmc. as a transcription factors, the expression of nurr1 can be affected by many factors including growth factors, neurotransmitters, and drugs. for example, injection of 6-hydroxydopamine into the striatum produced an increase in the number of cells expressing nurr1 in both substantia nigra compacta (snc) and substantia nigra reticulate. similar to our data, a series of evidence indicated that da receptor agonists may play an active role on the nurr1 expression. for instance, ropinirole has been documented to prevent the progression of pd in nurr1 deficient mouse, and pan. reported that sh - sy5y cells treated with d3 receptor agonist pramipexole enhanced the expression of nurr1 mrna and protein in vitro. studies on postmortem brains have been found that an age - related decline of da phenotypic markers was associated with down - regulation of nurr1 expression in human sn. reported the optical density of nurr1 immunofluorescence was significantly decreased in nigral neurons containing -synuclein - immunoreactive inclusions in pd patients. in addition, recent studies showed a significant decreased of nurr1 mrna in snc of d2 dopamine receptor / mice (d2r). however, our data showed that the expression of nurr1 mrna in pbmc was affected by drugs like da agonist, but was no significant correlation with the disease severity (h and y scale) or age of onset of pd. for further exploring the pattern of nurr1 expression in vitro, we found that nurr1 mrna relative levels in pbmc increased significantly after treated with 10 mol / l pramipexole for 2, 4, and 8 h, which in accordance with our clinical investigation from blood cells of pd patients, demonstrating da agonist plays an active effect on nurr1 gene in pbmc. reported that levels of nurr1 mrna and protein in sh - sy5y increased after pramipexole treatment, indicating that the biological pattern of nurr1 expression in pbmc might be different from the changes in degeneration of da neurons in midbrain. in summary, we observed that nurr1 mrna level in pbmc from our pd patients was significantly influenced by antiparkinsonism drugs da agonists but not l - dopa, and such effect may contribute to da agonists - mediated neuroprotection on da neurons. furthermore, we found a slight insignificant reduction of nurr1 level in our de novo pd patients, which might present a correlation with the disease. a larger sample of de novo pd patients may be needed to evaluate whether reduced expression of nurr1 in pbmc can be used as a biomarker for early pd.	background : nurr1 plays an essential role in the development, survival, and function maintenance of midbrain dopaminergic (da) neurons, and it is a potential target for parkinson 's disease (pd). nurr1 mrna can be detected in peripheral blood mononuclear cells (pbmcs), but whether there is any association of altered nurr1 expression in pbmc with the disease and da drug treatments remains elusive. this study aimed to measure the nurr1 mrna level in pbmc and evaluate the effect of nurr1 expression by da agents in vivo and in vitro.methods:the mrna levels of nurr1 in pbmc of four subgroups of 362 pd patients and 193 healthy controls (hcs) using real - time polymerase chain reaction were measured. the nonparametric mann - whitney u - test and kruskal - wallis test were performed to evaluate the differences between pd and hc, as well as the subgroups of pd. multivariate linear regression analysis was used to evaluate the independent association of nurr1 expression with hoehn and yahr scale, age, and drug treatments. besides, the nurr1 expression in cultured pbmc was measured to determine whether da agonist pramipexole affects its mrna level.results:the relative nurr1 mrna levels in da agonists treated subgroup were significant higher than those in recent - onset cases without any anti - pd treatments (de novo) (p < 0.001) and hc groups (p < 0.010), respectively. furthermore, the increase in nurr1 mrna expression was seen in da agonist and l - dopa group. multivariate linear regression showed da agonists, l - dopa, and da agonists were independent predictors correlated with nurr1 mrna expression level in pbmc. in vitro, in the cultured pbmc treated with 10 mol / l pramipexole, the nurr1 mrna levels were significantly increased by 99.61%, 71.75%, 73.16% in 2, 4, and 8 h, respectively (p < 0.001).conclusions : da agonists can induce nurr1 expression in pbmc, and such effect may contribute to da agonists - mediated neuroprotection on da neurons.
thermodynamic database for proteins and mutants (protherm) and thermodynamic database for protein nucleic acid interactions (pronit) are two comprehensive, integrated databases that document experimentally determined thermodynamic parameters published in the literature. both protherm (14) and pronit (5) include several thermodynamic parameters along with sequence and structural information, experimental methods and conditions, and literature information. recent years have seen tremendous progress in studies on proteins owing to the development of various experimental methods to analyze proteins at the genome scale. the correlation between structure and thermodynamics of these key molecules provides valuable insights into the way in which they function. even though the information is available in scientific journals, books (6,7) and literature databases, retrieving useful, specific data from these resources our major goal in developing these databases is to provide the scientific community a single, comprehensive data repository, where all the thermodynamic data related to protein stability and protein nucleic acid interactions are available. the availability of such thermodynamic databases would be a valuable resource for understanding the protein folding mechanism, protein stability, molecular recognition and gene expressions. this can lead to a wide spectrum of applications such as developing algorithms / methods for prediction systems, protein engineering and quantitative simulation of gene regulatory networks. the thermodynamic data available in protherm and pronit are widely used by researchers to study the underlying mechanisms of protein stability upon mutations and protein nucleic acid interactions (see the reference sections on both the websites). this paper describes the major updates and enhancements to these databases for the last few years. both the databases contain information on protein, mutational information, experimental methods and conditions, several thermodynamic parameters and literature information. previous publications (15) explain in detail the content and organization of the databases. protherm and pronit are implemented in 3dinsight (8), a relational database system for structure, function and property of biomolecules. this facilitates more efficient search and retrieval of data by flexible queries, and enables users to gain insight into the relationship among structure, thermodynamics and function of proteins. we have been collecting the thermodynamic data from published original articles, by searching the pubmed literature database with a combination of specific terms, as well as by searching online journals probably containing thermodynamic data. the input data are checked automatically by checking programs and also manually to avoid errors. then, we upload the data first to a test site, where expert curators check and verify the data. after this checking,, an email notification for each input entry is sent to the corresponding author, which enables the authors to check their own data and thereby improve the data validation. we update both the databases frequently. the current release, protherm 5.0 contains 17 113 entries from 771 proteins, retrieved from 1497 scientific articles, which is 20% increase in data from the last version (4). currently, the numbers of data for wild - type proteins, single, double and multiple mutants are 7014, 8202, 1277 and 620, respectively. based on the solvent accessibility of mutants, 4426 mutations are buried, 2687 partially buried and 2751 exposed. in terms of secondary structures, 3993 mutations are in helix, 2622 in strand, 1227 in turn and 2467 in coil regions. majority of data are obtained from cd (6825) and dsc (5294) experiments followed by fluorescence (3628). further, 10 154 data are obtained by thermal denaturation, 3890 and 2796 data from gdnhcl and urea denaturation, respectively. there are 158 different dna - binding proteins with 3489 wild - type entries and 1411 mutant entries. majority of data are obtained by gel shift (1316), fluorescence (1143) and filter binding (1053), followed by calorimetry (727), surface plasmon resonance (185) and footprinting (168). although proteins from a variety of organisms are present in pronit, majority of interaction data are from escherichia coli proteins (1625) followed by mus musculus (637) and homo sapiens (569). thus, we try to provide link from thermodynamic data in protherm to structural information. so far, protherm data are connected to sequence and structural information of proteins through 3dinsight. we have added a new cross - link between protherm and sting (9), a comprehensive analysis tool for proteins with many structural descriptors. for given protein mutations searched within sting, conversely, all the protherm data with available protein structure have pointers to the corresponding sting entry with detailed structural information. this cross - link will greatly facilitate the analysis of structure thermodynamic relationship of proteins. the protherm page also provides cross - reference tables necessary for creating cross - links with pdb (10), pir (11) and swiss - prot (12) databases. we have also added several new features in the search interface to make the search more efficient and convenient. in the current release of pronit 2.0, we have included 200 protein rna interaction data. to facilitate the retrieval of data based on dna and rna separately, we have added a new field called type_nuc, where we provide the information about whether the nucleic acid sequence is dna (single - stranded dna, ssdna, or double - stranded dna, dsdna) or rna. furthermore, a search option is added to retrieve data based on ssdna, dsdna or rna. hence, we have added a new field, synonyms, in order to address this problem. other additions of field are the swissprot i d of the protein and related_entries, which provides the list of entries that contain data from the same paper (the original paper usually contains multiple data and they are entered in different entries). we also provide a link to all homologous pdb codes with sequence identity of > 95%. also, display options and sorting options are significantly improved. we have supplied the lists of pronit entries, protein names, protein sources, pdb code, ndb codes (13), authors and references in the advanced search page, along with a new query help page to help users for easy retrieval of the data. several entries are deleted because of duplication, co - operative binding and so on, and the database entries are now renumbered. a mapping table, which relates the old and new entry numbers, is provided to help old users of pronit. the users of protherm and pronit are requested to cite the references (4) and (5), respectively, in their publication including the above urls. users who use both the databases for their work may cite this article in their publications. suggestions and other materials for inclusion in the databases are welcome and should be sent to either [email protected] or [email protected].	protherm and pronit are two thermodynamic databases that contain experimentally determined thermodynamic parameters of protein stability and protein nucleic acid interactions, respectively. the current versions of both the databases have considerably increased the total number of entries and enhanced search interface with added new fields, improved search, display and sorting options. as on september 2005, protherm release 5.0 contains 17 113 entries from 771 proteins, retrieved from 1497 scientific articles (20% increase in data from the previous version). pronit release 2.0 contains 4900 entries from 273 research articles, representing 158 proteins. both databases can be queried using www interfaces. both quick search and advanced search are provided on this web page to facilitate easy retrieval and display of the data from these databases. protherm is freely available online at and pronit at.
a balanced response to pathogens and other immune stimuli is important to maintain physiologic homeostasis. however, whereas infectious agents may cause organismal death if the immune response is not properly activated, hyperactivated or inappropriately timed immune responses can also lead to various pathologies. this issue is particularly relevant in autoimmune disorders such as lupus erythematosus (le), in which aberrant immune signaling and autoantibody development are associated with a broad spectrum of negative outcomes ranging from skin rashes and dermal scarring to more deadly systemic effects that include nephrotoxicity. the molecular mechanisms of autoimmune disease pathogenesis remain largely unknown but are thought to involve both genetic and environmental contributions. furthermore, recent data indicate that deregulation of cellular autophagic and apoptotic processes may contribute to immune disorders. thus, this review discusses possible molecular mechanisms by which aberrant crosstalk between various intracellular signaling pathways associated with cellular responses to environmental dna damaging agents and viral or microbial infection may exacerbate the phenotypes of autoimmunity. the type i interferons, which include ifn- and ifn-, are important regulators of the innate immune response following infection. interferons are cytokines that are secreted from viral- and microbe - infected cells and alert the immune system to the presence of infection. interferons act on the type i ifn receptor (ifnar) on target cells to initiate intracellular signal transduction pathways that lead to the expression of antiviral and immunomodulatory genes that stimulate various immune cells, inhibit cell growth, and promote apoptosis following infection. interestingly, alternations in ifn production and signaling are often found in patients with autoimmune disorders.1,2 indeed, the identification of interferon activity in the serum of autoimmune patients initially suggested an aberrant role for ifn in the pathogenesis of autoimmunity.3 moreover, the levels of serum ifn are generally found to be correlated with disease activity and severity.4,5 the notion that type i ifn may actively contribute to autoimmune phenotypes was based on the discovery that treatment of certain patients with type i ifn led to an increase in autoantibody production and to various autoimmune diseases,69 and it has since been proposed that excess ifn could lead to a break in immune tolerance mechanisms through the activation of myeloid dendritic cells that subsequently stimulate autoreactive t cells.1012 the importance of ifn signaling to autoimmunity is also highlighted by the observation that most patients with systemic lupus erythematosus show signs of a so - called ifn gene signature in their blood.1316 understanding the mechanisms of ifn activation and production under normal and pathologic conditions may therefore reveal novel approaches to limit the progression and severity of autoimmune diseases. important insights into the mechanisms of ifn production have been made over the past decade with the discovery of signal transduction pathways that respond to pathogen - derived nucleic acids and other factors to induce ifn production. the induction of ifns requires the recognition of pathogen - associated molecular patterns (pamps) produced by viruses and microbes by specific pattern recognition receptors (prrs) in the infected organism. this recognition can take place either outside the cell or within the cell and ultimately results in the activation of intracellular signaling pathways that induce ifn gene expression. although a variety of biological macromolecules can act as pamps, including viral nucleic acids and bacterial cell wall components, the response of cells to pathogenic dna and cyclic dinucleotides has attracted a great deal of attention over the past few years. classical nucleic acid sensing prrs include toll - like receptors (tlrs) that are present on the cell surface and within endosomes. however, tlr expression is typically restricted to specific cell types, such as plasmacytoid dendritic cells. given that cells that do not express tlrs are also thought to be capable of responding to viral and microbial dna to induce ifn production, it became clear that tlr - independent pathways must also exist in many diverse cell types to induce ifn and a subsequent innate immune response. indeed, the discovery of sting in 2008 (stimulator of interferon genes ; also known as tmem173, mita, myps, and eris) as an endoplasmic reticulum associated adaptor protein that facilitates ifn induction in response to nonself dna was a major breakthrough in the field.1719 however, the mechanism of sting activation by dna remained unresolved for a number of years. although sting has some direct affinity for dna,20 microbe - derived 3,3-cgamp, c - di - amp, and c - di - gmp had previously been shown to be ligands for sting and to induce ifn activation.2128 the important discovery in 2013 of the enzyme cyclic gmp - amp synthase (cgas),29 which generates the cyclic dinucleotide 2,3-cgamp in response to dna stimulation,21,22,30,31 appeared to reconcile these findings. thus, our current understanding of sting function is that it is activated by specific cyclic dinucleotides that are produced either directly by invading pathogens or indirectly by endogenous cgas. thus, the discovery of an endogenous ligand for sting, along with its corresponding native human biosynthetic enzyme, has provided important new insights into the mechanisms of innate immunity and generated great excitement in the field.32,33 the binding of cyclic dinucleotides to sting causes a conformational change in the protein that allows it to act as a scaffold or adaptor protein for the kinase tank - binding kinase 1 (tbk1) to phosphorylate a transcription factor known as interferon regulatory factor 3 (irf3),34 which had previously been shown to be critical for ifn induction by cytosolic dna.35 this phosphorylation event induces the dimerization of irf3 and allows it to enter the nucleus where it can function as a transcription factor to drive the expression of type i ifns and other gene targets.3537 a schematic summarizing the sting - dependent production of ifn in response to viral and microbial dna and cyclic dinucleotides is provided in figure 1. interestingly, a recent observation that rare gain - of- function mutations in sting contribute to autoimmunity and autoinflammatory diseases in human populations38,39 highlights the physiologic importance of sting in autoimmunity. furthermore, these findings suggest that the identification and characterization of additional genetic and environmental factors that impact the sting pathway may provide new insights into autoimmune pathologies and provide novel approaches to control innate immune responses. although the cellular autophagic machinery plays well- recognized roles in breaking down damaged proteins and organelles by sequestering and directing cargo to the lysosome, the same machinery is also required for host cells to cope with invading pathogens.40 indeed, viral infection and even synthetic dna transfection have been shown to induce canonical biochemical read - outs of autophagy, such as lc3 lipidation, and to lead to the co - localization of cytosolic dna with autophagic proteins.4144 recent findings further show that this response to infection is tightly coordinated with the sting - dependent innate immune signaling pathway. for example, sting was shown to be required for the efficient ubiquitin - mediated autophagic clearance of mycobacterium tuberculosis in macrophages,45 and additional studies with the double - stranded dna (dsdna) genome viruses hsv-1 and human cytomegalovirus similarly demonstrated a role for sting in the induction of the autophagic response.46,47 moreover, the enzyme cgas was demonstrated to be required to target cytosolic dna from bacterial pathogens to the autophagy pathway.44,4850 interestingly, other microbes can bypass this pathway by producing cyclic di - gmp that directly activates sting.49 the precise mechanism by which the cgas - sting pathway engages the autophagic machinery to degrade viral and microbial dna as well as cyclic dinucleotides remains to be better characterized. interplay between the cgas - sting innate immune response and the autophagic factors may improve the efficiency of viral pathogen recognition and removal from the infected cell. there is also evidence that autophagic proteins can directly interact with components of the cgas - sting pathway and influence its downstream signaling. for example, the proautophagic protein beclin-1, which plays important roles in the induction and maturation of the autophagosome, directly binds to cgas to negatively regulate its activity and suppress cgamp production.44,48 although this interaction is important for promoting efficient autophagy - mediated degradation of cytosolic pathogen dna through release of the negative autophagy regulator rubicon from the beclin-1 complex, direct and negative regulation of cgas activity by beclin-1 also serves to prevent excessive or persistent immune stimulation by cgas following dsdna stimulation or hsv-1 infection. interestingly, the autophagy regulatory kinase unc-51 like kinase 1 (ulk1) was reported to phosphorylate sting to suppress irf3 activation.51 this response occurred after the autophagy - dependent and sting - dependent delivery of tbk1 to endosomes or lysosomes and involved the dissociation of ulk1 from its repressor amp - activated kinase (ampk). this negative regulation of sting was shown to be dependent on cgamp produced by cgas, which indicates that cgamp not only directly activates sting but also stimulates a negative feedback loop that shuts off sting activity to prevent the persistent induction of ifn. together, beclin-1 and ulk1 provide 2 mechanisms by which the autophagic machinery may work to limit sting - dependent innate immune signaling in response to infection. a schematic summarizing this regulation of the cgas - sting pathway autophagy and apoptosis are well recognized as being 2 important cellular processes that allow cells and organisms to cope with and respond to cellular damage induced by a variety of stressors. whereas autophagy is thought to be the primary mechanism by which cells turnover damaged organelles and other smaller cellular substituents, apoptosis is utilized to get rid of whole cells. however, certain stimuli, such as nutrient deprivation or viral infection, can potentially lead to the activation of either pathway. indeed, both processes can occur in the same cell, though often with different kinetics in which autophagy is utilized prior to the induction of apoptosis. moreover, there are a variety of mechanisms by which the autophagic and apoptotic pathways can become intertwined to affect cell fate.52 various cell stressors, including nutrient deprivation, can stimulate an autophagic response to allow cells to adapt to altered cellular or environmental conditions.53,54 however, cells that are unable to cope with the stressor through autophagy may ultimately undergo apoptosis. under these conditions, it is expected that shutting off autophagic signaling may be important to conserve cellular resources for the process of apoptosis. consistent with this notion, a variety of autophagic proteins, including atg3, beclin-1, and ambra1, have been shown to be targeted for caspase - mediated destruction during apoptosis.5557 furthermore, the localization of the tumor suppressor protein p53 to the cytosol is associated with its interaction with fip200 (fak family kinase - interacting protein of 200 kda), which blocks the activation of the ulk1-fip200-atg13 complex that is necessary for autophagosome formation.58,59 thus, there is clear evidence that apoptotic signaling can suppress autophagy under conditions of extreme cellular stress. as will be discussed below interestingly, exposure to excessive amounts of uv wavelengths of sunlight has long been known to induce apoptosis in the skin and to exacerbate the symptoms of autoimmune disorders such as le in susceptible individuals.60,61 uv light induces photoproducts in dna that interfere with dna replication and transcription and therefore are potentially lethal to cells if the damage is not properly removed by the nucleotide excision repair system.62 indeed, clinical case studies have shown uv exposures to have serious consequences in individuals with a history of the autoimmune disorder lupus.63,64 the current paradigm for how uv - induced cell death in the skin may promote autoimmune disorders such as lupus is based on the notion that the defective clearance of uv - damaged, apoptotic keratinocytes leads to the development of antibodies against nuclear autoantigens that are released from dying cells.65 - 67 although this hypothesis has several attractive features, there have been criticisms that the methodologies used to measure cell death lack sufficient specificity.68 - 70 thus, the cell death - autoantigen release model remains to be fully tested and validated. moreover, there are likely other mechanisms that may explain how lethal or even sublethal uv exposures could influence the uv - associated pathogenesis of lupus. to examine the links between uv exposures and innate immune signaling further, a recent study using cultured keratinocytes and other human cells lines in vitro sought to clarify how uv radiation affected the sting - dependent innate immune signaling pathway.71 using dsdna and specific cyclic dinucleotides (cgamp, c - di - gmp) to mimic a viral or microbial infection, the irradiation of cells with uv light prior to, or soon after, dna / cyclic dinucleotide transfection was found to potentiate the stimulatory effect of the cytosolic dna / cyclic dinucleotides on the sting pathway. thus, increased irf3 phosphorylation, dimerization, and nuclear entry were observed in cells containing cytosolic dna or cyclic dinucleotides when the cells were exposed to uv radiation. interestingly, the maximal effect of uv radiation occurred at doses that saturated the ability of cells to remove genomic damage by the nucleotide excision repair machinery. moreover, the stimulation of the sting - dependent innate immune response was found to also occur with the dna damaging uv mimetic and environmental carcinogen benzo[a ] pyrene-7,8-dihydrodiol-9,10-epoxide, which indicates that other environmental agents of relevance to human health may also contribute to autoimmunity. to uncover the mechanism of this phenomenon, potential roles for dna repair intermediates and various dna damage and cell stress response kinases were initially considered.71 however, the results of these experiments indicated that some other aspect of the cellular response to dna damage was responsible for potentiation of the sting pathway. furthermore, it was noted that the kinetics of apoptotic signaling were closely correlated in time with a posttranslational loss in the expression of the autophagic proteins ambra1 and ulk1. because ulk1 is a negative regulator of sting,51 these results indicated that the stronger sting response that occurred following uv irradiation was likely due to the uv - dependent loss of ulk1 and a subsequent de - repression of sting function. moreover, given that ambra1 was previously shown to directly affect ulk1 protein stability and to be controlled by a caspase - dependent and calpain - dependent pathway,72 these various findings together suggested that uv light induced apoptotic signaling and the corresponding loss of the sting negative regulator ulk1 were responsible for potentiating the cellular response to cytosolic dna and cyclic dinucleotides (figure 2). consistent with this hypothesis, inhibiting caspase and calpain activation prevented both ambra1 and ulk1 loss and the ability of uv radiation to stimulate the cytosolic dna - dependent activation of sting.71 importantly, the timing of uv irradiation relative to the introduction of cytosolic dna or cyclic dinucleotides was critical to the response, as exposure to repair - saturating uv doses more than 1to 2 hours prior to or following the introduction of cytosolic dna failed to potentiate the sting response.71 because ulk1 is required to initiate autophagosome formation, the premature degradation of ulk1 by uv - induced apoptotic signaling might be expected to prevent proper activation of the sting pathway. similarly, the transient nature of the sting signaling pathway following the introduction of dna or cyclic dinucleotides into the cytosol would mean that a delayed loss of ulk1 would occur too late to affect the rapid cellular response to sting ligands. thus, these findings indicate that an optimal window of exposure to uv light and cytosolic dna is important in determining how apoptotic responses to uv affect the autophagic regulation of the sting pathway. the observation that apoptotic signaling induced by environmental agents such as uv light can affect the autophagic response to cytosolic dna and cyclic dinucleotides provides a new mechanistic basis for exploring how environmental exposures influence autoimmune diseases. human skin is constantly exposed to uv wavelengths of sunlight and to a variety of pathogens. although both uv exposure and infection are known risk factors that contribute to autoimmune phenotypes in human patients, experimental models of autoimmunity have thus far failed to consider a combinatorial interaction of both factors in autoimmune disease pathogenesis. thus, the results presented above suggest that aberrant crosstalk between the apoptotic and autophagic pathways in response to environmental carcinogens such as uv and viral and microbial infections may contribute to autoimmune disease phenotypes and should be explored in future studies.	autoimmune disorders constitute a major and growing health concern. however, the genetic and environmental factors that contribute to or exacerbate disease symptoms remain unclear. type i interferons (ifns) are known to break immune tolerance and be elevated in the serum of patients with autoimmune diseases such as lupus. extensive work over the past decade has characterized the role of a protein termed stimulator of interferon genes, or sting, in mediating ifn expression and activation in response to cytosolic dna and cyclic dinucleotides. interestingly, this sting - dependent innate immune pathway both utilizes and is targeted by the cell s autophagic machinery. given that aberrant interplay between the apoptotic and autophagic machineries contributes to deregulation of the sting - dependent pathway, ifn - regulated autoimmune phenotypes may be influenced by the combined exposure to environmental carcinogens and pathogenic microorganisms and viruses. this review therefore summarizes recent data regarding these important issues in the field of autoimmunity.
diabetes contributes strongly to the risk of cvd with a risk that is increased four to six times, both for stroke and myocardial infarction. the increase in risk for myocardial infarction seem to be more pronounced in women than in men. the prevalence of igt in the general population is 23 times higher than that of previously unknown diabetes, which, in turn, is as common as known diabetes. patients with impaired glucose tolerance have an increased risk of cvd that is independent of traditional risk factors such as hypertension, smoking and hypercholesterolemia and it has been suggested that slightly elevated glucose levels, even in the non - diabetic range, might be associated with increased macrovascular disease. several publications have focused on the relationship between igt and coronary heart disease (chd). the results have indicated that ethnicity, sex and age might be factors that modify the strength of the risk of chd with impaired glucose tolerance [6 - 11 ]. the aim of the present investigation was to further evaluate the possible relation between igt and chd. therefore, we examined ecg 's to identify signs of clinically undiagnosed myocardial infarction together with cardiovascular risk factors in a randomly selected population of men and women from northern sweden, a region with an ethnically homogenous population and a high incidence of cardiovascular disease. this study was performed within the framework of the northern sweden monica project which, in turn, is a part of the who monica project (monitoring of trends and determinants in cardiovascular disease). during january to april 1986, a population was screened for cardiovascular risk factors. a total of 2000 individuals in the 25 to 64 year range were invited. within each age group (2534, 3544, 4554, 5564 years) 250 men and 250 women were randomly selected from continuously updated population registers in norrbotten and vsterbotten, the two northernmost provinces of sweden. people who still did not come, were contacted by telephone to ascertain reasons for reluctance to attend and to get basic information on social background and risk factors. the participants were asked to complete a questionnaire with items on, inter alia, social background, smoking habits, medical history and intake of drugs. the questionnaire was returned on the site of the survey, which was performed by two mobile teams in local health centres (or corresponding) throughout the monica area. seven hundred ninety subjects from vsterbotten participated in the study (79 % of all invited). six hundred seventeen subjects, without known diabetes, underwent a 75 g oral glucose tolerance test (ogtt) with measurement of plasma glucose. anthropometric measurements and biochemical analyses were as previously described. clinically diagnosed myocardial infarction, or known mi (kmi), was defined by a positive answer to the question " have you ever had a myocardial infarction ? ". clinically diagnosed, or known diabetes, was defined by a positive answer to the question " do you have diabetes ? ". a validation study of 70 incident diabetes cases in this cohort using clinical case records show these answers to be highly accurate (unpublished data). hypertension was defined as systolic blood pressure greater than 160 mm hg or diastolic blood pressure greater than 95 mm hg or a positive answer to the question " are you being treated with pharmaceutical drugs for high blood pressure ". the northern sweden monica study has been approved by the research ethics committee of ume university and the data handling procedures by the national computer data inspection board. all electrocardiograms (ecg 's) were recorded with a cardiovit cs-6 microprocessor - based electrocardiograph (schiller ag, basel, switzerland). twelve leads were recorded for 10 seconds and used for interpretation by the cs-6 recorder. original readings were printed on paper (paper speed 50 mm / s) together with an averaged ecg and a diagnostic statement made by the computer. ecg 's were asessed according to the minnesota code by two trained observers and q / qs - wave ; codes 1.11.3 were designated as myocardial infarction. ecg 's from two subjects were coded differently by the observers and were therefore excluded from the mi group. students t - test and were used to test for differences between subjects with normal ecg and subjects with ukmi. in a stepwise logistic regression with ukmi as dependent variable, traditional risk factors (hypertension, smoking, hypercholesterolemia) and the occurrence of igt were entered as dichotomous independent variables and age as a continuous variable. all electrocardiograms (ecg 's) were recorded with a cardiovit cs-6 microprocessor - based electrocardiograph (schiller ag, basel, switzerland). twelve leads were recorded for 10 seconds and used for interpretation by the cs-6 recorder. original readings were printed on paper (paper speed 50 mm / s) together with an averaged ecg and a diagnostic statement made by the computer. ecg 's were asessed according to the minnesota code by two trained observers and q / qs - wave ; codes 1.11.3 were designated as myocardial infarction. ecg 's from two subjects were coded differently by the observers and were therefore excluded from the mi group. students t - test and were used to test for differences between subjects with normal ecg and subjects with ukmi. in a stepwise logistic regression with ukmi as dependent variable, traditional risk factors (hypertension, smoking, hypercholesterolemia) and the occurrence of igt were entered as dichotomous independent variables and age as a continuous variable. table 1 shows baseline characteristics of 790 subjects, 25 to 64 years of age. both sexes were evenly represented. previously known mi (kmi) was present in 2.7 %, hypertension in 18,5 %, hypercholesterolemia in 36.3 % and diabetes in 2.8 %. characteristics of the total study population, 25 to 64 years of age in the northern sweden monica population survey in 1986. cigarette smoking were more common in women but the use of smokeless tobacco was rare compared to men. characteristics of the study population, men and women, 25 to 64 years of age in the northern sweden monica population survey in 1986. an ogtt was carried out in 617 subjects, showing igt in 8.6 % and unknown diabetes in 2.3 %. a gender - specific analysis of this population did not differ significantly regarding age, anthropometric variables or biochemical markers compared to the total study population shown in table 1 and 2 (table 3). characteristics of the glucose tolerance tested subjects, men and women, 25 to 64 years of age in the northern sweden monica population survey in 1986. interestingly, there were twice as many subjects with ecg indicating mi (ukmi) that had not been clinically diagnosed than with known infarctions (kmi). comparison of clinically diagnosed myocardial infarctions and ecg indicating myocardial infarction (n = 771). mi = myocardial infarction a gender - specific analysis comparing subjects with normal ecg to ukmi subjects are shown in table 5. subjects of both sexes with ukmi were older and had a greater burden of previous cvd than did subjects with normal ecg 's. body mass index and waist - hip ratio were higher in subjects with ukmi, more so in women than in men. there was a tendency to higher lipid levels in the ukmi group, at least in women. the number of subjects with igt was significantly higher in the ukmi group in women, but not in men compared to the group with normal ecg. bmi = body mass index the association between ukmi and sex, age, smoking, hypercholesterolemia, hypertension and igt was analyzed in a multiple logistic regression model. none of the variables, except for age, showed significant association with ukmi (table 6). predictors of ukmi in a multiple logistic stepwise regression analysis ukmi = unknown myocardial infarction. or (= odds ratio) men and women were analyzed separately and the results are shown in table 7. in men, no variable was significantly associated with ukmi although the odds ratio for hypercholesterolemia was of borderline significance ; 3.22 (ci 0.92 ; 11.22). in women, the odds ratio of having ukmi was 4.14 (ci 1.13 ; 15.14) in women with igt compared to women with normal glucose tolerance. the odds ratio for hypertension was of borderline significance ; 3.33 (ci 0.97 ; 11.43). the results were similar if subjects with st / t changes and left ventricular hypertrophy were excluded from the " normal ecg " group. if, on the other hand, st / t changes are included in the mi group no significant association between any variable was seen (data not shown). predictors of ukmi in a multiple logistic stepwise regression analysis conditions and abbreviations were as in table 4 our findings indicate that previously unknown q - wave infarction is considerably more common in women 2564 years of age with impaired glucose tolerance than in women with normal glucose tolerance, even after adjustment for traditional risk factors. the total number of participants was rather small which is a limitation of the study but the attendence rate of 80 % is in accordance with other major population surveys. cross - sectional studies concerning resting ecg abnormalities indicating ihd in subjects with igt have reported varying results. in younger south asian men (4054 years) settled overseas, major q - waves were strongly associated with glucose intolerance and hyperinsulinemia. this association was less strong for european men in the same study. in a chinese population, igt was associated with ecg abnormalities in both men and women, but the ecg criteria used also included t - wave abnormalities and complete left bundle branch block, which differs from the stricter q - wave criteria used in our study. data from the rancho bernardo study, an older population of white subjects, showed that ecg abnormalities were more common in subjects with non - insulin dependent diabetes (niddm) but not in those with igt. also in this study, ecg criteria were wider than our criteria. in the san luis valley diabetes study, 2074-year - old hispanics and non - hispanic whites of both sexes were studied. an association between niddm, but not igt, with mainly q / qs waves was seen. a study of north american white males, aged 4059 years, could not show any association between blood glucose and major ecg abnormalities indicating mi. hence, no consistent data concerning association between igt and silent myocardial infarctions have been shown and only few studies have included women. to our knowledge mi 's were somewhat more common in subjects with igt, although not reaching conventional levels of significance. this indicates a problem of statistical power and perhaps differing impact of blood glucose abnormalities in different ethnic groups. some of these studies found that increases in risk were attenuated by adjustement for other known risk factors. our population - based study is thus the first to show that in middle - aged european women, there is a strong and independent relationship between igt and previously unknown mi, defined by strict criteria. major q - waves were shown to occur in only 43 % of subjects in whom old myocardial infarcts were detected at autopsy, and q / qs abnormalities were found to be rather common in non q - wave infarctions as well. twenty to thirty percent of major q / qs infarctions are known to be silent. moreover, q / qs abnormalities often disappear during recovery from an acute myocardial infarction. this was also evident in our study where only a minority of reported previous mi were classified as mi on ecg. interestingly, it was recently shown that left ventricular hypertrophy (lvh) mass and wall thickness increased with worsening glucose intolerance, an effect that was more striking in women compared with men. hence, since lvh can mimic mi this might lead to misclassification bias in the ecg interpretation. in the present investigation many kmi 's were seen in the group with normal ecg, a not surprising observation considering the findings mentioned. specificity for q / qs - infarctions was high but sensitivity was low when ecg was compared to autopsy - verified infarctions. thus, some infarctions might be undetected in our material which could lead to misclassification bias and a dilution of effect. an increased ihd mortality has been described in subjects with igt, particularly in women. in the decode study, which included monica data from our survey, igt predicted mortality from all causes, cvd and chd. previous studies indicate that the risk of cvd is increased at the time of diabetes diagnosis. the risk seem to be independent of the duration of diabetes suggesting that factors operating before the development of overt diabetes contribute to the risk of cvd. this was recently shown in women where the risk for mi or stroke was substantially increased before diagnosis of type 2 diabetes. hyperglycemia has also been suggested to be a direct cause of some of the changes associated with atherosclerosis, but several other factors could act as casual links in this association such as impaired fibrinolysis and high fibrinogen levels, high levels of leptin as well as others which have not been assessed in population studies. these factors could link igt to atherosclerosis and thereby to the q / qs abnormalities found in the present investigation although the diverging results in men and women are difficult to explain. in conclusion, we found that igt was associated with ecg findings indicating silent myocardial infarction in women in a middle - aged general population in northern sweden. as encouraging results have recently been published on the effect of life - style modification to prevent high risk individuals from developing diabetes, our study underlines that such efforts may also lead to decreased cardiovascular risks. also, more research is needed to improve our understanding of the pathogenesis of coronary artery disease in subjects with early glucose dysregulation dl participated in the design of the study, performed the statistical analyses and drafted the manuscript. me participated in the design of the study and in the final preparation of the manuscript. we are grateful to dr per bjerle for participating in the sampling and coding of the ecg 's. this study was supported by grants from the swedish medical research council, (grant no. 27x-07192 to ka), the council for worklife and social research, the heart and chest fund, the foundation for strategic research, king gustaf v 's and queen victoria 's foundation and vsterbotten and norrbotten county councils.	backgroundpatients with impaired glucose tolerance (igt) have an increased risk of cardiovascular disease (cvd) that is independent of traditional risk factors. hence, slightly elevated glucose levels, even in the non - diabetic range, might be associated with increased macrovascular disease.methodswithin the northern sweden monica project a population survey was performed in 1986. electrocardiograms (ecg 's) were recorded for half of the survey (n = 790) and oral glucose test was carried out in 78 % of those. the association between subjects with ecg 's indicating previously unknown myocardial infarction (ukmi), igt and conventional risk factors were analyzed by logistic regression for men and women separately, adjusting for age, smoking, hypercholesterolemia and hypertension.resultsimpaired glucose tolerance was significantly more common among women with ukmi, but not in men, compared to the group with normal ecg. in men, no variable was significantly associated with ukmi although the odds ratio (or) for hypercholesterolemia was of borderline significance, 3.2 (95% confidence interval (ci) 0.9 to 11). the or of having ukmi was 4.1 (ci 1.1 to 15) in women with igt compared to women with normal glucose tolerance after multiple adjustment. the or for hypertension was of borderline significance ; 3.3 (ci 0.97 to 11).conclusionwe found that igt was associated with ecg findings indicating silent myocardial infarction in women in a middle - aged general population in northern sweden. the results persisted even after adjusting for known risk factors.
thoracic disk herniation is a rare surgical entity that is estimated to account for 0.25 to 1.8% of all spinal herniated disks,1 2 3 4 with an estimated incidence of 1 per million patient - years.2 they usually present between the third and sixth decades with a female predilection.5 hott described giant calcified thoracic disks (gctds) as a specific subgroup of herniated thoracic disks with a distinct clinical presentation, work - up, and management.6 gctds were defined as occupying more than 40% of the spinal canal based on preoperative computed tomography (ct), myelography, magnetic resonance imaging (mri) or both.6 gctds offer a neurosurgical challenge owing to their size, consistency, and degree of spinal cord compression. some diversity in the surgical management of gctds still exists among neurosurgeons. here, we present 18 cases of gctds, which represents the second largest cohort in the literature, and we describe the trench vertebrectomy via a thoracotomy as a safe and effective surgical technique. twenty - nine patients who underwent surgical treatment in our unit for herniated thoracic disks between the years 2000 and 2013 were reviewed. following radiologic review, 18 patients were found to have gctds as defined by hott.6 retrospective data was collected on patient demographics, presentation, operative details, and imaging findings including the fusion rates. the modified japanese orthopaedic association (mjoa) score was used to assess the outcomes. there was a female preponderance in our series with a male - to - female ratio of 4:14. the median age at diagnosis was 51.2 years with a range of 37 to 70 years. the mean duration of presenting symptom(s) was 9.9 months, with a range of 2 weeks to 3 years. seventeen (94.4%) patients in this series presented with signs and/or symptoms of myelopathy, with the remaining 1 (5.6%) patient presenting with symptoms more in keeping with a radiculopathy. the commonest presenting symptom was lower limb weakness in 66.7% of the series, and the commonest clinical sign was hyperreflexia in 83.3% (table 1). all patients in our center had both a preoperative computed tomography (ct) scan and mri (figs. 1 and 2). a ct scan defines the degree of calcification, disk morphology, and aids in preoperative planning (fig. the commonest affected level in our cohort was at t8t9 in 6 patients (33.3%), followed by t7t8 in 22.2% (table 1). (a) preoperative axial computed tomography (ct) showing the giant calcified thoracic disk (gctd) within the spinal canal. (b) (a) preoperative axial magnetic resonance imaging (mri) of the same disk space as fig. 1. (b) the access was through a thoracotomy in all but one patient, in whom a costotransverse approach was used. the ultrasonic bone cutter was used to facilitate the vertebrectomy in the last three patients in our series. the patient was positioned in the lateral position with the side of the thoracotomy uppermost. for localization of the affected level the thoracotomy was usually from the left side, because the chest cavity is larger on this side and it is also easier to mobilize the aorta than the great veins. in the upper thoracic levels (above t5), a right - sided thoracotomy may be used to avoid the arch of the aorta. to gain access to the affected level the lung was collapsed at this stage and the pleura opened. once the affected level was identified and confirmed with an x - ray, the parietal pleura was opened over the vertebral bodies below and above and reflected away from the spine. to allow better visualization of the lateral disk, the costovertebral and costotransverse ligaments were divided, and up to 2 cm of rib head were removed with bone nibblers or a drill if necessary. a trench vertebrectomy was performed with half of vertebra on either side excised ; it was not necessary to extend this procedure to remove the anterior part of the vertebral body (figs. 3 and 4). the ultrasonic bone cutter was useful here as an alternative to piecemeal resection or using a pneumatic drill. the groove was deepened until enough bone was removed to access the disk and normal dura on either side (fig. (a) intraoperative image using the misonix bone cutter (msb ; misonix, farmingdale, new york, united states) to drill the giant calcified thoracic disk. (b) intraoperative image of the bone graft being placed. (a) postoperative axial computed tomography (ct) outlining decompression using the trench technique. the base of the disk was eased away from the posterior longitudinal ligament, a layer of which may be left if adherent to the dura. after complete excision, the dura will move forward. in 8/18 cases (44.4%), the disk material was noted to be adherent to the cord. in such instances, a remnant layer of disk 6a, b. it shows fragments of calcified material within the degenerate disk associated with peripheral ossification. fifteen (83.3%) patients experienced a postoperative improvement of at least 1 point in the mjoa score on last follow - up. six (33.3%) patients required a blood transfusion during their hospital stay, which ranged from 2 to 5 u. one (5.6%) patient was transferred to the intensive care unit for ventilatory support due to development of adult respiratory distress syndrome. the average length of stay was 10.7 days, with a range of 4 to 35 days. the mean duration of follow - up was 19.8 months (range 7 months to 2 years). postoperative imaging was performed on all patients but was heterogeneous in modality including plain films, ct scans, or mris at 6 to 12 months. fusion was reported in all patients on the follow - up radiology reports ; the criteria used were the documentation of bony trabeculation and absence of bony lucency at the graft / vertebral end plate interface (fig. the postoperative imaging was not standardized compared with the preoperative imaging, and formal sagittal alignment measures were not calculated ; however, no patient developed a clinically or radiologically evident kyphotic deformity. all the bluish areas in right side of the slide represent calcium ; this tissue has been decalcified to be able to cut it, hence the rest of the right side is pink. there was a female preponderance in our series with a male - to - female ratio of 4:14. the median age at diagnosis was 51.2 years with a range of 37 to 70 years. the mean duration of presenting symptom(s) was 9.9 months, with a range of 2 weeks to 3 years. seventeen (94.4%) patients in this series presented with signs and/or symptoms of myelopathy, with the remaining 1 (5.6%) patient presenting with symptoms more in keeping with a radiculopathy. the commonest presenting symptom was lower limb weakness in 66.7% of the series, and the commonest clinical sign was hyperreflexia in 83.3% (table 1). all patients in our center had both a preoperative computed tomography (ct) scan and mri (figs. 1 and 2). a ct scan defines the degree of calcification, disk morphology, and aids in preoperative planning (fig. the commonest affected level in our cohort was at t8t9 in 6 patients (33.3%), followed by t7t8 in 22.2% (table 1). (a) preoperative axial computed tomography (ct) showing the giant calcified thoracic disk (gctd) within the spinal canal. (a) preoperative axial magnetic resonance imaging (mri) of the same disk space as fig. 1. the access was through a thoracotomy in all but one patient, in whom a costotransverse approach was used. the ultrasonic bone cutter was used to facilitate the vertebrectomy in the last three patients in our series. the patient was positioned in the lateral position with the side of the thoracotomy uppermost. for localization of the affected level the thoracotomy was usually from the left side, because the chest cavity is larger on this side and it is also easier to mobilize the aorta than the great veins. in the upper thoracic levels (above t5), a right - sided thoracotomy may be used to avoid the arch of the aorta. to gain access to the affected level once the affected level was identified and confirmed with an x - ray, the parietal pleura was opened over the vertebral bodies below and above and reflected away from the spine. to allow better visualization of the lateral disk, the costovertebral and costotransverse ligaments were divided, and up to 2 cm of rib head were removed with bone nibblers or a drill if necessary. a trench vertebrectomy was performed with half of vertebra on either side excised ; it was not necessary to extend this procedure to remove the anterior part of the vertebral body (figs. 3 and 4). the ultrasonic bone cutter was useful here as an alternative to piecemeal resection or using a pneumatic drill. the groove was deepened until enough bone was removed to access the disk and normal dura on either side (fig. 3). (a) intraoperative image using the misonix bone cutter (msb ; misonix, farmingdale, new york, united states) to drill the giant calcified thoracic disk. (b) (a) postoperative axial computed tomography (ct) outlining decompression using the trench technique. (b) postoperative sagittal ct clearly delineating the trench vertebrectomy. at this stage, the base of the disk was eased away from the posterior longitudinal ligament, a layer of which may be left if adherent to the dura. after complete excision, the dura will move forward. in 8/18 cases (44.4%), the disk material was noted to be adherent to the cord. in such instances, a remnant layer of disk 6a, b. it shows fragments of calcified material within the degenerate disk associated with peripheral ossification. fifteen (83.3%) patients experienced a postoperative improvement of at least 1 point in the mjoa score on last follow - up. six (33.3%) patients required a blood transfusion during their hospital stay, which ranged from 2 to 5 u. one (5.6%) patient was transferred to the intensive care unit for ventilatory support due to development of adult respiratory distress syndrome. the average length of stay was 10.7 days, with a range of 4 to 35 days. the mean duration of follow - up was 19.8 months (range 7 months to 2 years). postoperative imaging was performed on all patients but was heterogeneous in modality including plain films, ct scans, or mris at 6 to 12 months. fusion was reported in all patients on the follow - up radiology reports ; the criteria used were the documentation of bony trabeculation and absence of bony lucency at the graft / vertebral end plate interface (fig. the postoperative imaging was not standardized compared with the preoperative imaging, and formal sagittal alignment measures were not calculated ; however, no patient developed a clinically or radiologically evident kyphotic deformity. delayed postoperative sagittal computed tomography showing fusion at t8t9. all the bluish areas in right side of the slide represent calcium ; this tissue has been decalcified to be able to cut it, hence the rest of the right side is pink. gctds are rare and present a technical neurosurgical challenge. because of their size and degree of calcification, a significant proportion of gctds display some degree of thecal involvement, with penetration into the subarachnoid space via tears of the posterior longitudinal ligament and the dura mater.7 8 presentation may be with clinical signs and symptoms of a compressive myelopathy, radiculopathy, and/or back pain.1 6 8 9 10 in our series, the median age and female preponderance is a finding echoed in other studies,11 12 13 14 which is speculated to be secondary to altered calcium - phosphate metabolism that occurs postmenopausally.8 the pathophysiology of the calcification process is also poorly understood, although it has been suggested that calcium deposition originates from the posterior longitudinal ligament with extension into the disk space.15 all of the lesions in our cohort occurred at the lower thoracic levels, which is consistent with the findings by other authors.1 6 8 10 it is estimated that 75% of herniated thoracic disks occur below the level of t8, which is a probable combined consequence of the greater weight load in the lower thoracic spine and its greater range of movement.6 12 14 16 myelopathy was present in 17 (94.4%) of our patients. the other large case series in the literature describing gctds reported similar high rates of myelopathy,1 6 8 10 with hott comparing these high rates to 47% in smaller, nongiant thds.6 various surgical techniques and approaches to manage gctds are advocated in the literature. the choice of surgical approach has evolved over the last century and has included laminectomy, transpedicular, costotransversectomy, and transthoracic methods. posterior approaches are more or less obsolete owing to extremely poor results, particularly from laminectomy.7 17 18 19 20 despite the refinement of several approaches, the optimal operative treatment for thoracic disk herniations remains open to debate and may in part be dictated by the skills of the operating surgeons, whether the disk is central, and the presence of calcification.4 14 17 21 in their review, mulier and debois compared 7 of their own patients between 1986 and 1993 with 384 others to compare transthoracic, lateral, or posterolateral approaches to thds ; their results illustrated that transthoracic surgeries were associated with better neurologic outcomes.21 most authors advocate an open thoracotomy approach, in particular for centrally located calcified disk herniations.1 4 6 21 22 technical variations in the anterior transthoracic approach exist, with moran recommending a mini - open retropleural transthoracic approach with comparable results,10 although some authors advocate a two - level vertebrectomy followed by instrumented fusion.6 22 barbanera suggest using a mini - vertebrectomy, although instrumentation was still required in 2 (28.5%) cases of their series.1 in our view, a trench vertebrectomy has the advantage of visualizing the dura on either side of the herniated disk, allowing a safe excision, especially in the cases of a broad, central, heavily calcified disk prolapse with severe cord compression. in addition, this technique enables minimal bone removal compared with a complete two - level vertebrectomy. a costotransverse approach may be an alternative and can be utilized if the anatomy of the prolapsed disk is suitable (e.g., a more lateral cord compression) and good visualization of the anterolateral dura above and below the compressed segment can be safely attained. it enables direct visualization of the ventral spinal column, which is essential in anterior spinal pathologies such as gctds. the approach enables the greatest access and avoids manipulation of the spinal cord, which is already compromised in gctds.23 24 a left - sided thoracotomy evades the vena cava and liver on the right, though a right - sided approach may be undertaken if the pathology is predominately on that side or to evade poor visualization from structures such as the aortic arch around the level of t3 and t4.24 disadvantages of this approach are that it may require a longer hospitalization and longer intensive care stay than with other procedures, with a greater risk of pulmonary complications ; also, postoperative pain may be greater.4 17 21 thoracic excision of gctds is known to be associated with possible significant blood loss,10 particularly where vertebrectomy is conducted,25 which can mandate the ligation of two radicular arteries above and below the disk space. the use of the bone cutter in our experience allows all radicular arteries to be preserved, even those at the disk space level, thereby reducing the risk of spinal cord stroke secondary to ligation of the radicular artery of adamkiewicz. spinal cord injury and dural tears are also less likely due to the selective tissue - cutting properties of the ultrasonic bone cutter. bone is cut in preference to soft tissue, which responds elastically in contact with the blade and therefore moves and vibrates, causing dissemination of the energy.26 27 28 the complications associated with anterior approaches to the spine vary depending upon the specific approach, surgical indication, and patient factors. a large meta - analysis by mcdonnell reviewed 447 patients undergoing anterior surgery to the thoracic, thoracolumbar, or lumbar spine.25 indications included correction of deformity, tumor, vertebral osteomyelitis, or diskitis. the authors ' aim was to determine the perioperative complications, which were divided by the authors into major and minor as per table 3. others have also used this system to report their complication rates.22 in a large meta - analysis of thoracic spine surgery, fessler and sturgill reported a complication rate of 11% in transthoracic approaches.17 ayhan reported a complication rate of 21.4%, with two of these complications arising secondary to instrumentation.22 in utilizing the criteria for perioperative complications from mcdonnell,25 we report a major complication rate of 5.6% with one of our patients being intubated and ventilated for adult respiratory distress syndrome. minor complications occurred in 2 (11.1%) patients ; both had an intraoperative cerebrospinal fluid leak, which was repaired at the time with no sequelae. intraoperative blood transfusion occurred in 6 (33.3%) patients, which is an expected complication of transthoracic surgery with vertebrectomy as previously mentioned. in our series, 15 (83.3%) patients had a functional improvement of at least one grade on the mjoa scoring system, with the remaining patients ' mjoa scores remaining unchanged. these results compare favorably with those from the other gctd series.1 6 10 22 none of the major series on gctds reported on fusion rates or development of kyphosis.1 6 10 22 in our case cohort, all patients had evidence of fusion on postoperative imaging as defined by documentation of bony trabeculation and absence of bony lucency at the graft / vertebral end plate interface on radiology reports. no patient developed a clinically or radiologically evident kyphotic deformity on follow - up ; we acknowledge the limitations of our cohort study in this regard as cobb angles were not recorded and compared pre- and postoperatively. however, others have also noted that when the amount of bone removed is minimal in an anterior approach, it is not likely to produce frank spinal instability, and instrumentation is not required in these cases.4 10 better long - term data from cohort studies is required to make firm conclusion. thoracic vertebrectomy for a variety of indications have reported worsening of the kyphotic angle postoperatively, albeit the change was minimal.29 30 31 32 a trench vertebrectomy via a thoracotomy allows safe identification of normal dura on either side of the compressed segment prior to performing a diskectomy. a rib autograft has achieved 100% fusion rates with no kyphotic deformities reported in our series. overall, 83% of patients showed postoperative improvement in their mjoa score with none of our cohort experiencing neurologic decline.	study design case series and review of the literature.objective to review the management of giant calcified disks in our large cohort and compare with the existing literature. we discuss our surgical technique.methods twenty - nine cases of herniated thoracic disk between 2000 and 2013 were reviewed. eighteen patients were identified as having giant calcified thoracic disks, defined as diffusely calcified disks occupying at least 40% of the spinal canal. demographic data was collected in addition to presentation, imaging findings, operative details, and outcomes using the modified japanese orthopaedic association (mjoa) scale.results giant calcified thoracic disks (gctds) are unique clinical entities that require special neurosurgical consideration owing to significant (40%) involvement of the spinal canal and compression of the spinal cord, often leading to myelopathy. the median age at diagnosis was 51.2 years (range 37 to 70) with the mean duration of presenting symptoms being 9.9 months (range 2 weeks to 3 years). seventeen (94.4%) patients presented with at least one sign of myelopathy (hyperreflexia, hypertonia, bladder or bowel dysfunction) with the remaining 1 (5.6%) patient presenting with symptoms in keeping with radiculopathy. thoracotomy was performed on 17 (94.4%) patients, and 1 (5.6%) patient had a costotransverse approach. mean follow - up was 19.8 months (range 7 months to 2 years). mjoa score improved in 15 (83.3%) patients. mjoa scores in the other patients remained stable.conclusions gctds are difficult neurosurgical challenges owing to their size, degree of spinal cord compression, and consistency. we recommend a trench vertebrectomy via a thoracotomy in their surgical management. this procedure safely allows the identification of normal dura on either side of the compressed segment prior to performing a diskectomy. excellent fusion rates were achieved with insertion of rib head autograft in the trench.
having an almost unlimited capacity to regenerate tissues lost to age and injury, planarians have long fascinated naturalists. in the western hemisphere alone, their documented history spans more than 200 years. planarians were described in the early 19th century as being immortal under the edge of the knife (1). among planarians, free - living freshwater hermaphrodite schmidtea mediterranea has emerged as a suitable model system because it displays robust regenerative properties and, unlike most other planarians, it is a stable diploid (2n=8) with a genome size of about 4.810 basepairs (nearly half that of other common planarians). moreover, a robertsonian translocation (that is, the fusion of a whole arm of chromosome 1 to chromosome 3) has produced an exclusively asexual biotype. both sexual and asexual forms have proven easy to rear in the laboratory (2). the capacity of regeneration in planarians is mediated by a proliferative cell population that contains pluripotent stem cells, named neoblasts, a term first used by harriet randolph to describe a particular cell type in the annelid lumbriculus. randolph reported that some species of earthworms contain large, undifferentiated, embryonic - like cells with a high nuclei - to - cytoplasm ratio, and she named them neoblasts (3). this word later adopted to describe similar cells in planarians. by morphology, neoblasts represent ~25~30% of all planarian cells. the progeny of neoblasts have been shown to produce epidermis, rhabdite cells, muscle, and germ cells, among others. considering that neoblasts are the only dividing cell type in planarians neoblasts express about 4000 genes, including genes important for pluripotency in escs, including regulators as well as targets of oct4 (5). they also express transcripts for the piwi proteins smedwi-1 and smedwi-2, the bruno - like protein bruli, and a tudor protein. these proteins are typically found in association with nuage, an electron - dense perinuclear organelle present in germ cells, which plays a role in transposon silencing and maintenance of genome integrity (5), suggesting the existence of a common ancestral state of germ cells and neoblasts (6). techniques for studying gene function in planarians, such as rnai (7) and in situ hybridizations (8), combined with the characterization of a large number of cdnas from the species schmidtea mediterranea (9), and recent rna sequencing approaches have allowed the initiation of molecular genetic studies of planarian biology (4). given that 85% of the genes that yielded regeneration phenotypes in the s. mediterranea rnai screen are conserved in other animals, the results of regeneration screening in planarians could serve as a good point of departure to determining the extent of conservation of regeneration mechanisms among the metazoans (10). category 1 of neoblasts that posit in the most middle parts of the planarian mesenchyme are the only neoblasts capable of self - renewality. using an asymmetrical division, category 1 of neoblasts generate category 2, which they posit more peripherally to the category 1 of neoblasts. this category of neoblasts is not dividing, as it only can differentiate to category 3 of neoblasts, the most peripheral category of neoblasts in the planarian mesenchyme. in the next step, category 3 of neoblasts can differentiate to differentiated cells that lost during age or injury (11). wagner and colleagues were able to show that if planaria were irradiated at a dosage such that nearly all neoblasts were destroyed, there would be some individuals in which a single neoblast survived and made a colony. from this neoblast, dividing progenitor cells formed, ultimately producing cell types of all germ layers and demonstrating the presence of pluripotent cells in the adult body. in addition to that, even a colonogenic neoblast, when transplanted from an asexual biotype to a lethally irradiated sexual biotype, can generate a colony and convert the sexual biotype in to an asexual biotype (12). historically two models for explaining neoblast specification in planarians were considered. in the nave neoblast model, neoblasts produce non - dividing, multipotent blastema cells. cells in the neoblast population are essentially all the same, responding like drones to wounds by simply migrating and dividing, producing the blastema cells. the action would then happen in blastema cells, with these multipotent and nave postmitotic cells adopting appropriate identities based on the external signals that they receive ; for example, as a consequence of their position in a blastema. in the these specialized neoblasts produce different lineage - committed and nondividing blastema cells (13). scimone and his coworker s propose that cneoblasts (directly, or via their descendants) begin expressing numerous transcription factors of specific lineages in distinct neoblast cells. in this model, almost all of the lineages formed during development could be reconstituted during regeneration, with progenitors that generate and comprise planarian blastemas being a heterogeneous patchwork of lineage - specified cells. most studies, however, analyzed neoblasts at the population rather than the single - cell level, leaving the degree of heterogeneity in this population unresolved (14). using single cell approaches wolfswinkel and his coworkers identified two prominent neoblast classes which they named zeta and sigma classes. zeta - neoblasts encompass specified cells that give rise to an abundant postmitotic lineage, including epidermal cells, and are not required for regeneration. by contrast, sigma - neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta - neoblasts. historically, planarian regeneration has been subdivided into two types of processes defined by the terms epimorphosis and morphallaxis (15). amputation elicits a series of responses that ultimately result in a minimization of tissue loss. first, the animal pulls away from the wounding agent, possibly reflecting a predator avoidance reflex. a strong muscular contraction at the site of wounding occurs within seconds and minimizes the surface area of the wound (16). specialized planarian cells, referred to as rhabdites, release their contents at the wound site producing a protective mucosal covering, with possible immunological functions (17). a head fragment containing the brain will continue to locomote, possibly to escape the fate its body might have befallen to a hungry predator. although trunk fragments can move and typically keep their ventral sides down, they remain relatively stationary during regeneration. a thin layer of epithelium covers the wound within 30 min, a process that occurs by cell spreading rather than proliferation. the spreading involves both dorsal and ventral epithelial cells, which lose their characteristic morphologies as they cover the wound. in contrast to wounds produced in humans, scarring (i.e., deposition of dense collagenous fibers) does not seem to occur in planarians. as a result, because amputation and wound healing provide a context in which dorsal and ventral epidermis come into direct contact with each other, it has been suggested that this dorsal / ventral (d / v) interaction may trigger the regenerative response (4). neoblasts can sense this heterogeneity in dorsoventral positional information and response to it by proliferation and migration to the amputation site. in the next step neoblast progenies can take participate in the structure of the regeneration blastema, an unpigmented epithelial / mesenchymal bud. during the following days the differentiated cells can generate from blastema differentiation and morphallaxis regeneration happens, during which the anatomical proportion of the animal is restored. in the last stage of planarian regeneration the behavioral characteristic of the differentiated cells restored (10). as with any stem cell, neoblast thus, either blocking the capacity of neoblasts to proliferate or depleting their population leads to the loss of regenerative capacity. in contrast, an excess of neoblast proliferation can lead to the formation of overgrowths or tumors. deciphering how this regulation is achieved is essential in order to understand not only the cellular basis of planarian regeneration but also the role of stem cells in processes such as tumorigenesis (18). it has been suggested, for instance, that animals with high regenerative capabilities (such as planarians) appear to be more refractory to the development of spontaneous or induced tumors (19). in spite of regeneration, injury induces immune response in planarians, in which a phagocytic, mesenchymal cell, named reticular cell is activated. within 10 h of injury in the presence of bacteria, a recently identified group of molecules secreted by macrophages, maresins, enhance macrophage phagocytosis. when exposed to human maresin mar1 these data indicate that key planarian signaling components can respond to human mar1, and thus conservation exists between human and planarian maresins and their signal cascades (20). about a century ago thomas haunt morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative head stuff and tail stuff along the anterior - posterior axis. when a planarian is amputated transversely, two fragments are generated and are capable of regenerating. the term polarity has been used to describe the fact that an anterior - facing wound will regenerate a head and a posterior - facing wound will regenerate a tail. morgan suggested that something in the piece itself determines that a head shall develop at the anterior cut surface and a tail at the posterior cut surface. recently it has been cleared that muscle cells and neurons express positional control genes (pcgs) and neoblasts can sense its positional information via these positional control cells (pccs) around them (21). nowadays the identities of substances that make this polarity in the planarian body are clarified to some extent. the nervous system of planarians is composed of two hemispheres that form the conversed u shaped brain of the animals. this conversed u shaped brain, connects with two ventrally nerve cords that extend toward the most posterior tip of the animal (22). within these nerve cells from the end of the u shaped brain, hedgehog (hh) granules are composed and transformed to the posterior part of the body, using microtubules associated proteins. these gradients of hh accumulated in the most posterior part of the body triggers the wingless signaling in the differentiated muscle cells around the neoblasts. neoblasts can sense this posteriorzing gradient of wingless, which makes them have posterior identity. in the case of planarian amputation, hh granules are always accumulated in the most posterior parts of the amputation surface, make these parts more potent for tail formation. wnt/-catenin signaling also regulates tissue homeostasis and regeneration in metazoans (23). in planarians, wnt ligands are thought to control tissue polarity by shaping a -catenin activity gradient along the anterior - posterior axis. a teashirt (tsh) ortholog is induced planarian and vertebrate regeneration in a -catenin - dependent manner (24). on the other hand planarian wnt/-catenin signaling has been shown to act upstream to the lim - homeobox genes at the posterior end (25). like vertebrates, formation of anterior parts of the planarian body is due to fgf signaling (26). there are two fgf receptor families in planarians ; fgfrl1 and fgfr3, and a fgf receptor related molecule named nou - darake (ndk), which is expressed in the head region. ndk lacks the cytoplasmic kinase domains characteristic of the fgf receptor families and only restricts fgf signaling to the anterior parts of the body (27). the interaction of fgf and wnt signaling within the planarian body, along the anterior - posterior body makes different parts of the body, including head, pre - pharynx, pharynx and tail (28). recently, it was proposed that subepidermal muscle cells are the cellular source of positional information by expressing position control genes (pcgs) such as genes encoding wnts and their inhibitors (21). in addition to anterior - posterior axis, planarian body has polarity along its dorsal - ventral axis. just as in vertebrate and flies, the dorsal - ventral axis of regenerating cells in planarians is regulated by bmp and its inhibitors (26), like admp (29) as well as noggin like genes (30). planarians are able to regenerate a complete animal, including the brain and reproductive system, after any kind of amputation. although their striking regenerative capacity has attracted generations of biologists, recent years have seen them transformed into an essential model for the study of regeneration and stem cell biology using modern molecular and genomic tools. the impressive plasticity of planarians is based upon the presence in adult animals of a unique type of totipotent stem cells named neo - blasts (4). planarians represent an ideal system for molecular and cellular investigations because their gene function can readily be studied in adults via rnai and in situ hybridizations (7). on the other hand a variety of genes and cell signaling molecules related to neoblast self - renewality and stemness molecular genetic studies in planarians have revealed that orthologs of numerous embryonic patterning genes in other organisms have roles in adult planarian tissues for instructing tissue turnover and regeneration (31). recently a planarian ortholog of morn2 has identified as a key component in lc3-associated phagocytosis and resistance to bacterial infection (32). a variety of characteristics make planarians an extraordinary model for regeneration studies ; first, the animal is one of the simplest metazoans in which regeneration is patently manifested. second, the organism is relatively easy to manipulate experimentally. indeed, there exists more than 100 years of scientific literature reporting experimentation with planarians (4). pronounced limitations of somatic tissue turnover and regenerative properties in current invertebrate models, coupled with the difficulty of studying vertebrate somatic stem cells in vivo, are compelling reasons to examine and test the suitability of planarians to inform both regeneration and stem cell biology (2). in this review we attempted to show some important aspects of planarian characteristics make this animal an extraordinary model for regenerative medicine researches. on the other hand a variety of genes and signaling molecules conserved between this animals and higher vertebrates as well as human makes this animal a good model for study about diseases as well as parkinson (33) and may provide new ideas for handling mouse and human es cells for therapeutic use in the near future (34).	the emergence of regenerative medicine has raised the hope of treating an extraordinary range of disease and serious injuries. understanding the processes of cell proliferation, differentiation and pattern formation in regenerative organisms could help find ways to enhance the poor regenerative abilities shown by many other animals, including humans. recently, planarians have emerged as an attractive model in which to study regeneration. these animals are considering as in vivo plate, during which we can study the behavior and characristics of stem cells in their own niche. a variety of characteristic such as : simplicity, easy to manipulate experimentally, the existence of more than 100 years of literature, makes these animals an extraordinary model for regenerative medicine researches. among planarians free - living freshwater hermaphrodite schmidtea mediterranea has emerged as a suitable model system because it displays robust regenerative properties and, unlike most other planarians, it is a stable diploid with a genome size of about 4.8108 base pairs, nearly half that of other common planarians. planarian regeneration involves two highly flexible systems : pluripotent neoblasts that can generate any new cell type and muscle cells that provide positional instructions for the regeneration of anybody region. neoblasts represent roughly 25~30 percent of all planarian cells and are scattered broadly through the parenchyma, being absent only from the animal head tips and the pharynx. two models for neo - blast specification have been proposed ; the naive model posits that all neoblasts are stem cells with the same potential and are a largely homogeneous population.
a current trend in catalysis sees a refocus on the catalytic action of the individual parts of a more complex catalyst entity. as many catalyst systems represent a combination of (noble) metals and (oxidic) supporting materials, the latter are increasingly studied with respect to their intrinsic surface reactivity. however, due to the inherent structural and electronic complexity of oxides, the identification of e.g. a such complex systems may either be two - phase systems such as mechanically mixed oxide powders(e.g., being promising methanol - to - gasoline catalysts) or single - phase binary oxides adopting a distinct crystallographic structure. a well - known example of the latter are perovskite - type materials with the general formula abo3, where a and b are cationic metal species with variable composition. their use as ferroelectrica, high - temperature superconductors, or solid oxide fuel cell (sofc) cathodes is already well - known. potential applications in catalysis are reported for environmentally relevant denox processes, diesel exhaust catalysis, polymerization of olefins, total oxidation of hydrocarbons, or dry reforming of methane. specifically, also the water - gas shift reactivity has recently been in the focus of research. a comprehensive review of the salient aspects of the structure and known catalytic performance of perovskites has been given by e.g. by pena. or royer. a rather new field of catalytic research deals with the replacement of the nowadays common ni / ysz sofc anodes by acceptor - doped mixed conducting perovskite - type anodes. for this application a combination of good ionic and electronic conductivity, as well as high (electro)catalytic activity and high resilience against carbon deposition, is necessary. acceptor - doped oxides under reducing conditions, however, often exhibit a relatively high ionic conductivity, but their rather low electronic conductivity may be problematic for an application as sofc anode materials. in the case of model - type thin film electrodes, this poor electronic conductivity can lead to an electronic sheet resistance and thus to inhomogeneous electrochemical polarization. to avoid artifacts in electrochemical measurements, metallic thin film current collectors can be applied on such thin film electrodes, where the electrochemical surface activity of perovskite - type electrode materials can be separated from resistances due to poor current collection. moreover, catalytic activity in carbon - based fuel reforming and hydrogen oxidation on ni / ysz electrodes is provided by the ni phase. however, also perovskite - type lanthanum chromite / ferrite - based sofc anodes have been shown to exhibit good catalytic activity for methane steam reforming and methane oxidation. the understanding of the surface catalytic properties of such acceptor - doped perovskites in reducing atmospheres, however, is still in its infancy. with respect to fuel cell research and oxide surface chemistry, investigations on water - gas shift reactivity and hydrocarbon reforming are hence an obvious research field. the water - gas shift reaction is known to be catalyzed by redox - active oxidic materials, in particular also by more complex mixed systems such as cuo / zno / al2o3, fe2o3/cr2o3, or even perovskite materials. although there appears to be increasing interest in the general use of perovskite materials as valuable catalysts, also including hydrocarbon conversion reactions, direct correlations of catalytic properties and associated structural changes for eventual determination of the catalytically active site still remain scarce due to the inherent complexity of these materials. this is a particular pity since e.g. hydrocarbon conversion is usually carried out at very high temperatures (t > 600 c), which might give rise to an array of structural changes, including (but not limited to) surface reconstruction, altering bulk and/or surface terminations, and/or chemical segregation of individual atom species. especially the surface structure and chemistry, e.g., the cation concentration or the formation and amount of oxygen vacancies, are in a dynamical state depending on the experimental conditions. for oxide and sofc - related research this basically refers to the hydroxylation degree of the surface, high temperatures, and the oxygen partial pressure and reactivity. thus, surface and bulk structure and composition might significantly deviate from one another and need to be separately assessed. regarding chemical alterations of perovskite - type materials, cation segregation is already a well - known phenomenon especially under operational conditions of an sofc. moreover, reversible in situ segregation of electrolysis - promoting metallic fe species under reducing (cathodic) conditions was recently reported as well as growth of defined nanoparticles by controlled perovskite nonstoichiometry. in order to fill this knowledge gap and to bridge the gap between sofc - relevant reactivity and conventional catalysis, which also includes a cross - correlation between catalytic application and electrocatalysis, the present contribution aims at (i) characterizing the electrochemical surface activity of perovskite - type materials (in particular lsf) in both oxidizing and reducing atmosphere by means of impedance spectroscopy. to account for the low electronic conductivity of acceptor - doped mixed conductors in reducing atmosphere (see above), model - type thin film electrodes with a current - collecting platinum thin film grid are used in these investigations. (ii) as the specific oxygen reactivity is suspected to play a major role in eventually steering the catalytic reactivity, the main goal of this study is to highlight reactivity differences for the (inverse) water - gas shift ((i-)wgs) and methane conversion on two representative perovskite - type materials, la0.6sr0.4feo3 (lsf) and srti0.7fe0.3o3 (stf), respectively. the latter two are deemed relevant materials due to their different iron content. as iron is mostly suspected to be the center of catalytic reactivity due to its pronounced redox behavior, we aim at providing data to what extent this is true. in fact, combined structural characterization (mostly by tem) and catalytic testing will indicate that this is only part of the truth, and a more complex behavior, especially with respect to the quality of oxygen vacancies, must be assumed. in due course, the stability of the perovskite (surface) structure during a full catalytic cycle including preoxidation and reduction will be monitored. therefore, catalytic measurements in a recirculating batch reactor setup especially designed for the measurement of small reaction rates, structure - determining methods (analytical high - resolution electron microscopy and x - ray diffraction), and raman spectroscopic measurements were jointly applied. la0.6sr0.4feo3 was prepared via the pecchini route : respective amounts of la2o3, srco3, and metallic fe (all three at least 99.95% pure, sigma - aldrich) were dissolved in hno3 (double distilled, sigma - aldrich). all three solutions were mixed, and citric acid (99.9995% pure, sigma - aldrich) was added to the solution in a 20% excess with respect to the cations. the solution was heated, and after the evaporation of water the mixture formed a highly viscous foam, which finally decomposed in a spontaneous combustion. the obtained powder was calcined for 2 h at 1000 c in air ; the product was ground in a mortar and checked for phase purity by x - ray diffraction (xrd). the srti0.7fe0.3o3 powder was obtained by a solid state reaction from srco3, tio2, and fe2o3 (all three at least 99.98% pure, sigma - aldrich). the educts were thoroughly mixed, calcined at 1000 c for 2 h, ground in a mortar, and calcined a second time at 1250 c for 2 h. the product was again ground and also checked for phase purity by xrd. surface areas using the bet method were determined for both samples to be around 0.4 m g. bet surface areas were measured with a quantachrome nova 2000 surface and pore size analyzer. for catalytic measurements the powders were used as obtained by the procedures described above. for pulsed laser deposition (pld) of thin films, targets of lsf were prepared by isostatically pressing the powders and sintering the green bodies at 1250 c for 5 h in air. thin films of lsf were deposited on yttria - stabilized zirconia (100) single crystals (ysz, 9.5 mol % y2o3, crystec, germany) by pld. for electrochemical measurements in reducing conditions a current collecting thin film grid was deposited prior to the pld process by sputtering 10 nm ti on ysz followed by 100 nm of pt. this thin film was micropatterned by a photolithography process and ar - ion beam etching. for the subsequent pld process a krf excimer laser (lambda compexpro 201f) with 248 nm wavelength was used to ablate the lsf target. the deposition was done in 4 10 mbar of pure oxygen with a laser pulse repetition rate of 5 hz, a nominal laser pulse energy of 400 mj, and a ysz substrate temperature of 650 c (controlled by a pyrometer ; heitronics, germany). current collectors circular microelectrodes were produced by again carrying out a photolithography process and an ar - ion beam etching step. a scanning electron microscopy (sem) image of a circular lsf electrode with buried ti / pt current collector is shown in figure s1, and a schematic depiction of the thin film electrode setup is shown in figure s2. in a previous impedance study on stf electrodes with buried pt current collectors, it was shown that the surface exchange resistance and the chemical capacitance of the electrode can be extracted from the low - frequency part of the impedance spectra. impedance spectroscopy measurements on these thin film electrodes were carried out in a home - built microcontact setup. it allows contacting of individual electrodes by pt / ir tips, which can be accurately positioned by piezoelectric high - precision actuators (newport agilis). the atmosphere in the setup was either synthetic air or 2.5% h2/2.5% h2o / balance ar, and the electrode temperature was about 610620 c. impedance measurements were conducted with an alpha - a high performance frequency analyzer with pot / gal 30 v 2a test interface (both novocontrol, germany) in a frequency range between 10 mhz and 1 mhz at an ac voltage of 10 mv (root - mean - square, rms). impedance data analysis was done by the complex nonlinear least - squares fitting software z - view 3.1 (scribner, usa). the catalytic measurements on lsf and stf powder samples were exclusively performed in a recirculating batch reactor (ca. detection of the reactants and products was carried out by a quadrupole mass spectrometer (qms) attached to the reaction chamber via a capillary leak. for a typical inverse water - gas shift experiment, a 1:4 mixture of co2 and h2, delivering the right stoichiometry for a potential methanation reaction, as well as 25 mbar of h2o to ensure thermodynamically stable conditions, was filled into the reaction chamber of the batch reactor. for the water - gas shift reaction, a 1:1 ratio of co and h2o (25 mbar each) was used. subsequently, the mixture was heated with 5 c min from room temperature to 600 c, followed by isothermal periods of variable duration before the final cooldown. ar (7.5 mbar) was additionally admitted to all reaction mixtures to correct all other signals for the continuous gas withdrawal through the leak. the mass - to - charge ratios m / z = 2 (h2), 15 and 16 (ch4), 18 (h2o), 28 (co), 40 (ar), and 44 (co2) were routinely monitored. the co signal was corrected for the contribution from co2 fragmentation in the mass spectrometer. the qms intensities were calibrated (i.e., converted into partial pressure given in mbar) using external calibration standards. a brief account of the mass and heat transport limitations should be given at this point. we exclude effects of mass transport and pore diffusion limitation, since the samples are used in quite small amounts (100200 mg) of loose powder, which can be quickly penetrated by the reaction gas. note that the same catalyst setup that has been used for the studies on comparable systems, where much higher reaction rates were accurately measured. heat transfer limitation is also judged to play a minor role since local temperature effects are excluded due to the low reaction rates, and generally, heat transfer via the gas phase is enhanced due to the deliberate admission of he to the reaction mixture (1 bar total pressure). the volumetric adsorption measurements were conducted in an all - quartz apparatus described in detail elsewhere (reactor volume : 32 ml), additionally using a balzers qma125 quadrupole mass analyzer and a linn furnace operating up to 1220 c. all perovskite powder samples were subjected to a pretreatment in flowing oxygen up to 600 c, with an isothermal period at 600 c for 1 h before finally cooling down. after this oxidation step, the system was evacuated at room temperature to a base pressure of about 5 10 mbar after the aforementioned oxidation treatment, and a defined amount of 100 mbar of h2 was admitted. subsequently, the samples were heated to 600 c at a rate of 10 c min, followed by an isothermal period at 600 c for 1 h before cooling to room temperature. for better comparison also to literature - reported data, all mass spectrometer data (in mbar) were converted into micromoles on the basis of the ideal gas equation, with subsequent normalization to sample mass and surface area. cleaning of the gases was performed using either a liquid n2 (for h2) or a liquid n2/ethanol cooling trap (for o2). note that the term adsorption (better viewed as uptake) in this case also includes actual hydrogen consumption reactions, such as bulk reduction or hydrogen insertion into the perovskite lattice. as a zeolite trap is used to quantitatively remove reaction - formed water from the volumetric chamber, accurate absolute quantification of the second mechanism is presently not possible. however, as will be shown later, different behavior of both stf and lsf in their oxidized and reduced states, respectively, is an experimental matter of fact. although the exact bulk oxygen and hydrogen stoichiometry of the samples in different states of reduction is not known due to the above - mentioned obstacle, we nevertheless decided to use a specific partially reduced prereaction state prior to catalytic testing (see section 2.3). prereduction at 873 k in dry hydrogen was on the one hand done gently enough to avoid reductive decomposition of the samples. on the other hand, substantial formation of additional oxygen vacancies (beyond those already induced by the prereduction) during e.g. a subsequent temperature - programmed water - gas shift reaction experiment could be largely suppressed by the reductive pretreatment and by performing the catalytic experiment at t tprereduction. thus, true catalytic experiments with complete reactant / product mass balance became feasible. structural characterization of the samples was carried out by two types of electron microscopes. bright - field (high - resolution) imaging and high - angle annular dark - field imaging were carried out using a 200 kv fei tecnai f20 stwin analytical (scanning) transmission electron microscope ((s)tem) equipped with a tridiem energy filter. high - resolution, high - angle annular dark - field imaging eel and edx spectroscopy were performed using an aberration - corrected fei titan microscope operating at 300 kv. electron - energy loss spectra are background - corrected and corrected for plural scattering. prior to imaging, the samples were sputter - cleaned to remove surface carbon impurities. for the high - resolution images, the stf and lsf samples were suspended in acetone and cleaned in an ultrasonic bath prior to mounting on a holey carbon film. in the case of haadf images to be taken, the samples were heated to 80100 c under high - vacuum conditions (4 1010 mbar) prior to imaging. x - ray powder diffraction data were collected at ambient conditions with a bruker axs d8-advance powder diffractometer using cu k1 and cu k2 radiation (1 = 1.5406 ; 2 = 1.5444 ; 40 kv ; 40 ma). data acquisition was performed in the 2 range between 2 and 90 with a step width of 0.02 and a counting time of 3 s under sample rotating conditions. the sample itself was prepared on a si single crystal holder with suppressed background intensity. confocal raman spectra of the polycrystalline samples in the range of 503800 cm were recorded with a horiba jobin yvon labram - hr 800 raman microspectrometer. the samples were excited using the 532 nm (2.33 ev) emission line of a frequency - doubled 25 mw nd : yag laser under an olympus 100 objective lens with a numerical aperture of 0.9 and additionally he ne laser (633 nm) for detection of luminescence effects. the size of the laser spot on the surface was approximately 1 m in diameter. the scattered light was dispersed by an optical grating with 1800 lines mm and collected by a (peltier - cooled) 1024 256 open - electrode ccd detector confocally coupled to the focal point of the sample. the spectral resolution, determined by measuring the fwhm of the rayleigh line, was below 2 cm (using a slit width of 100 m). the accuracy of the raman line shifts, calibrated by measuring a silicon standard (522 cm), was in the order of 0.5 cm. spectra were recorded from a representative grain of the samples as a single measurement (10 s integration time). impedance spectra obtained on lsf electrodes with and without buried ti / pt current collectors are depicted in figure 1. both types of electrodes were characterized in air (see figure 1a) as well as in reducing 2.5% h2/2.5% h2o / ar atmosphere (cf. the impedance spectra consist of three relatively well separated features. in the high - frequency range (see inset in figure 1a) a real axis intercept can be observed, which can be attributed to the spreading resistance of ion conduction in ysz. this resistance, together with the size of the circular microelectrodes, was used to obtain the true temperature of the measured microelectrodes (for details regarding this calculation please refer to refs (77 and 78)). the arcs in the medium- and low - frequency region can be related to an ionic transfer resistance at the electrode / electrolyte interface and to the oxygen incorporation resistance at the electrode surface, respectively. in the case of the electrode with buried current collector the interfacial resistance and the dominating surface resistance are increased, which can be easily explained by the ion blocking character of the current collectors. thus, only a smaller part of the electrode / electrolyte interface as well as of the electrode surface is available. taking the geometry of the grid into account (3/4 of the interface covered with metal, 1/4 free interface), the electrode kinetics of both types of electrodes (with and without grid) in air can be regarded as very similar. in reducing atmosphere figure 1b). here, the spectrum measured on lsf without current collector is both qualitatively and quantitatively significantly different than the spectra obtained in oxidizing atmosphere. first, the high - frequency axis intercept is about 2 orders of magnitude larger than one might expect from the nominal size of the electrode. second, in the medium - frequency regime an additional shoulder appears, and third, the total polarization resistance of the electrode is more than 1 order of magnitude larger than in air (cf. the finding that the high - frequency intercept which is expected to be the spreading resistance of ion conduction in ysz is much too large for the nominal electrode size is a strong indication that owing to a high sheet resistance in the lsf film only a rather small part of the electrode around the tip contact is electrochemically active. this interpretation is further supported by the fact that in the case of lsf electrodes with buried current collector the high - frequency real axis intercepts are close to those of spectra measured in air (see inset in figure 1c). moreover, the total electrode impedance of lsf with current collector measured in h2/h2o is within the same order of magnitude as the electrode resistance in air (see figure 1c). considering the active areas of the two electrodes (0.79 10 cm for lsf in air and 3.14 10 cm for lsf in h2/h2o), one can even calculate almost identical area - specific polarization resistances of 16 and 15 cm for lsf under oxidizing and reducing conditions, respectively.a since the inverse polarization resistances are a direct measure for the exchange current and thus the exchange rates at the electrode surfaces, at ca. 620 c the catalytic activitiy of the lsf surface for oxygen reduction and hydrogen oxidation is rather similar. in a previous impedance study on stf model electrodes in reducing atmosphere a surface resistance of ca. this comparison reveals that lsf is significantly more active for h2 oxidation than stf. corresponding differences in the conventional catalytic behavior of the two materials will be discussed below on the basis of their specific oxygen vacancy / lattice oxygen reactivities. in particular, vacancies in lsf are shown below (section 3.2) to be much more reactive with respect to quenching by water splitting (the reverse process of hydrogen oxidation by lattice oxygen), and the associated water affinity of the vacancies is clearly higher than on stf. taking into account the above - mentioned area - related polarization resistance, an application of lsf in porous sofc anodes may lead to electrodes with polarization resistances comparable to nowadays used porous paste cathodes. this potential applicability, however, raises the question of compatibility of lsf anodes with carbon based fuels such as methane. thus, besides the fundamentally interesting questions regarding surface activity of perovskite - type catalysts (as discussed in the introduction), the catalytic activity of lsf for reactions such as water - gas shift and methane activation needs to be investigated in detail as shown in the following sections. impedance spectra (nyquist plots) measured on 200 m thick lsf microelectrodes with and without buried ti / pt current collectors. the insets in the top right corners show magnifications of the high - frequency region of the spectra. the measurements were performed in air (a) as well as in reducing atmosphere, 2.5% h2/2.5% h2o / balance ar (b). the spectra measured on lsf with current collectors in air (from a) and in reducing atmosphere (from b) are compared in (c). before focusing on the structural changes occurring on the stf and lsf perovskite - type systems upon oxidative and reductive pretreatments or during a catalytic cycle, the activities for the water - gas shift and methane conversion reaction are outlined. this also includes a discussion of temperature - programmed hydrogen uptake experiments, which very much adds to the understanding of the associated temperature - programmed catalytic reaction profiles. the term adsorption in this respect includes all hydrogen uptake or consumption processes lowering the hydrogen concentration in the gas, including adsorption, absorption in the oxide, but also water formation by oxide reduction. figure 2 shows two sets of temperature - programmed inverse water - gas shift reaction runs on stf with different sample pretreatments. a preoxidation at 400 c in oxygen for 1 h was applied in both experiments to ensure identical starting conditions. the experiment shown in panel a was done directly after this oxidation step ; i.e., it refers to the fully oxidized stf catalyst. in the case of the catalyst shown in panel b, a reductive treatment at 600 c (1 h, 1 bar hydrogen) was performed for the above - mentioned reasons (see section 2.4.) after the preoxidation step and before the catalytic experiment. the mass spectrometer signals of h2, ch4, co, and co2 were recorded. as can be clearly seen in both panels, the reduction / reaction starts in the case of the fully oxidized sample at 430 c, and on the prereduced one at around 500 c, as indicated by the pronounced decrease of the h2 signal. as the inverse water - gas shift reactionalso consumes co2 and produces co in a fixed stoichiometric amount, the profile shown in panel b is that of a true inverse water - gas shift reaction (i - wgsr) with stoichiometric reactant consumption and product formation (the water signal is not displayed since a zeolite trap was used in the tpr experiments ; see section 2.4). in contrast, stoichiometric co formation is apparently not the case for the fully oxidized sample, given the substantially higher hydrogen consumption shown in panel a, largely exceeding the stoichiometric amount with respect to co. neither is co2 simultaneously consumed nor sufficient co produced alongside hydrogen being reacted off. this behavior can only be consistently interpreted in terms of a reduction of the perovskite catalyst by hydrogen forming oxygen vacancies in the perovskite lattice, which remain mostly unreactive toward co2, and releasing h2o into the reaction mixture ; in krger vink notation this reaction readsfrom a catalytical point of view, the water - gas shift equilibrium is obviously disfavorably shifted toward co2 due to the ongoing hydrogen depletion of the reaction gas mixture at these elevated temperatures. this behavior can be regarded as a strong indication that a suitable prereduction of the perovskite - type catalyst is essential for generating sufficiently reactive vacancies capable of stoichiometric co2 reduction and thus stoichiometric turnover of the reactants toward the water - gas shift equilibrium. a similar catalytic inverse water - gas shift reaction profile was observed for the corresponding lsf powder sample after a reductive pretreatment : under comparable experimental conditions, the reaction of h2 and co2 to co (and h2o) starts at around 500 c. as observed for stf, also the prereduced lsf shows a stoichiometric inverse water - gas shift reaction profile (cf. figure 6 a) ; further details of the lsf catalyst will be discussed below. temperature - programmed reaction profiles of the inverse water - gas shift reaction on stf without (panel a) and with (panel b) prereduction in hydrogen at 600 c (1 h, 1 bar). heating rate : 5 c min, 1:4 starting mixture of co2 and h2 including 25 mbar of h2o, 7.5 mbar of ar, and he added to 1 bar total pressure. to further shed light on the hydrogen reducibility of lsf and stf, temperature - programmed hydrogen consumption measurements after oxidation at 600 c were performed (see figure 3). according to these measurements, on lsf hydrogen starts to be steadily consumed at around 200 c with a notable rate increase around 350 c. at least two discernible steps in the hydrogen consumption are visible. a very similar tpr pattern was observed on stf, although the onset temperature is shifted by about 60260 c. the h2 consumption appears to proceed at a slower rate compared to lsf, and no clearly discernible steps were observed. saturation of hydrogen uptake is apparently not even reached at the highest studied temperatures for both perovskite samples, as can be deduced from the quite pronounced isothermal uptake, accounting for yet another 200300 mol m hydrogen consumption at 600 c within 60 min. most notable, however, is the outstanding amount of consumed hydrogen in the course of the experiment. after the isothermal period, a total amount of 3500 mol m consumed hydrogen on lsf and about 2000 mol m on stf has been determined, which is approximately 300 times the amount measured on highly reducible simple oxides, such as in2o3, under comparable experimental conditions. volumetric hydrogen uptake measured on stf and lsf. heating rate from room temperature to 600 c at 10 c min followed by an isothermal perion at 600 c for 1 h. initial hydrogen pressure : 100 mbar. the measurement was performed using a zeolite trap in the cold part of the reaction chamber for effective removal of water resulting from the oxide reduction. as already mentioned above, in the case of both perovskite - type catalyst powders the reductive treatment leads to an oxygen nonstoichiometry, i.e., to a formation of oxygen vacancies and the corresponding modification of the electronic charge carrier concentrations. from the above - mentioned amounts of consumed hydrogen the degree of reduction of the two perovskite - type catalysts (i.e., their oxygen nonstoichiometry in the prereduced state) can be estimated. for la0.6sr0.4feo3 and srti0.7fe0.3o3 values of ca. 0.1 were obtained, respectively (only in rough approximation, i.e., upon excluding the formation of bulk hydrogen species obscuring this mass balance ; compare section 2.4). it is already worth to mention that effects of pronounced reduction of perovskites via reaction of lattice oxygen with h2 are also visible in the respective raman spectra and x - ray diffractograms (cf. this indicates that the perovskite structural entity as a whole is stable, but nevertheless structurally slightly altered by the hydrogen treatment, at least at temperatures t 600 c. in previous studies on simpler oxides, like in2o3 or ga2o3, the reactivity in the (inverse) water - gas shift equilibrium was determined to be strongly dependent on the prereduction degree of surface and bulk regions. thus, associated tests on the oxygen vacancy quenching ability were performed on lsf and stf powders by exposing the prereduced catalysts to 25 mbar of h2o vapor and performing a tpr experiment (figure 4). interestingly, no reaction of h2o on a hydrogen prereduced sample has been observed for stf (inset : the small water uptake at low temperatures is an experimental artifact), in contrast to lsf (main panel), where a clear hydrogen signal, likely arising from the quenching of the introduced vacancies via dissociative decomposition of h2o, is observed. the potential implications of this result on the promotion of electrochemical water splitting are already mentioned in section 3.1 ; those regarding the activity toward attaining the (inverse) water - gas shift equilibrium are jointly discussed in the context of figure 6 (see below). note that under the chosen experimental reduction conditions (static reduction), no signs of segregation of metallic iron are visible, although we can not exclude the formation of undetectable modest amounts of metallic iron. tem experiments show no pronounced iron oxide segregation after room temperature exposure of a reduced sample to ambient (redissolution of ionic iron in the perovskite lattice is therefore excluded). reactivity of water with oxygen vacancies introduced by hydrogen reduction as shown in figure 3. both samples were prereduced in hydrogen at 600 c (1 bar, 1 h), recooled to room temperature, and exposed to about 25 mbar of h2o vapor, before a temperature - programmed reaction (tpr) was started. the heating rate was set to 10 c min. in figure 5 the activity of lsf and stf in the inverse water - gas shift reaction is depicted (after prereduction at 600 c). in both panels a and b, the time - dependent conversion of co2, normalized to the sample surface, as well as the reaction temperature is plotted versus the reaction time. the latter is composed of a heating period (5 c min) followed by a prolonged isothermal period in order to quantify the respective isothermal reaction rate at a given conversion from the respective slope of the conversion curve. panel a shows isothermal experiments at 500 c and panel b those at 450 c. for both temperatures and at low conversion values, the normalized co2 conversion rate on lsf is always higher compared to that on stf. at conversions approaching the water - gas shift equilibrium, a readout of the slope makes no more sense anyway. as during the isothermal periods increasing conversion is observed, this allows the determination of surface area - normalized and thus comparable reaction rates (at the same co2 conversion), given in mbar min m. the extracted reaction rates from the isothermal range of the conversion vs time plots are summarized in table 1. note that the co2 conversion was calculated as the ratio of the co2 pressure and total pressure of co2 and co, which is equivalent to the co2 pressure in the beginning. the temperature - dependent water - gas shift equilibrium for the chosen experimental conditions is shown in figure s3 of the supporting information and very much coincides with the experimental values. the rates were determined in both cases between 20 and 30% conversion. at 500 c, co2 conversions of about 40% and 60% are obtained on stf and lsf, respectively, whereby the isothermal reaction time of 1 h at 500 c is sufficient to approach the (i-)wgsr equilibrium only on lsf. a qualitative comparison with the water - gas shift reaction on other perovskite - type oxides, namely la1xcexfeo3 and la0.9sr0.1cr1+xmnxcoxo3, reveals that especially on the latter (again measured at 450 and 500 c) much shorter contact times (of a few minutes) with the catalyst are sufficient to achieve equilibrium conversion, especially if the chromium content is high. we therefore conclude that our specific activities may rather compare to the respective mn / co - rich samples. note also that in combination with the hydrogen tpr experiments shown in figure 3 the reaction profiles in figures 2 and 4 generally suggest a potential influence of the vacancies (formed by reduction) on the catalytic properties in the water - gas shift equilibrium. in general, wgsr activity of different perovskite materials is well - known and almost always directly linked to the number of oxygen vancancies, as derived from chemical looping pulse experiments with sequential pulsing of reactants. note in this respect, however, that the wgsr reaction mixture is therefore not admitted simultaneously, and generally, no difference is made regarding the qualitative nature of the oxygen vacancies. vacancy formation is in due course usually connected with the amount of the iron content of the material. however, correlating the reactivities to the amount of the initial number of vacancies introduced by the hydrogen treatment (and therefore to the amount of iron) is not feasible : in doing so, one has to assume that (i) all the hydrogen consumption leads to water formation (hence, the amount of vacancies represents only the upper limit) and (ii) the amount of vacancies does not change during the reaction. as discussed above, (i) is clearly not fulfilled. in view of figure 4, it is also obvious that (ii) does not hold, since the reactivity toward water is different on stf and lsf. as for the direct correlation to the bulk iron content, note also that the surfaces of both perovskites after the catalytic runs are predominantly sro - terminated (cf. in particular, our results illustrate the qualitative differences of surface (near) defect / vacancy reactivity on these materials and the necessity to link the apparent activation barriers (see below) to the specific oxygen affinity of such (to some extent likely fe- or ti - containing) redox - active centers. moreover, it appears mandatory to link the surface area normalized macroscopic catalytic activity to their effective (i.e., not bulk - proportional) surface concentration, which is determined by the specific segregation chemistry of the surfaces under realistic reaction conditions. eventually, an arrhenius estimation of the apparent activation energy for the i - wgsr based on the initial rates at low conversions yielded ca. 90 and ca. 130 surface - area - normalized temperature - programmed reaction profiles of the inverse water - gas shift reaction shown as co2 conversion vs time plots at two different temperatures (450 c : panel b ; 500 c : panel a) with extended isothermal periods for both fully oxidized stf and lsf to reveal the inherently different catalytic activity. heating rate : 5 c min, 1:4 starting mixture of co2 and h2 with 25 mbar of h2o, 7.5 mbar of ar, and he added to 1 bar total pressure. figure 6 summarizes a set of reactivity data necessary for a thorough understanding of the (inverse) water - gas shift and methane reactivity. for direct comparison, panels a and e again show the inverse water - gas shift reactivity profiles on lsf and stf after prereduction (note that figure 6e corresponds to figure 2a). for comparison to the inverse water - gas shift experiments, panels b and f experiments have been conducted both on fully oxidized (o2, 400 c, panels d and h) and prereduced (h2, 600 c, panels b and f) samples. as for the water - gas shift activity, co is reacted off with simultaneous co2 and h2 production on both perovskites, if the perovskite powder is prereduced (cf. the onset temperature of reaction is at about 300 c, therefore between the one typically observed for conventional low - temperature copper - based wgs catalysts like cu / zno / al2o3 and iron - based catalysts used for higher temperatures. on fully oxidized samples only co2 formation without h2 production was observed (cf. figure s2). apparently, in the latter case, total oxidation of co to co2 is the only observed reaction channel, occurring via reduction of reactive lattice oxygen by co, but not primarily via a catalytic water - gas shift reaction cycle. this is directly proven by the simple reduction of the fully oxidized perovskite systems by co without water (figure 6, panels d and h), which both show direct transformation of co into co2. note in this respect that the start temperature of the water - gas shift reaction on both catalysts is lower as compared to the inverse direction (compare panels a and b as well as e and f in figure 6). additional low - temperature prereduction by co (thus forming co2) takes place (see also the two - step wgsr reaction profile on stf, figure 6f). obviously, the catalyst adapts itself to the equilibrium state in a kinetically different manner depending on the starting conditions (co2 and h2 for the inverse ; co and h2o for the water - gas shift reaction). as it is well - known from experiments on simpler oxide systems (e.g., ga2o3 or in2o3), the (inverse) water - gas shift reaction can in principle follow different distinct reaction pathways, depending on the temperature - dependent relative contribution of surface adsorbate and defect chemistry (i.e., oxygen affinity of vacancies / reduced surface centers vs stability of adsorbed intermediates) : a vacancy - mediated mechanism (vmm), a formiate - mediated mechanism (fmm), or following a pathway including co dissociation. on ga2o3, fmm and vmm pathways were observed, depending on the degree of reduction (i.e., the number of vacancies), degree of surface hydroxylation, and temperature. on in2o3, due to its outstanding reducibility, the vm mechanism was strongly dominating already at low temperatures. in due course, the question arises, which pathway is predominantly followed on the more complex perovskite systems ? our results suggest that on lsf the vm mechanism is predominantly observed but appears to play a minor role on stf, as can be deduced from the water - quenching experiments shown in figure 4. at the present stage of experiments, we can not exclude a possible contribution of an fm mechanism on lsf, even more on stf, where future directed in situ ftir experiments will definitely show if, and in which temperature regime, the reaction profile observed in panel f follows the latter mechanism. as can be deduced from the inability of the vacancies to dissociate water (figure 4), the vm mechanism is at least not predominantly prevailing on stf. most important in this respect is a comparison of the depth of oxide reduction and further reducibility in h2 at 600 c, i.e., a prereduced state compared with the fully oxidized state in figure 2 : upon deep reduction higher temperatures are required to (re)establish reactive reduced centers in course of the catalytic inverse water - gas shift reaction cycle (figure 6a, e). in line with our results, recent studies by diebold. on the wgsr mechanism on fe3o4 revealed that indeed the fmm is dominant at low temperatures via oh groups and formates from oh and co. at higher temperatures (t > 520 k), a defect - mediated water - splitting mechanism (mars van krevelen type) is shown by stm. co dissociation, on the contrary, is certainly correlated with the presence of metallic iron. if the hydrogen partial pressure and the temperature are high enough, the presence of iron carbide / metallic iron can not be excluded, but if then the mechanism is still the same, remains an open question. note that after prereduction at 600 c the hydrogen is pumped off and the sample recooled in vacuum this treatment most probably removes the major part of the adsorbed hydrogen. admission of the inverse water - gas shift reaction mixture and heating to about 300 c for lsf results in a readsorption of hydrogen between 300 and 470 c, without simultaneously observing inverse water - gas shift reactivity. only above 470 c, a similar behavior, albeit at slightly different temperatures and less pronounced, is also observed for stf (figure 6e). in order to determine the rate - limiting step of the reaction, thus the depth of reduction in the active working state of the catalysts and the associated specific reactivity of the reduced centers at the surface has to be considered. these appear clearly different from those obtained after prereduction of a fully oxidized sample. in this respect, the water - gas shift reaction on fully oxidized perovskite samples only yields total oxidation to co2 (cf. comparative experiments on the methane reactivity clearly indicate that on fully oxidized catalyst samples in the absence of water as well as under steam reforming conditions methane is always totally oxidized to co2 ; the corresponding catalytic results are depicted in figure 6, panels c and g, as well as in figure s5 of the supporting information, respectively. most importantly, on prereduced perovskite catalysts (h2, 600 c, 1 h) no methane reactivity at all was observed under otherwise comparable experimental conditions (cf. supporting information figure s6). this indicates that the fully oxidized states of lsf and stf exhibit reactive oxygen species capable of fully oxidizing methane in contrast to a deeply reduced state, where the remaining oxygen species obviously are unreactive toward methane. hence, also no methane reforming activity is observed. especially for lsf, partial reoxidation by water has been observed (see cf. figure 4), which is anyway not enough to set in methane oxidation. this can be understood on the basis that water obviously dissociatively adsorbs on the surface through partial refilling of vacancies. as this does not lead to methane reactivity, we might infer that methane reactivity is hindered because of missing methane activation. this might be partially connected to the iron content of the sample and the associated redox chemistry (cf. the results on methane oxidation are in good agreement with reports by belessi for the reaction of co and no and spinicci. for the total oxidation of methane using lafeo3, who observed a significant influence of the nonstoichiometry on the activity between compositions of la0.95feo2.85 and la0.7feo2.55. (partial) oxidation of methane is generally a reaction that has been vastly studied on many different perovskite systems, but almost always using a chemical looping pulse technique at higher reaction temperatures. summarizing in close correlation to the results presented here oxygen nonstoichiometry has been elucidated as the key parameter governing the activity and stability. however, structure activity correlations appear scarce especially regarding reforming reactions, and research is therefore focused on methane combustion. it is clear, nevertheless, that upon presence of water the complexity is much higher and qualitative differences in the oxygen vacancies between perovskite systems may arise as can be seen in figures 4 and 6. h). panels a and e : i - wgsr, co2 + h2 co + h2o, 20 mbar of co2 and 80 mbar of h2 including 25 mbar of h2o (preoxidation at 400 c, prereduction at 600 c). panel e, for the sake of clarity, again shows the reactivity profile of figure 2a ; panels b and f : wgsr, co + h2o co2 + h2 25 mbar co and 25 mbar h2o (preoxidation at 400 c, prereduction at 600 c). panels c and g : methane oxidation on initially fully oxidized samples, ch4 co2 + h2o, 20 mbar of ch4 (preoxidation 400 c). panels d and h : co oxidation on initially fully oxidized stf and lsf, co co2 + v, 25 mbar of co (preoxidation at 400 c). for establishment of unambiguous structure activity correlations, a clear picture of structural and morphological changes occurring upon catalyst pretreatments as well as during reaction must evolve. this is even more imperative given the complexity of the perovskite systems under investigation. in due course, the catalysts were characterized by electron microscopy, x - ray diffraction, and raman spectroscopy after each step of a full inverse water - gas shift reaction cyle including preoxidation and prereduction. scanning high - resolution transmission electron microscopy was used to determine the surface termination of both perovskites to detect eventual irreversible structural changes in the catalytically relevant surface regions. x - ray diffraction and raman spectroscopy were then used to investigate perovskite lattice stability and to eventually corroborate the above - discussed tpr profiles, especially regarding the influence of prereduction. this in due course also raises the question of full reversibility of the changes introduced both by the specific pretreatments and the subsequent catalytic reaction. in order to serve as a valuable (electro)catalyst, all structural changes must be fully reversible upon reoxidation after reduction and reaction. especially for more complex systems, this is by no means a trivial question and needs to be accurately assessed. figure 7 highlights the morphology of a representative stf grain in the as - grown state (corresponding high - resolution tem images are highlighted in figure s7 of the supporting information). panel a shows a bright - field transmission electron microscopy (tem) image of a micrometer - sized grain with some internal structure being mostly due to mass thickness contrast, whereas panel b reveals that stf is basically chemically homogeneous after the catalytic reaction and no pronounced chemical segregation takes place. for this purpose, the corresponding ti k (a), fe l (c), and o k edges are mapped over the indicated squared region by energy - dispersive x - ray spectroscopy (edx). before performing the edx experiments, a high - angle annular dark - field (haadf) image of the grain was taken, which also shows no substantial variation in chemical composition. note that the haadf contrast is basically proportional to z (with z denoting the atom number), thickness t, and material density. thus, regions with greater z or higher thickness appear brighter in the corresponding images. by combination of haadf images and edx measurements, the contrast variations in the former image bright - field overview tem image of a representative stf grain (panel a) and stem - haadf image of an extended region on the edge of a stf grain to emphasize the chemical homogeneity of the sample (panel b). l (c), and o k (d) edges are shown as false color insets and were collected in the squared region as indicated. to further shed light on the structural changes on the atomic level during each step of a catalytic cycle, (scanning) high - resolution electron microscopy imaging was performed for visualizing changes in the ordering of the samples, but most importantly, for changes in the bulk structure and surface termination. the corresponding surface termination after performing the active inverse water - gas shift state is subsequently shown in the atomically resolved stem - haadf images of figure 8 (panel a, stf ; panel b, lsf). ball models of the respective structures are shown as large insets, with the different colors denoting the different atoms present in the structures. furthermore, due to the resolved atom rows, it is possible to assign the individual atoms to the intensity profile in the haadf image. it should be noted that, despite difficulties to distinguish fe from ti and sr from la, the a and the b cations can clearly be distinguished. the sr atoms, as the heaviest atoms, appear correspondingly bright in these images. thus, the surfaces can clearly identified to be predominantly (sr, la)o - terminated after the catalytic run, and no fe / ti segregation was observed for any of the perovskite structures. in figure 8, panel b, the (sr, la)o termination of the lsf surface is best visible at the bottom left side, where a side edge of the crystallite is shown. these results are in line with recent results on the surface composition of lsf in different gas atmospheres, obtained by in situ x - ray photoelectron spectroscopy studies, which also strongly indicate a sr high - resolution stem - haadf images of stf (above) and lsf (below) with ball models of both crystallographic structures as overlayers to visualize the exact position of each atom in the structure and at the surface. x - ray diffraction has already been shown to be a valuable tool to investigate the bulk structural changes of several perovskite systems, especially regarding changes in crystallite size, lattice parameters, oxygen vacancy concentration, or valence state as well as structural transformations upon oxidative and reductive treatments at elevated temperatures. in due course, we will show if and how the bulk structure is affected by the reductive or catalytic (pre)treatments. the red diffractogram in panel a, corresponding to the initial, untreated sample, is in agreement with the cubic structure of srfe1xtixo3 reported e.g. by adler., whose structure data can be used as starting values for refining. the obtained lattice parameter of a = 3.899 (from fitting of the peaks including the split peaks of k1 and k2 at higher angles see also the inset) corroborates this conclusion. after reduction, however, a significant shift of the peaks to lower diffraction angles is observed, which can be most clearly seen in the inset highlighting the group of peaks around 2 = 77. this shift has also been observed previously and has, in combination with xanes spectra, been assigned to formation of oxygen vacancies and the corresponding changes in the fe valence state, both causing a change in the lattice parameter. most notably, as highlighted in the green xrd pattern, this shift is not reversed during prolonged contact to the inverse water - gas shift reaction mixture but can only be reversed by reoxidative treatment at 400 c (see also the corresponding raman spectroscopy experiments in the following section). for lsf the xrd experiments show a very similar behavior as can be seen by comparing the red and black xrd pattern in figure 9, panel b, denoting the initial state and the state after final reoxidation at 400 c with oxygen, respectively. the initial diffractogram (black) again corresponds well to literature - reported data on the rhombohedral sr0.4la0.6feo2.8 structure. using co / co2 mixtures of varying content, corresponding to different reduction potentials, a slight shift of the peaks as well as a corresponding intensity increase of the peak at lower 2 the same features are observed : the intensity of the peak at higher 2 (at 68.4) can not be distinguished from the background noise upon reduction (blue trace) but more or less re - established after the catalytic reaction (green trace) and after reoxidation. in summary, also the xrd experiments allowed for monitoring the structural changes after each step of the catalytic cycle and yielded direct information with respect to the formation and reactivity of oxygen vacancies after the prereduction procedures. our xrd experiments also corroborated the above - mentioned differences between stf and lsf with respect to the reactivity of oxygen vacancies toward water (cf. the changes introduced by stf prereduction could not be reversed by either water formed during catalytic reaction or a direct treatment but only by reoxidation in molecular oxygen. in contrast, in the case of lsf the initial state is almost re - established even by water formed during the catalytic reaction (and even more by a direct treatment, cf. consequently, this again implies a different extent of reduction of stf and lsf under comparable reaction conditions and also the presence of more reactive defects toward h2o in the case of lsf. (a) x - ray diffraction patterns of stf collected after different steps of a catalytic cycle. initial state (red), after reduction at 600 c in 1 bar of hydrogen (blue) and after catalysis as described before (i - wgsr, green). the inset shows a close - up view of the peaks at around 2 = 77. (b) x - ray diffraction patterns of lsf collected after different steps of a catalytic cycle. initial state (black), after reduction at 600 c in 1 bar of hydrogen (blue), after i - wgsr (green) and after reoxidation at 400 c in 1 bar of oxygen (red). the inset shows a close - up view of the peaks at around 2 = 68. raman spectroscopy has already been proven to be an indispensable tool to characterize the structure of various perovskites, including stf and lsf, not only in the initial state but also after various annealing treatments. moreover, the raman spectrum of the archetypical srtio3 perovskite is already well - understood, as are the effects of temperature, grain boundaries, and sizes or phase transitions. stf and lsf have also been characterized regarding the effects of anodic or cathodic polarization or charge - disproportionation phase transitions. hence, as the method is already well - established and a sufficiently large data set is already available, it is a promising methodical approach to study the catalytic effects on the structure of the perovskite systems described in the preceding sections. the experiments have been in this case restricted to the stf material, as lsf shows pronounced luminescence effects, rendering the measurements of real raman shifts difficult. in close correlation to the other experiments, figure 10 now summarizes the raman spectra acquired after different steps of catalyst pretreatment and reaction. the black spectrum refers to the initial, untreated stf perovskite, which in due course will be used as reference material for all structural changes. for full appreciation of the spectral changes, it is useful to give a brief overview of the raman spectrum of the untreated stf state. the spectrum between 200 and 2000 cm reveals several pronounced peaks at 125, 180, 325, 480, 540, and 770 cm and a number of very broad signals above 1000 cm. except the one at 700 cm, these correspond (with increasing wavenumber) to the to1, lo1/to2, lo2/to3, lo3, to4, and lo4 transversal and longitudinal optical phonon modes, respectively. most importantly, the lo4 mode at 770 cm is hardly present in the spectrum of untreated stf. the most pronounced feature at 700 cm and the associated shoulder at lo4 770 cm have been observed by vracar. to be strongly affected by variations in the iron content of the sample. the latter was found to be very pronounced for low iron contents but getting progressively weaker in relation to the feature at 700 cm with increasing iron content. hence, peak shifts or intensity variations of these peaks might be used as an indicator for local changes in the structural environment of fe - containing centers. after reduction at 600 c in hydrogen (blue spectrum) the overall features do not change substantially, with the one exception of an even more suppressed intensity of the lo4 mode at 770 cm. however, after the catalytic reaction (green spectrum), the changes are much more pronounced. this basically refers to intensity changes of the to4 and lo4 modes and to the appearance of several new peaks, especially in the high wavenumber region. new features are now found at 1050 and 1500 cm, and the lo4 mode, previously only present as a suppressed shoulder, has now gained intensity and exceeds the corresponding to4 mode. o stretching vibration and is connected with the features at 1050 and 1500 cm. the peak at 1500 cm (a 2lo4 overtone) gains only intensity, if the associated lo4 mode gets progressively more pronounced. the peak at 1050 cm most probably is a superposition of the overtones of the 2lo2 and 2to4 modes. note also the pronounced peak shift between the spectrum of the untreated perovskite and the reduced state, which is practically reversed upon treatment in the catalytic inverse water - gas shift reaction mixture. after reoxidation at 400 c (red spectrum), all previously introduced changes are completely reversed and the initial state is clearly re - established. taking the xrd result into consideration, the following correlation arises : from the xrd measurements after prereduction, the creation of an increasing number of oxygen vacancies has been inferred after reduction, which is now accompanied by the almost total suppression of the lo4 mode and the red - shifted peaks. after catalysis, the xrd measurements did not indicate reversal of the bulk structural changes introduced by the reduction but showed that the inverse water - gas shift reaction atmosphere is not able to fully replenish especially the bulk vacancies. in contrast, the raman experiments after catalysis show that part of the introduced changes can be indeed reversed, as indicated by the shift of lo4 peak position back to the oxidized state (note, however, that there are still some differences between the raman spectra of the untreated and oxidized stf sample, which indicate that some irreversible structural changes are still present). most importantly, as the changes in the spectrum mainly affect the peaks sensitive to the fe content, we might conclude that these changes not only mainly affect surface - near regions but also appear to be at least in part associated with a local concentration change of fe - containing centers. although the stem images showed that these changes obviously do not alter the surface termination of stf, we nevertheless may assume that the stf surface - near regions are in a highly dynamic state, adapting easily to the reductive or oxidative environments of catalyst pretreatment or reaction. raman spectra collected on stf after various catalytic treatments : black, untreated sample ; blue, after reduction at 600 c in hydrogen (1 h) ; red, after reoxidation at 400 c (1 h) ; green, after i - wgsr up to 600 c (1 h). vertical lines have been added to distinct features as a guide to the eye. in the present study the (electro)catalytic properties of the reducible perovskite - type oxides la0.6sr0.4feo3 and srti0.7fe0.3o3 were investigated. the activity of lsf for the (electro)oxidation of hydrogen was studied by means of impedance spectroscopy experiments on model - type thin film electrodes. interestingly, the surface resistance (which is a measure of the surface activity) at ca. 610 c in a h2/h2o atmosphere was very similar to the surface resistance in synthetic air. compared to stf, the activity of lsf for the h2 oxidation reaction is significantly higher. catalytic - wise, the presented results allow the correlation of a catalytic profile measured on a perovskite system to bulk and surface structural changes occurring during each step of catalytic pretreatments and catalytic reaction. for this correlation two different types of experiments were necessary : (1) catalytic measurements identifying the activity of the perovskite - type catalysts. exemplarily shown for the inverse water - gas shift equilibrium, a pretreatment of the perovskite powders in hydrogen is a prerequisite for a successful use of lsf and stf as wgsr catalysts. using this pretreatment and combining xrd and catalytic experiments (for stf also raman measurements), a clear difference in the surface and bulk reactivity of the two perovskite - type oxides thus, as the most important parameter highly influencing the reactivity, the reduction degree and depth could be determined : it controls the oxygen reactivity and the specific oxidation and reduction capability of the active sites in the working state of the catalysts. especially, the methane activation / oxidation ability strongly depends on the chosen reduction depth. (2) by means of high - resolution tem measurements on both catalyst powders a predominant sro or (sr, la)o surface termination could be verified after a catalytic reaction, indicating that a bulk - proportional surface contribution of e.g. redox - active iron centers in both catalysts is unlikely and a more complex scenario, especially with regard to qualitatively differently oxygen - affine surface - near vacancies and a reaction- and segregation - specific concentration of such redox - active sites at the surface, must be assumed.	comparative (electro)catalytic, structural, and spectroscopic studies in hydrogen electro - oxidation, the (inverse) water - gas shift reaction, and methane conversion on two representative mixed ionic electronic conducting perovskite - type materials la0.6sr0.4feo3 (lsf) and srti0.7fe0.3o3 (stf) were performed with the aim of eventually correlating (electro)catalytic activity and associated structural changes and to highlight intrinsic reactivity characteristics as a function of the reduction state. starting from a strongly prereduced (vacancy - rich) initial state, only (inverse) water - gas shift activity has been observed on both materials beyond ca. 450 c but no catalytic methane reforming or methane decomposition reactivity up to 600 c. in contrast, when starting from the fully oxidized state, total methane oxidation to co2 was observed on both materials. the catalytic performance of both perovskite - type oxides is thus strongly dependent on the degree / depth of reduction, on the associated reactivity of the remaining lattice oxygen, and on the reduction - induced oxygen vacancies. the latter are clearly more reactive toward water on lsf, and this higher reactivity is linked to the superior electrocatalytic performance of lsf in hydrogen oxidation. combined electron microscopy, x - ray diffraction, and raman measurements in turn also revealed altered surface and bulk structures and reactivities.
animals : the research was approved and overseen by the animal experiments committee of riken (wako, japan) and was conducted in accordance with the institutional guidelines for experiments using animals. common marmosets were reared at the riken brain science institute (wako, japan), maintained at 27c and 50% humidity on a 12/12-hr light / dark cycle. all marmosets in this study were chosen from animals between 2 and 6 years old. marmosets were allowed ad libitum access to water and food pellets (cms-1 m, clea japan inc., tokyo, japan) with added vitamin c, d, calcium and acidophilus. hot water and comb honey were also added to soften the pellets and to improve the animals preference for the food. animals were given a piece of calorie mate (otsuka pharmaceutical co., ltd., tokyo, japan) or castella (castella, yamazaki baking co., ltd., briefly, individuals with body weights less than 325 g with 0.05% body weight loss per day were defined as the wms group (n=7). individuals with body weight higher than 375 g (a median value of 350400 g was reported as the average weight of adult marmosets), with 0.036% weight gain per day (average weight gain ratio of normal marmosets reported in), were defined as the control group (n=7). body weight, the highest body weight, current body weight / the highest body weight ratio and gain / loss ratio per day are listed in table 1table 1.sex, age, body weight, the highest body weight, current body weight / the highest body weight ratio and body weight gain / loss ratio per day in individual animals are representedsexage(years)bw (g)the highestbw (g)bw / the highestbw ratio (%) bw gain or lossratio per day (%) wms 1female5274.3437.062.80.63wms 2female3220.4351.162.80.10wms 3female4207.8348.159.71.23wms 4female4201.6290.069.50.35wms 5female4220.3375.258.70.44wms 6female4250.0320.078.10.40wms 7male4255.0360.070.80.30control 1female4455.0464.598.00.04control 2male3397.1476.483.40.04control 3female4387.9391.599.10.04control 4female2439.7470.893.40.07control 5female4461.2461.21000.18control 6female4434.3434.31000.14control 7male2432.8432.81000.29. appearance check : first, all animals underwent an appearance check, including fur and posture (with or without unkempt fur, pale face, undervitalized appearance, alopecia, curved back, pigeon - toed and stiff movement). blood collection : blood samples were drawn from an individual s femoral vein using 26-gauge needles. for the duration of blood collection, animals were under manual retention. the rest of the blood was centrifuged (1,800g, 20 min and 4c) after standing for 1 hr at room temperature. the purified serum was stored at 80c until used for serum chemistry and serum mmp9 concentration tests. cbc and serum chemistry tests : a cbc analysis, including white blood cells, red blood cells, hematocrit, hemoglobin, platelets, mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch) and mean corpuscular hemoglobin concentration (mchc), was performed using celltac (mek-6450, nihon kohden co., ltd., tokyo, japan). a serum chemistry panel (albumin and calcium) was performed using a drychem 4,000 system (fujifilm co., ltd., serum mmp9 concentration test : we performed serum tests of mmp9 concentrations using commercial elisa kits (quantikine elisa human mmp-9, smp900 ; r&d systems, inc., anatomical study : marmoset colons were dissected after perfusion with saline followed by 4% paraformaldehyde under deep anesthesia. the dissected colons were fixed with tissue fixative (genostaff, co., ltd., tokyo, japan), then embedded in paraffin wax and sectioned at 6 m for hematoxylin and eosin (he) staining. statistical analysis : two - tailed mann - whitney u - tests were used to compare the wms and control groups, and spearman s rank coefficient (two tailed) was used to measure correlations between body weight and each data point (graphpad prism, ver. 6 for windows ; graphpad software inc., la jolla, ca, u.s.a.). data are presented as means standard error of the mean (sem). appearance check : pigeon - toe, which was considered to be caused by stiff movement in the hind limb (fig. 1a and 1bfig. (b) magnification of the white square in a. hind limbs are pigeon - toed. red arrow indicates an area of alopecia. the black square indicates tail - base alopecia. (d) magnification of the black square in c. red arrow indicates an area of alopecia, and the black arrow indicates ulceration.), and alopecia (fig. 1d), were observed in all seven animals in the wms group. neither abnormality was observed in any of the seven animals in the control group. (b) magnification of the white square in a. hind limbs are pigeon - toed. (d) magnification of the black square in c. red arrow indicates an area of alopecia, and the black arrow indicates ulceration. cbc : the hematocrit and hemoglobin values differed significantly between the animals in the two groups (p0.05) between the groups in mcv (mean 67.8 0.82 fl in the wms group and 65.8 1.74 fl in the control group), mch (mean 22.4 0.87 pg in the wms group and 21.0 0.34 pg in the control group), mchc (mean 33.2 1.42 g / dl in the wms group and 31.9 0.16 g / dl in the control group) or white blood cell values (mean 71.4 28.23 10/l in the mws group and 57.9 7.30 10/l in the control group). serum chemistry test : the levels of serum albumin differed significantly between animals in the two groups (p<0.01, fig. 3afig. (a) serum albumin values and (b) serum calcium values of marmosets in the wms and control groups.. correlations between (c) body weight and serum albumin value and (d) body weight and serum calcium value in individual animals. the dotted lines represent best - fit lines of the data points.). the mean albumin levels in the wms and control groups were 3.49 0.221 g / dl and 5.53 0.300 g / dl, respectively. a significant correlation between body weight and serum albumin level was observed (p<0.01, rs=0.879, fig. (a) serum albumin values and (b) serum calcium values of marmosets in the wms and control groups. correlations between (c) body weight and serum albumin value and (d) body weight and serum calcium value in individual animals. similarly, the levels of serum calcium differed significantly between animals in the two groups (p<0.01, fig. the mean calcium levels in the wms and control groups were 8.8 0.40 mg / dl and 11.3 0.56 mg / dl, respectively. a significant correlation between body weight and serum calcium level serum mmp9 concentration : there was a significant difference in serum mmp9 concentration between the two groups (p<0.01, fig. (a) blood mmp9 values of marmosets in the wms and control groups. (b) correlations between body weight, and blood mmp9 values in individual animals. the mean mmp9 concentrations in the wms and control groups were 91.7 21.79 ng / ml and 17.4 2.73 ng / ml, respectively. a significant negative correlation between body weight and serum mmp9 concentration was observed (p<0.05, rs=0.660, fig. (a) blood mmp9 values of marmosets in the wms and control groups. (b) correlations between body weight, and blood mmp9 values in individual animals. bowel section of animals in the wms group : there were no polyps or cancer in the colons in either the wms or control group (data not shown). epithelial damage and disruption of crypt architecture were observed in the transverse colon samples (fig. 5afig. (c) magnification of the black square in a. scale bar : 50 m. (d) magnification of the black square in b. scale bar : 50 m. red arrows in c and d indicate goblet cells. and 5b) and ascending colon samples (fig. the colonic tissues revealed inflammatory cell infiltration in the lamina propria of the mucosa, atrophy of intestinal villus and decreased numbers of goblet cells (fig. 5b and 5d). there were no abnormalities in the colons in the control group (data not shown). (c) magnification of the black square in a. scale bar : 50 m. (d) magnification of the black square in b. scale bar : 50 m. common marmosets are often used for long - term research experiments, such as behavioral analysis, in the field of brain science. a long duration is needed for the production of transgenic marmosets, at least 5 months for the duration of pregnancy and a litter size of 24 infants, so each animal is very valuable. wms can occur in any animal used for ongoing research. due to this morbidity and mortality it was reported that some treatments, such as long - term administration of steroids with relatively few side effects, such as budesonide, can cause temporary remission. however, because steroid treatment is relatively ineffective in animals in the terminal stage and is not persistent, steroid treatment can not be said to be adequate. thus, early identification of diseased animals, removal from experiments and prompt treatment of these animals are important. as other reports have mentioned, alopecia [18, 25 ], pigeon - toe [18, 25 ], anemia, thrombocytosis, hypoalbuminemia [2, 18 ] and hypocalcemia were also observed in the wms group in this study. these results support the hypothesis that hematocrit, hemoglobin, platelets, serum albumin and serum calcium can be used for the diagnosis of wms. it was suggested that the anemia and hypoalbuminemia seen in the animals reflect poor nutritional condition, caused by an uptake disorder or protein and/or iron absorption defect. however, because no significant difference or even trend was seen in mcv, mch or mchc between the wms and control groups, we suggest that the anemia and hypoalbuminemia observed in this study were due to other factor(s) depending on the individuals. anemia is a major symptom of ibd in humans [13, 29 ], and iron deficiency is the largest contributing factor. however, it has been suggested that erythropoietin depression, vitamin insufficiency and autoimmune hemolytic disease in ibd patients could also cause anemia. given that several factors other than iron deficiency could have caused anemia in wms, similar to the situation in human ibd, further investigation is needed. it is known that mmps function to process several inflammatory cytokines, including tumor necrosis factor alpha (tnf-). mmp9 is thought to be the main metalloproteinase implicated in the development of ibd, and the increase in mmp9 in mouse and rat ibd has been reported [4, 22 ]. although there are other biomarkers used for diagnosis of ibd, such as c - reactive proteins (crp) or erythrocyte sedimentation rate (esr), it was reported that mmp9 is a more useful biomarker for diagnosis of human ibd than other biochemical markers in terms of that mmp9 can reflect disease state because mmp9 has much higher specificity in distinguishing between active ibd and inactive ibd than other parameters. in our study, the wms group showed significantly increased serum mmp9 concentrations, suggesting that mmp9 is connected with the pathological condition of wms. our results suggest that serum mmp9 might have an important role in the mechanism of wms and could be useful for the diagnosis of wms. a mmp9-targeted inhibitor was developed in rats and is hoped to be a specific medicine for ibd. to our knowledge, this is the first report of increased mmp9 levels in marmosets with wms. we previously reported that fecal calprotectin can be a marker for detecting colonic inflammation, and it was reported that fecal mmp9 can be used for detecting human ibd as well as fecal calprotectin. so, further investigations of fecal mmp9 as a marker for detecting wms should be performed. we propose that the wms marmoset could be an animal model of human ibd with respect to mmp9. further investigations of other molecules involved in human ibd need to be performed in marmosets to explore this possibility. tnf- and interleukin-6 (il-6) are major cytokines in inflammation, and there is a report regarding the involvement of tnf- and il-6 in human autoimmune diseases, including ibd. although tnf- and il-6 might be involved in wms, little has been reported on the behavior of this molecule. because it is known that mmp9 is regulated by il-6 and affects tnf-, future experiments aimed at investigating the relationship between mmp9 and tnf-, or il-6 in wms are needed.	use of the common marmoset (callithrix jacchus) as a non - human primate experimental animal has increased in recent years. although wasting marmoset syndrome (wms) is one of the biggest problems in captive marmoset colonies, the molecular mechanisms, biochemical markers for accurate diagnosis and a reliable treatment remain unknown. in this study, as a first step to finding biochemical marker(s) for the accurate diagnosis of wms, we conducted blood cell counts, including hematocrit, hemoglobin and platelets, and examined serum chemistry values, including albumin, calcium and levels of serum matrix metalloproteinase 9 (mmp9), using a colony of marmosets with and without weight loss. mmp9 is thought to be an enzyme responsible for the degradation of extracellular matrix components and participates in the pathogenesis of inflammatory conditions, such as human and murine inflammatory bowel disease, which, like wms, are characterized histologically by inflammatory cell infiltrations in the intestines. the values of hematocrit and hemoglobin and levels of serum albumin and calcium in the wms group were significantly decreased versus the control group. the platelet values and serum mmp9 concentrations were increased significantly in the wms group compared with the control group. mmp9 could be a new and useful marker for the diagnosis of wms in addition to hematocrit, hemoglobin, serum albumin and calcium. our results also indicate that mmp9 could be a useful molecular candidate for treatment.
determinar a influncia de trs sistemas distintos de instrumentos rotatrios para alargamento cervical na determinao do comprimento real de trabalho. material e mtodos : trinta primeiros molares inferiores foram submetidos ao acesso endodntico convencional e odontometria inicial pelo mtodo visual, utilizando - se para irrigao / aspirao da cmara pulpar o hipoclorito de sdio a 5%. procedeu - se a ampliao cervical dos canais msio - vestibulares empregando - se diferentes alargadores cervicais. de acordo com o sistema de ampliao cervical empregado, as 30 amostras foram divididas aleatoriamente em 3 grupos de 10 dentes cada. no grupo 1 as brocas de gates - glidden foram utilizadas para a ampliao cervical ; no grupo 2, orifice openers foram empregados ; e, no grupo 3, foi usado o sistema la axxess. foram utilizadas em ordem crescente, duas numeraes do sistema rotatrio correspondente a cada grupo e, aps, realizou - se a odontometria final. a leitura das duas tomadas odontomtricas foi realizada com paqumetro digital, em milmetros, considerando - se duas casas decimais para averiguar a provvel discrepncia entre ambas as tomadas. a anlise de varincia (anova) e o teste de tukey (p 0,05) demonstraram diferena estatstica entre o grupo 1 e os grupos 2 e 3. todos os grupos apresentaram encurtamento do comprimento de trabalho aps alargamento cervical ; os grupos preparados com instrumentos la axxes e orifice opener apresentaram os melhores resultados. during the last years, endodontics has been searching for new instruments, instrumentation techniques, irrigants and drugs to achieve the best cleaning and disinfection of root canals12. within this context philippas16 mentioned that physiological or pathological continuous and progressive dentin formation in the pulp chamber floor created interferences that reduced the root canal diameter, especially at the cervical third, and thus it should be removed during endodontic treatment. leeb7 investigated the effect of cervical flaring on the establishment of apical diameter of different groups of teeth and observed that the use of a single gates - glidden bur removed the cervical constriction allowing larger files to reach the apical portion. lopes and costa filho10 described that, in addition to the advantages of using specific instruments to prepare the root canal openings, this clinical step also provides better action of irrigation and suction, since auxiliary chemical substances may be placed close to the apical third, thereby allowing elimination of a larger amount of smear layer. estrela,.5 mentioned the main advantages of cervical flaring in endodontics : reducing pressure during irrigation due to the possibility of overflow ; deeper penetration of irrigant ; effective removal of cervical contamination ; reduced possibility of ledges, apical deviation or instrument fracture ; better control of the active tip of the instrument, thus overcoming the influence from apical curvature by a straighter access ; and an improvement of the obturation steps, especially lateral condensation. the anatomical complexity of molars, especially the mandibular molar and its mesial root, increases the difficulty of routine endodontic procedures, due to the number of root canals, curvatures or even the apical anatomical configuration with several isthmi.21 travassos,.19 concluded that preparation of the cervical third of curved root canals before the use of endodontic instruments allowed straighter access to the apical region, reducing the possibility of interferences or apical deviation. tan and messer18 reported that continuous dentin formation reduces the volume of the pulp chamber in different types of teeth due to the progressive narrowing of root canals, besides the appearance of cervical constrictions. these constrictions should be removed by accomplishment of cervical preparation, thus allowing more accurate working length determination and more precise establishment of the initial apical instrument diameter (iai). lopes and siqueira jr.11 stated that cervical flaring allowed the introduction of endodontic files without locking into the root canal on the pulp chamber, until the first root canal curvature is reached, thus eliminating cervical interferences to allow better establishment of the initial apical instrument (iai). tan and messer18, investigated the effect of instrument type and the influence of cervical flaring on the establishment of the iai of molars and premolars, which pose the greatest challenge in endodontic treatment. these authors concluded that the removal of cervical dentin projections improved the insertion of larger files to the working length, thus providing criteria that should allow the achievement of a adequate biomechanical preparation. macedo,.13 evaluated the wear of the cervical third of mesiobuccal canals of maxillary molars submitted to different procedures for preparation of the root canal openings and concluded that this clinical step provides excellent configuration of the cervical root canal surface of these roots, thus allowing better uprighting of the endodontic instrument for root canal shaping. barroso,.3 investigated the influence of different methods of cervical flaring on the establishment of the iai in maxillary premolars and concluded that larger instruments may be introduced to the real working length after cervical flaring. pcora,.15 analyzed the influence of cervical preflaring on establishment of the iai and concluded that this step allowed the introduction of larger files to the real working length (rwl). therefore, the objective of this study was to investigate the influence of three different rotary systems for cervical flaring on establishment of the rwl, testing the hypothesis that there is no difference between the systems studied. the present study was approved by the ethics committee of the university of passo fundo (upf). the study used 30 permanent lower first molars obtained from the tooth bank of the dental school (fo - upf)., the teeth were washed in tap water for 24 hours to remove any remnants of thymol solution. conventional endodontic access was performed removing and contouring the pulp chamber roof and using irrigation / suction with 5% sodium hypochlorite. the working length was measured by introducing a number 10 k file into the mesiobuccal root canal of each teeth until the apical foramen, followed by 1-mm stepback to establish the real working length (rwl). teeth were randomly divided into 3 groups (n= 10) and the cervical flaring of the mesiobuccal canal was performed using three different rotary instruments. group 1 (g1) was prepared with gates - glidden burs in a conventional low - speed handpiece at 5,000 rpm. preparation was initiated with number 1 bur, which was introduced 3 times until resistance was found and then removed. irrigation / suction was performed with 5% sodium hypochlorite, followed by utilization of number 2 bur as used for number 1 bur. group 2 (g2) was prepared using nickel - titanium cervical flaring instruments (orifice openers, sybronendo, glendora, usa) in a tc 3000 handpiece (nuvag, tcm endo, goldach, switzerland) set at 315 rpm, at configurations 25:08 and 25:10, in increasing order of introduction, until resistance was found. cervical flaring in group 3 (g3) was performed with nickel - titanium la axxes instruments (sybronendo, glendora, usa), connected to a low - speed handpiece at 5,000 rpm. the system was also used in increasing order, at configurations 20:06 and 35:06, until resistance was found. the working length measurements were performed as previously mentioned. a digital calyper (mitutoyo, tokyo, japan) was used to measure the working length, in millimeters, investigating a possible discrepancy between initial and final measurements. the differences between both working length measurements were calculated and statistically analyzed using analysis of variance (anova) and tukey 's multiple range tests with a significance level of 5% (=0.05). analysis of variance (anova) revealed statistically significant differences, which were identified by the tukey 's multiple range test (p0.05) and are presented in table 1. no significant differences were found between the mean values of g2 and g3, which were different from the mean values of g1. this procedure aims to remove cervical interferences from the root canal openings, which represent an obstacle to free access of endodontic instruments to the apical portions of the root canal. removal of these anatomical interferences allows free access, thus enhancing root canal shaping at the apical third2. root curvatures, especially at the apical third, represent an additional difficulty to the professional, since they may be difficult to observe. the curvatures in thin, flattened root canals, as the mesial roots of molars, which represent a remarkable difficulty for preparation and several different techniques have been advocating in an attempt to solve this problem. therefore, this study was conducted on the mesiobuccal root of the mandibular first molar because it presents difficult access in the oral cavity and it may have more than one canal20. teeth storage using 0.1% thymol at 9c maintained the specimens hydrated and structurally stabilized, with no need of tissue fixation and absence of bacterial proliferation, thus simulating the clinical conditions and enhancing the intervention1. this study investigated different systems used for cervical flaring to examine the effect of cervical interference removal on the establishment of the anatomical diameter of the root canal. thus, rotary instruments were used for cervical flaring because they allow root canal preparation with larger instruments and reduce the risk of failures as ledges, perforations and zips17. in addition, the advantages of nickel - titanium instruments (ni - ti) and rotary instrumentation include more effective removal of debris due to the possibility of overflow and continuous rotation of instruments, faster preparation and reduction of root canal transportation6. the instruments selection criteria for this study include the following : 1- gates - glidden (n. 1 and 2) are widely used as rotary instruments for cervical flaring8 ; 2- orifice opener (sybronendo, glendora, usa) (25.08, 25.10) has recognized performance for cervical preparation of root canals9 ; and 3- la axxess (20.06, 35.06), has recently been introduced into the market with almost no literature available and, therefore, should be investigated. the rotary instruments used during cervical preparation following the aforementioned sequence steps, revealed to be an adequate, fast and safe procedure, as also observed by aun,. cervical flaring was performed following the " free tip preparation " technique, using an instrumentation sequence from the lowest to the highest taper. thus, the instrument tip is free and often act as a guide, significantly reducing the torsional fracture and providing more efficient action of the instrument, in agreement with pcora,.14, yet in disagreement with tan and messer18, who performed cervical pre - flaring based on the " crown - down " technique, adapted from manual instrumentation. neither root perforation nor instrument fracture occurred in this study, which can be explain by the linear limit of instrument penetration, instrument introduction until resistance was found, observation of manufacturers ' instructions, experience of the operator and standardization of the degree of curvature of each canal. therefore, the specimens submitted to pre - flaring with la axxess instruments (g3) produced the highest mean value difference between the first and second working length measurements, which was not statistical different form the mean value of g2 (p>0.05), in accordance with pcora,.15. this finding may be due to the characteristics of la axxess instruments, such as the metal alloy properties, the taper (0.06), the inactive tip and the flute design of the active tip that allows the removal of cervical interferences without occurrence of deviations or perforations. along with other research results, this study contributes with the improvement of the endodontic therapy, considering the importance of a proper apical flaring of root canals. all groups exhibited shorter working length after cervical flaring ; considering the methods of cervical flaring to establish of the working length investigated in this study, the specimens prepared with instruments la axxes (g3) and orifice opener (g2) presented the best results.	objective : to investigate the influence of three different rotary systems for cervical flaring on establishment of the real working length. material and methods : thirty mandibular first molars were submitted to conventional endodontic access and initial working length measurement, followed by irrigation / suction of the pulp chamber with 5% sodium hypochlorite. teeth were randomly divided into 3 groups (n=10) and cervical flaring of the mesiobuccal canals were performed using one of the following instrument systems : group 1 gates - glidden burs ; group 2 orifice openers ; group 3 la axxess system. two subsequent numbers of instruments of each rotary system were used and the final working length was recorded. a digital calyper was used to record the working length, in millimeters, to investigate a possible discrepancy between initial and final measurements.results:analysis of variance (anova) na tukey test revealed statistical difference between group 1 and groups 2 and 3 (p 0.05).conclusions : all groups presented shorter working length after cervical flaring ; groups prepared with instruments la axxes and orifice opener presented the best results among the systems studied.
lobular capillary hemangioma (lch), also known as pyogenic granuloma, is a benign, rapidly growing, hypervascular lesion. its usual locations are the skin and mucous membranes of the head and neck, the nasal cavity being a rare site of origin.1 2 its etiopathogenesis is not fully established, although an association with traumatic and hormonal factors has been identified. these lesions tend to occur more frequently after nasal surgery or nasal packing and in women during pregnancy or taking oral contraceptive pills.3 here, we present the first reported case of a nasal lch following an endoscopic transsphenoidal resection of a gonadotrophin - producing pituitary adenoma, reflecting a possible but rare complication of this procedure. the neurologic examination revealed a bitemporal hemianopsia and the laboratory studies, including endocrinological investigation were normal. she was diagnosed with a pituitary cystic macroadenoma and underwent an endoscopic transsphenoidal removal of the lesion in our institution by a team of two neurosurgeons experienced in endoscopic procedures. nasal packing was done bilaterally with merocel (medtronic) (polyvinyl - alcohol sponge) nasal tampon lubricated with antiseptic cream, maintained for 5 days and then removed uneventfully. the histopathologic review established the diagnosis of a gonadotropin (luteinizing hormone and follicle - stimulating hormone)-producing adenoma. one month after the surgery, the patient presented with a progressively growing right nasal mass (fig physical examination revealed a reddish, smooth - surfaced mass, filling the right nasal cavity. the mass was mobile and appeared pedunculated with its base attached to the anterior portion of the nasal septum. the polyp was removed surgically, under general anesthesia and using nasal forceps and electrical cautery. her blood pressure was normal, hemoglobin levels were discretely low, and coagulation tests were normal. the bleeding was controlled with the insertion of a nasal inflatable balloon catheter and cautery with silver nitrate. nasal endoscopy was repeated after 1 week, 6 months, and 1 year, showing no signs of lesion recurrence. grossly, it appears as a friable polypoid mass, which can present ulcerations on its surface. on histopathological examination its typical pattern is characterized by multiple, varying - sized capillaries in a lobular arrangement, surrounded by a loose fibromyxoid stroma.4 the most common locations for these lesions are the skin and mucous membranes of head and neck, particularly the gingiva of the oral cavity, the nasal cavity being a rare site of origin.1 2 within the nasal cavity, these lesions have been described to arise more frequently from the anterior portion of the nasal septum (little area) or the anterior portion of the inferior turbinate.5 6 it can occur at all ages and affects both the sexes equally.2 it presents usually by unilateral recurrent epistaxis and nasal obstruction, though in some cases smell alterations can be involved.1 the lesion is usually identified on direct examination of the anterior nasal cavity.7 the exact etiopathogenesis of this lesion is unclear, although some predisposing factors have been implied, such as local trauma or hormonal influences. regarding traumatic factors, the most commonly reported are nose picking and nasal packing, which are thought to trigger an inflammatory process in response to traumatic injury to the surface exposed.2 7 also, hormonal stimulation has also been implicated, with some cases reported in association to pregnancy and oral contraception.8 in this case, the lesion developed after an endoscopic transsphenoidal pituitary access, which induces traumatic injury to the nasal mucosa both through intraoperative manipulation and postoperative nasal packing. the pituitary adenoma removed was gonadotrophin - producing which can also provide a favorable hormonal environment. the differential diagnosis of an exophytic hypervascular mass in the nasal cavity presenting with intermittent episodes of epistaxis includes juvenile nasopharyngeal angiofibroma, which occurs typically in adolescent male patients, angiomatous polyp and rare malignant lesions, such as nasopharyngeal carcinoma or metastasis.1 7 imaging studies might be required in larger lesions which pose diagnostic difficulties and require an evaluation of extent and relation to adjacent structures.6 ct - scanning reveals an intensely enhancing mass and an iso- or hypoattenuating cap of variable thickness. bony changes (erosion or displacement) are not uncommon.3 treatment consists of total surgical excision that can be achieved through direct visualization endoscopically without significant complications. this technique might also be used in follow - up and early detection of recurrences, which are uncommon after complete excision.1 7 pregnancy - related lesions might regress spontaneously after delivery.8 this clinical case combines two predisposing factors to the development of a nasal lch hormonal influence (gonadotrophin - producing pituitary adenoma) and local traumatic injury (surgery and postoperative nasal packing). nasal lch is a rare but possible complication of endoscopic transsphenoidal resection of pituitary adenomas.	background lobular capillary hemangioma is a rare benign tumor, most frequently located in the head or neck region, the nasal cavity being uncommonly affected. its etiopathogenesis is not fully established, although traumatic and hormonal factors have been implied. case description a 50-year - old female patient underwent an uneventful endoscopic transsphenoidal removal of a pituitary cystic macroadenoma at our institution. nasal packing was used in postoperative hemostasis. histopathology was compatible with a gonadotrophin - producing adenoma. one month after the surgery, the patient presented with frequent episodes of epistaxis and a progressively growing nasal mass, which was removed endoscopically. its pathological examination confirmed a lobular capillary hemangioma. conclusions the authors present a clinical case combining two possible predisposing factors to the development of a nasal lobular capillary hemangioma : local traumatic injury through surgery and postoperative nasal packing and hormonal influence. this lesion is a rare complication of endoscopic transsphenoidal resections of pituitary adenomas.
the major aim of root canal therapy is to prevent and treat periradicular inflammation by eliminating microorganisms from the root canal system. the methods commonly used for this purpose include root canal preparation using different instruments and irrigants, adequate filling, and coronal restoration. chemical irrigants are essential for successful debridement of root canals during cleaning and shaping procedures. they are used during chemo - mechanical procedures not only as antimicrobial agents, but also to lubricate the dentinal walls, flush out debris and dissolve organic and inorganic components of the smear layer, thus cleaning the dentin surface. different irrigants have been proposed and used, including : 5.25% sodium hypochlorite, 2% chlorhexidine, 17% edta, 10% citric acid and 37% phosphoric acid solution. chlorhexidine has been used as irrigant during root canal therapy because of its antibacterial effects, substantivity, and relative absence of cytotoxicity, even though this solution is unable to dissolve the tissue. additionally, chlorhexidine has been suggested as a final irrigant. regarding its use as final irrigant, a final flush with 2% chlorhexidine favors the wettability of ah plus and real seal se sealers on the dentin surface. furthermore, it was verified that the bond strength of activ gp, a glass ionomer based system, was improved by using 2% chlorhexidine in the final irrigation after 17% edta. the development and maintenance of the sealing of the root canal system is the key to the success of root - canal treatment. the resin - based adhesive material has the potential to reduce the microleakage of the root canal because of its adhesive properties and penetration into dentinal walls. moreover, the irrigation protocols may have an influence on the adhesiveness of resin - based sealers to root dentin. a variety of experimental models are used to detect and measure any leakage along endodontic fillings, such as dye penetration, clearing of the teeth, radioisotope tests, bacterial penetration, electrochemical tests, fluid filtration, and glucose penetration model. the aim of the present study was to evaluate the effect of different irrigant protocols on coronal bacterial microleakage of gutta - percha / ah plus and resilon / real seal self - etch systems. one hundred ninety single - rooted pre - molars with straight roots, mature root apices and similar anatomical characteristics were used in this study. all instruments used in the root canal preparation were sterilized previously to the procedure. the teeth were positioned on a metallic apparatus that allowed for all procedures to be carried out without manual contact with the roots. a size 10 k - file (dentsply maillefer, petrpolis, rio de janeiro, brazil) was used to verify the patency of the canals and to determine the total length of the root canal, i.e. the work length. next, the foramina were standardized by using a size 20 k - file and root canals were shaped by using mtwo niti rotary system (vdw, mnich, bavaria, germany). the sequence employed was the following : 10/.04, 15/.05, 20/.06, 25/.06, 30/.05, 35/.04, 40/.04, and 25/.07. the teeth were divided into groups of ten according to the irrigation regimen (figure 1) and root canal filling. protocols for irrigation before the insertion of each file, 2% chlorhexidine (chx) gel (drogal, piracicaba, so paulo, brazil) or 5.25% sodium hypochlorite (naocl) (drogal, piracicaba, so paulo, brazil) were used as chemical - auxiliary substance. once the preparation was finished, 10 ml of distilled water (dw) was used to remove the chemical - auxiliary substance. next, 17% edta or 37% phosphoric acid solution (drogal, piracicaba, so paulo, brazil) was used for 3 minutes to remove the smear layer, with changes every 1 minute (1 ml per minute). finally, 2% chlorhexidine solution (drogal, piracicaba, so paulo, brazil) was used for final flush. during the chemo - mechanical preparation, all teeth had their apices sealed with utility wax (technew, rio de janeiro, rio de janeiro, brazil) to prevent flow through them. the root canals were dried with sterilized medium - sized paper points (endopoints, paraba do sul, rio de janeiro, brazil). groups 1 to 9 had the canals filled with gutta - percha cones (odous, belo horizonte, minas gerais, brazil) associated with ah plus sealer (dentsply, petrpolis, rio de janeiro, brazil), whereas groups 10 to 18 had the canals filled with resilon associated with real seal se (sybronendo, orange, california, usa). the cones packages were opened and they were used immediately, and the sealers were manipulated in sterile plates according to the manufacturer 's recommendations. a system - b endodontic heat source unit (sybronendo, orange, california, usa) was used to down - pack and obtura system (j morita, so paulo, so paulo, brazil) to backfill. subsequently, teeth were radiographed mesiodistally and buccolingually to assess the quality of the filling. all roots were kept on gauzes at 37c and 100% humidity for 2 weeks before leakage measurement in order to allow the materials to set properly. next, the external root surface of all specimens was sealed with two layers of red nail varnish (revlon, new york, new york, usa), except the last 1 mm of the apex. glass vials with rubber stoppers were adjusted for use. by using a shear, a hole was made at the center of each rubber stopper (figure 2b), through which each tooth was inserted under pressure up to the cementoenamel junction, so that its crown was outside the vial and its root inside (figure 2c). cyanoacrylate glue (cg) was applied at the interface between tooth and stopper for sealing. cylinders prepared from 10 ml plastic syringes were adapted to the outer surface of the stoppers to create a chamber around the crown of the tooth (figure 2d). again, cg was used at the interface between syringes and stoppers, followed by a parafilm layer (american national cantm, menasha, wisconsin, usa) to help in the sealing. the syringe / stopper / tooth sets were submitted to sterilization by gamma - rays (embrarad, so paulo, so paulo, brazil)., a hole was made at the center of the rubber stopper (b), through which the tooth was inserted (c). a cylinder prepared from a 10 ml plastic syringe was adapted to the outer surface of the stopper to create a chamber around the crown of the tooth (d). the glass flask filled with bhi broth was connected to the syringe / stopper / tooth set (e). turbidity in the bhi broth was evaluated (f) the sterilized glass flasks were then filled with sterile brain heart infusion broth (bhi ; oxoid, so paulo, so paulo, brazil) so that a 2-mm length of root apex was immersed in the broth. cg and parafilm were used to seal the interface between stopper and flask (figure 2e). in all samples, in order to ensure the efficiency of the seal, 2 ml of 1% sterile methylene blue dye was placed into the tube until the coronal portion of the sample was reached. after the third day, the methylene blue was removed with sterile distilled water by using a pipette. when a green medium was observed the specimen was discarded. the green color was due to the blue dye association with the yellow medium. for preparation of microbial medium, enterococcus faecalis (atcc 29212) was grown on bhi agar plates (brain heart infusion agar ; oxoid, so paulo, so paulo, brazil) and supplemented with 5% sheep blood for 24 hours at 37c in co2. then, the enterococcus faecalis was inoculated into tubes containing 5 ml sterile bhi suspension, which were adjusted spectrophotometrically at 800 nm (od800) to a turbidity of 1.5x108 colony - forming units (cfu)/ml. with the aid of a pipette, 5 ml of the suspensions were placed into the syringe apparatuses (in the upper region, removing the gauze stop), which were left at 37c for 90 days in co2 and checked daily for turbidity in the bhi broth. every 2 days, 3 ml of the suspension (bhi+microorganisms) were removed from the chamber and replaced by 3 ml of bhi to avoid saturation and to confirm the growth of enterococcus faecalis. positive cultures were confirmed by using gram staining (gram - positive), colony morphology on blood agar plates (cocci) and biochemical identification kits (rapid i d 32 strep, biomrieux sa, marcyl'etoile, charbonnieres - les - bains, france). ten samples were used as positive (n=5) and negative (n=5) controls. the positive controls consisted of instrumented teeth without obturation, while negative controls consisted of sound teeth, both with no contamination. the results were analyzed with kaplan - meier survival test, kruskal - wallis and mann - whitney tests (p<0.05). one hundred ninety single - rooted pre - molars with straight roots, mature root apices and similar anatomical characteristics were used in this study. all instruments used in the root canal preparation were sterilized previously to the procedure. the teeth were positioned on a metallic apparatus that allowed for all procedures to be carried out without manual contact with the roots. a size 10 k - file (dentsply maillefer, petrpolis, rio de janeiro, brazil) was used to verify the patency of the canals and to determine the total length of the root canal, i.e. the work length. next, the foramina were standardized by using a size 20 k - file and root canals were shaped by using mtwo niti rotary system (vdw, mnich, bavaria, germany). the sequence employed was the following : 10/.04, 15/.05, 20/.06, 25/.06, 30/.05, 35/.04, 40/.04, and 25/.07. the teeth were divided into groups of ten according to the irrigation regimen (figure 1) and root canal filling. protocols for irrigation before the insertion of each file, 2% chlorhexidine (chx) gel (drogal, piracicaba, so paulo, brazil) or 5.25% sodium hypochlorite (naocl) (drogal, piracicaba, so paulo, brazil) were used as chemical - auxiliary substance. once the preparation was finished, 10 ml of distilled water (dw) was used to remove the chemical - auxiliary substance. next, 17% edta or 37% phosphoric acid solution (drogal, piracicaba, so paulo, brazil) was used for 3 minutes to remove the smear layer, with changes every 1 minute (1 ml per minute). finally, 2% chlorhexidine solution (drogal, piracicaba, so paulo, brazil) was used for final flush. during the chemo - mechanical preparation, all teeth had their apices sealed with utility wax (technew, rio de janeiro, rio de janeiro, brazil) to prevent flow through them. the root canals were dried with sterilized medium - sized paper points (endopoints, paraba do sul, rio de janeiro, brazil). groups 1 to 9 had the canals filled with gutta - percha cones (odous, belo horizonte, minas gerais, brazil) associated with ah plus sealer (dentsply, petrpolis, rio de janeiro, brazil), whereas groups 10 to 18 had the canals filled with resilon associated with real seal se (sybronendo, orange, california, usa). the cones packages were opened and they were used immediately, and the sealers were manipulated in sterile plates according to the manufacturer 's recommendations. a system - b endodontic heat source unit (sybronendo, orange, california, usa) was used to down - pack and obtura system (j morita, so paulo, so paulo, brazil) to backfill. subsequently, teeth were radiographed mesiodistally and buccolingually to assess the quality of the filling. all roots were kept on gauzes at 37c and 100% humidity for 2 weeks before leakage measurement in order to allow the materials to set properly. next, the external root surface of all specimens was sealed with two layers of red nail varnish (revlon, new york, new york, usa), except the last 1 mm of the apex. glass vials with rubber stoppers were adjusted for use. by using a shear, a hole was made at the center of each rubber stopper (figure 2b), through which each tooth was inserted under pressure up to the cementoenamel junction, so that its crown was outside the vial and its root inside (figure 2c). cyanoacrylate glue (cg) was applied at the interface between tooth and stopper for sealing. cylinders prepared from 10 ml plastic syringes were adapted to the outer surface of the stoppers to create a chamber around the crown of the tooth (figure 2d). again, cg was used at the interface between syringes and stoppers, followed by a parafilm layer (american national cantm, menasha, wisconsin, usa) to help in the sealing. the syringe / stopper / tooth sets were submitted to sterilization by gamma - rays (embrarad, so paulo, so paulo, brazil). the apparatus used to evaluate coronal leakage (a). by using a shear, a hole was made at the center of the rubber stopper (b), through which the tooth was inserted (c). a cylinder prepared from a 10 ml plastic syringe was adapted to the outer surface of the stopper to create a chamber around the crown of the tooth (d). the glass flask filled with bhi broth was connected to the syringe / stopper / tooth set (e). turbidity in the bhi broth was evaluated (f) the sterilized glass flasks were then filled with sterile brain heart infusion broth (bhi ; oxoid, so paulo, so paulo, brazil) so that a 2-mm length of root apex was immersed in the broth. cg and parafilm were used to seal the interface between stopper and flask (figure 2e). in all samples, in order to ensure the efficiency of the seal, 2 ml of 1% sterile methylene blue dye was placed into the tube until the coronal portion of the sample was reached. after the third day, the methylene blue was removed with sterile distilled water by using a pipette. when a green medium was observed the specimen was discarded. the green color was due to the blue dye association with the yellow medium. for preparation of microbial medium, enterococcus faecalis (atcc 29212) was grown on bhi agar plates (brain heart infusion agar ; oxoid, so paulo, so paulo, brazil) and supplemented with 5% sheep blood for 24 hours at 37c in co2. then, the enterococcus faecalis was inoculated into tubes containing 5 ml sterile bhi suspension, which were adjusted spectrophotometrically at 800 nm (od800) to a turbidity of 1.5x108 colony - forming units (cfu)/ml. with the aid of a pipette, 5 ml of the suspensions were placed into the syringe apparatuses (in the upper region, removing the gauze stop), which were left at 37c for 90 days in co2 and checked daily for turbidity in the bhi broth. every 2 days, 3 ml of the suspension (bhi+microorganisms) were removed from the chamber and replaced by 3 ml of bhi to avoid saturation and to confirm the growth of enterococcus faecalis. positive cultures were confirmed by using gram staining (gram - positive), colony morphology on blood agar plates (cocci) and biochemical identification kits (rapid i d 32 strep, biomrieux sa, marcyl'etoile, charbonnieres - les - bains, france). ten samples were used as positive (n=5) and negative (n=5) controls. the positive controls consisted of instrumented teeth without obturation, while negative controls consisted of sound teeth, both with no contamination. the results were analyzed with kaplan - meier survival test, kruskal - wallis and mann - whitney tests (p<0.05). no differences were found following the use of chlorhexidine or naocl associated with edta or phosphoric acid for smear layer removal. however, it was clearly observed that groups receiving a final flush with chlorhexidine showed a lower number of contaminated samples. graph showing the number of contaminated samples according to the irrigation protocol different letters indicate statistically significant values (p<0.05) regarding the root canal filling system (gutta percha / ah plus and resilon / real seal se), there was no statistically significant differences in relation to the coronal microleakage. statistic analysis of the contamination days revealed difference in the groups receiving the final flush with chlorhexidine. chlorhexidine groups started to contaminate only in the 6week, while in the others the microbial growth was verified in the 1or 2week. the control apparatuses showed broth turbidity within 1 day of incubation in all samples, whereas no microbial growth was found in the negative control throughout the experiment. number of contaminated samples per week dw- distilled water ; chx- chlorhexidine ; gpah- gutta - percha / ah plus ; rrs- resilon / real seal se leakage of the root canal has been defined as the passage of bacteria, fluids, and chemical substances between the dentinal wall and the root canal filling material, and results from the presence of space at the interface of the filling material and the root canal wall. this space can result from deficient adaptation of the filling material to the root dentin, solubility of the sealer, or sealer expansion or shrinkage. there are 2 possibilities of leakage : at the interface between the main filling material and sealer, or between the sealer and root canal wall. in the present study, this methodology is reproducible and has clinical relevance, presenting reliable data and simulating clinical conditions. this methodology allows the observation of the exact day of the sample contamination, showed by the broth turbidity. the 37% phosphoric acid was evaluated in comparison with edta because this solution is effective for smear layer removal, showing better results than edta in the apical third during 3 minutes. additionally, the protocols associating naocl with phosphoric acid showed higher bond strength values when compared with naocl associated with edta. although phosphoric acid had showed better performance in removing the smear layer from the apical third, it did not influence the results of coronal leakage. the 2% chlorhexidine, a cationic bisbiguanide, was used here for the final flush after smear layer removal. this substance has a broad - spectrum mmp - inhibitory effect that improves the integrity of the hybrid layer6 and the resin - dentin bond stability. additionally, the use of chx increases the wettability of endodontic sealers on dentin, which can be explained by the presence of surface surfactant in chx, increasing the surface energy and promoting higher wetting ability to dentin. our results showed that a final flush with chlorhexidine significantly reduced the coronal microleakage, when compared to the other experimental groups. it might be explained by the fact that chlorhexidine is adsorbed onto dentin and prevent microbial colonization, increasing the time required for recontamination of filled rootup to 12 weeks due to its substantivity. regarding the effect of a final flush with chlorhexidine on adhesion, previous studies showed that this solution did not affect the bond strength of resin - based sealersand improved the adhesion of hydrophilic bonded materials such as activ gp and epiphany. naocl was not used as a final irrigant because a previous study showed that this solution decreased the bond strength between epoxy resin and dentin and increased the leakage. regarding the root canal filling system (gutta percha / ah plus and resilon/ real seal se), there was no statistically significant differences in relation to the coronal microleakage, agreeing with previous findings. however, some studies reported that resilon / epiphany sealer was more efficient than gutta - percha / ah plus, whereas others found the opposite. in conclusion, a final flush with 2% chlorhexidine after smear layer removal reduces coronal microleakage of teeth filled with gutta - percha / ah plus or resilon / real seal self - etch. the bacterial leakage methodology used made possible the verification of this behavior.	the development and maintenance of the sealing of the root canal system is the key to the success of root canal treatment. the resin - based adhesive material has the potential to reduce the microleakage of the root canal because of its adhesive properties and penetration into dentinal walls. moreover, the irrigation protocols may have an influence on the adhesiveness of resin - based sealers to root dentin.objectivethe objective of the present study was to evaluate the effect of different irrigant protocols on coronal bacterial microleakage of gutta - percha / ah plus and resilon / real seal self - etch systems.material and methodsone hundred ninety pre - molars were used. the teeth were divided into 18 experimental groups according to the irrigation protocols and filling materials used. the protocols used were : distilled water ; sodium hypochlorite (naocl)+edta ; naocl+h3po4 ; naocl+edta+chlorhexidine (chx) ; naocl+h3po4+chx ; chx+edta ; chx+ h3po4 ; chx+edta+chx and chx+h3po4+chx. gutta - percha / ah plus or resilon / real seal se were used as root - filling materials. the coronal microleakage was evaluated for 90 days against enterococcus faecalis. data were statistically analyzed using kaplan - meier survival test, kruskal - wallis and mann - whitney tests.resultsno significant difference was verified in the groups using chlorhexidine or sodium hypochlorite during the chemo - mechanical preparation followed by edta or phosphoric acid for smear layer removal. the same results were found for filling materials. however, the statistical analyses revealed that a final flush with 2% chlorhexidine reduced significantly the coronal microleakage.conclusiona final flush with 2% chlorhexidine after smear layer removal reduces coronal microleakage of teeth filled with gutta - percha / ah plus or resilon / real seal se.
the therapeutic value of medicinal plants has long been exploited for the management of various disease conditions in traditional practice. this practice has gained appreciable acceptance in health care delivery in developing and developed nations with the notion that they are relatively harmless, but research is beginning to show that some of them may be toxic. khaya senegalensis (desr.) a. juss (family : meliaceae) is a tree that is widely distributed in the sub - saharan savannah from senegal to sudan and uganda. it is a round evergreen crown of dark shiny foliage, pinnate leaves, and characteristic round capsules that grows up to 40 m high. the therapeutic value of khaya senegalensis has been recognized in different systems of traditional medicine for the treatment of various conditions. the decoction of the stem bark extract is commonly used for treating jaundice, dermatoses, malaria, fever, mucous diarrhea, and venereal diseases as well as for hookworm infection and a taeniacide remedy [2, 3 ]. khaya senegalensis extracts have been reported to exhibit anti - inflammatory effects as well as anti - bacterial, antihelmintic, antitumor, antioxidant and antiplasmodial activities. the stem bark extract and the chemical constituent profile have been the subject of extensive phytochemical and pharmacological investigations since the 1960s [911 ]. despite the extensive research on the stem bark extract of khaya senegalensis, a dearth of information on the toxicological profile of this plant still exists. this work is therefore designed to evaluate the acute and subchronic toxicity effects of the extract on the liver and kidney. stem bark of k. senegalensis was collected from the main campus of usmanu danfodiyo university, sokoto, (130 21 16 n and 50 5 37 e) in the month of june 2011. a.m. umar from the botany unit, department of biological sciences, usmanu danfodiyo university, sokoto, and a voucher specimen (udus / vs/2011/21) was prepared and deposited in the herbarium of the same department. albino male rats weighing 150200 g were purchased from the animal house, usmanu danfodiyo university, sokoto, nigeria. they were housed in metal cages and allowed free access to a standard laboratory pellet diet (ecwa feed nig ltd.) and water ad libitum under a 12-hour light - dark cycle throughout the experimental period. plant materials were washed and air - dried at room temperature until a constant weight was obtained. the samples were ground using a pestle and mortar and the powdered samples were then bagged and stored in plastic bags until required. the plant sample was separately placed in an erlenmeyer flask and one liter of distilled water was added into the flask and it was allowed to soak for a minimum of 24 hours. the mixture was filtered using a piece of clean, sterile muslin cloth to remove debris and then filtered on whatman filter paper. the resultant filtrate was evaporated to dryness and the percentage recovery was calculated (13.3%). acute oral toxicity was performed using the up- and down - procedure of the organisation for economic co - operation and development. this procedure involves the limit and main test, which was also used for the selection of a starting dose as well as determination of ld50 of the testing material. the test substance was administered orally to the rats in groups ii iv in a gradation of 400, 800, 1200, 1600, and 2000 mg / kg body weight per day for 28 days, while group i (control group) animals were given an equivalent volume of normal saline orally for the same period of 28 days under the same conditions. the body weight changes were monitored weekly throughout the experimental period. on the 29th day, after an overnight fast, the rats were anaesthetized in a chloroform vapour and blood samples were collected from the animals by cardiac puncture into labeled vacutainers for biochemical and haematological assays. subsequently, the animals were sacrificed by decapitation ; the organs (kidney, liver, and heart) were removed, weighed, and then fixed in 10% formalin for histopathological examinations. serum total protein was determined using the biuret method ; cupric ions in the biuret reagent react with the peptide bonds of the protein molecule in an alkaline solution to form a blue colored complex. albumin was determined using the bromocresol green method ; albumin binds with bromocresol green in an acidic medium to form a blue - green complex whose colour is proportional to the concentration of albumin in the serum. transaminase activity (aspartate aminotransferase (ast) and alanine, aminotransferase (alt)) was determined using the reitman and frankel method ; alt was measured by monitoring the concentration of pyruvated hydrazone that is formed with 2,4-dinitrophenylhydrazine while ast was measured by monitoring the concentration of oxaloacetate hydrazone that is formed with 2,4-dinitrophenylhydrazine. alkaline phosphatase (alp) activity was determined using the colorimetric method of rec ; the hydrolysis of 4-nitrophenyl phosphate at alkaline ph to 4-nitrophenol and inorganic phosphate is catalysed by alkaline phosphatase and the intensity of the colour is directly proportional to the activity of alp. urea was investigated using the urease - berthelot colorimetric method of fawcett and scott ; urea in serum is hydrolysed to ammonia in the presence of urease. creatinine was measured by the picrate method ; creatinine reacts with picric acid in an alkaline medium to form a yellow - red colored complex which can be measured spectrophotometrically at 520 nm. serum electrolytes na and k were measured using a flame emission spectrophotometer ; na and k are atomised using a flame ; the amount of atomisation is proportional to the quantity of the element in the solution, while hco3 was estimated using a titrimetric method. packed cell volume (pcv) of samples was determined using microhematocrit centrifuge at 12000 g for 5 min ; white blood cell (wbc) count and differentials were determined by mechanical expansion and optical magnification, augmented by supravital cell staining. three animals from each group were selected randomly and dissected through a central abdominal incision. the kidney, heart, and liver samples were collected and immediately fixed in 10% formal saline in labeled sample plastic bottles. the tissues were dehydrated in graded concentrations of xylene, embedded in molten paraffin wax, and sectioned at 5. tissue sections were fixed on glass slides and stained with hematoxylin and eosin for light microscopy at 400x. photomicrographs of some of the tissues were taken using a microscope fitted with a camera unit. the results were analyzed using one - way analysis of variance (anova), followed by dunnett 's multiple comparison test using graphpad instat software. the ld50 was found to be greater than 5000 mg / kg body weight. during the course of the toxicity experiment, the animals were observed for any change in behavioural pattern, physical characteristics, and activity. the rats in all the steps displayed no appreciable change in physical or behavioural activities ; they fed well and moved about in their cages well. the histological preparations of the liver after treatment with the extract are presented in figures 1(a) and 1(b) ; both the normal and experimental preparations appear within the range of a preserved histological architecture. the effects of oral administration of aqueous extract of khaya senegalensis stem bark on the ratio of organ weight : body weight of experimental rats and percentage change in body weight of the first and last days of the experimental period are shown in table 1. prolonged administration of the extract for 28 days resulted in significant (p 0.05) effect on the ratio of kidney weight : body weight as well as ratio of heart weight : body weight when compared to the values of their corresponding control group. however the ratios of liver weight : body weight and spleen weight : body weight were observed to be significantly different at the higher doses of 1600 mg / kg (p 0.05) effect on the haematological indices. the livers of animals in the control group (0 mg / kg) showed typical hepatolobular architecture, consisting of a central vein with radiating cords of hepatocytes separated by sinusoids ; portal areas composed of the portal vein, hepatic artery, and bile duct were situated at the periphery. the hepatocytes were polygonal in shape, with central, lightly stained nucleus and clear nucleolus ; few binucleated cells were also present, and the cytoplasm was regularly distributed without vacuolations. in the treated dose groups (400, 800, 1200, 1600, and 2000 mg / kg bw) the general hepatolobular architecture of the liver was deranged ; there was loss of radial arrangement of hepatocytes and sinusoids ; the number of binucleated cells was increased ; inflammatory cells, especially lymphocytes, were found infiltrating around the portal track ; areas of necrosis were found around the central vein. examination of the liver histological preparations obtained from all the experimental treated groups showed comparable changes as observed in figures 2(a)to 2(f). the histological preparations of the kidney and heart of the rats based on the subchronic toxicity study of aqueous stem bark extract of k. senegalensis indicated that they had normal architecture (figures 3 and 4). the observed changes, however, in all the treated groups were restricted to only mild congestion and were not dose dependent. the ld50 of aqueous stem bark extract of k. senegalensis was found to be greater than 5000 mg / kg ; it may therefore be considered nontoxic ; although this does not predict the lethal dose in humans, it however provides a guide for choosing the dose for use in sub - chronic studies. body weight is normally investigated as a sensitive indicator of chemically induced changes to organs. the comparison between the ratiometric differences of organ : body weight of the control group and the treated groups has been used to evaluate the toxic effect of the test substance. the result of this study shows that sub - chronic administration of k. senegalensis does not affect the anatomical proportion (ratios of kidney : body weight and heart : body weight) as compared to normal control ; this may be that the defensive mechanism of the animal has not been overcome and/or may be that the dose has not accumulated sufficiently to manifest any significant change. however, the significant increase of the ratio of liver : body weight (hepatomegaly) following the administration of the extract may be attributed to tissue oedema (oedematous) and ballooning resulting in necrosis ; this may be due to the role of the liver in detoxification and excretion. the spleen at the dose group of 2000 mg / kg body weight was significantly (p < 0.05) increased (splenomegaly) : this could result from any one of the following : hemolytic anemia, cirrhosis, hepatic vein obstruction, and portal vein obstruction. it could also have been a result of an induced alpha - mannosidosis by the extract, that is, if the extract was able to penetrate into the lysosome. it is therefore worthy to mention that the higher dose levels (1600 and 2000 mg / kg body weight) should be used with caution. the possibility of renal insufficiency or dysfunction occurring as a result of the toxic effect of the extract may be eminent. the observed (table 2) significant increase in serum sodium ions with a concomitant significant increase in potassium ion levels may be due to renal dysfunction resulting from the inability of the kidney to regulate an electrolyte balance. the extract may also interact with specific hormone receptors resulting in increased production of aldosterone and mineralocorticoids which may be responsible for the observed hypernatraemia. furthermore, the marked elevation in serum urea level in this study may suggest that the extract caused renal impairment in the rats or it may be a result of increased protein catabolism. however since urea is not a complete estimation of renal function, the observed significant increase in creatinine may further buttress the extract as toxic to the kidney at the doses used in this study. serums alt and ast considered in this study are important and play significant role in the diagnosis of liver cytolysis. the estimation of the levels of protein and bilirubin is used to examine the synthetic and excretory function of the liver, respectively. measurement of the activities of enzymes in tissues and body fluids plays a significant and well - known role in disease investigation and diagnosis. tissue enzyme assay can also indicate tissue cellular damage long before structural damage can be picked up by conventional histological techniques. such measurement can also give an insight to the site of cellular tissue damage as a result of assault by the plant extract. in this study, the resultant increase in the activities of serums alt and ast may be an indication of liver cytolysis, through which they are released into circulation. alt is located in the cytosol of hepatocytes and the enzyme is considered a more sensitive marker of hepatocellular damage than ast. ast is found in the cytoplasm and mitochondria in different tissues of the heart, liver, kidney, skeletal muscle, pancreas, and erythrocyte. the aminotransferases are significant in amino acid metabolism as they help in retaining amino groups during the degradation of amino acids which are further used for the synthesis of new amino acids, and hence they are involved in the biochemical regulation of the intracellular amino acid pool. also, when these enzymes are mobilized from the liver into the free circulation, glutamate concentration may be affected with a resultant decrease in glutathione, one - third of which is formed from glutamate. detoxification is the essential role of glutathione, and cells are exposed to an attack by the active metabolite when it is depleted. also, a significant (p < 0.05) change in the level of total bilirubin was observed (table 2). this may indicate that the functional integrity of the liver has been affected by the damage caused to the liver by the plant extract. bilirubin is assayed to measure the binding, excreting, and conjugating ability of a hepatocyte. hence, the observed increase may indicate that a component of the plant extract, might have competed and displaced the binding of bilirubin on albumin or the uptake of bilirubin is inhibited by the plant extract ; this may also account for the increased level of unconjugated bilirubin. extract administration produced a significant decrease in the total protein concentration in all the dose groups with the exception of 400 mg / kg body weight. hence the aqueous stem bark extract might inhibit the synthesis of some proteins, thereby resulting in the decrease in serum total protein. it has been reported that low protein level results when there is extensive liver damage. the result of this study revealed significant (p < 0.05) increase in the activity of serum alkaline phosphatase (alp). alp is often employed to assess the integrity of the plasma membrane of the liver. the significant increase in the serum alp following administration of the plant extracts may be due to disruption of the liver plasma membrane. the result of this study therefore demonstrates that k. senegalensis at the experimental doses is nephrotoxic and should be used with caution when administered over a long period of time. the possibility of the extract giving rise to haemopoietic damage, malignancy, allergy, or compromise to the immune status could be ruled out since all haematological parameters assayed did not show any significant changes when compared with their corresponding controls. the liver being an organ of detoxification is the first organ that encounters all absorbed materials from the gastrointestinal tract : it has been shown to respond to toxicological insults in a number of ways including cellular degeneration and necrosis and bile duct hyperplasia and fibrosis. ; its protective property by detoxification may explain the preserved architecture of the kidney and heart. the mild to moderate congestion as well as hemorrhage observed on the histological slides could be attributed to surgical handling of the organs since these conditions were also observed in the normal groups. these observations suggest that long term administration of k. senegalensis may be toxic and its toxicity may be organ specific.	the subchronic effect of aqueous stem bark extract of khaya senegalensis on some biochemical, haematological, and histopathological parameters of rats was investigated. the rats were divided into six groups of five rats per group. groups i to vi were administered graded doses of 0, 400, 800, 1200, 1600, and 2000 mg / kg bw, respectively. the result of study revealed that administration of the khaya senegalensis for twenty - eight days at the experimental dose resulted in significant (p < 0.05) increase in urea, electrolytes (na+, k+), and creatinine levels. the extract also significantly (p < 0.05) increased serum activity of alt, ast, and alp. the levels of protein, albumin, and bilirubin were significantly changed when compared to their control values, but they were not dose dependent. the hematological indices assayed in this study were not significantly affected at the experimental dose when compared to the control values. histological studies of the liver showed cellular degeneration and necrosis and bile duct hyperplasia and fibrosis with lymphocytic infiltration of the hepatocyte, providing supportive evidence for discussing the biochemical findings, indicative of functional derangement. the histological architecture of the kidney and that of the heart were however preserved. the result of this study indicates that the aqueous stem bark extract of k. senegalensis may affect the cellular integrity of vital organs of the body.
colorectal cancers (crcs) are among the most common cancers in the world. in developed countries, 6% of general population are at risk for crc and it accounts for 10% of newly diagnosed cases in male as well as 11% in female. surveys by a federation of european gastroenterology announced crc as the most common cancer in 2000. the incident rate of crc varies in different parts of the world with a higher incidence in developed countries, such as us, australia, western europe, and new zealand compared with southern america, africa, and asia. in iran, the incident rate of crc was 51.7%, 87.7%, and 12.8% in 2007, 2008, and 2009, respectively. crc is the most common cancer after skin, breast, and gastric cancer in the iranian population. despite preventive interventions, crc exhibit increasing prevalence in recent years. the most common prevalence is observed in sporadic group and individuals without any familial or hereditary past history. lifestyle, nutrition, and physical activities play a key role in increased susceptibility to crc. studies in animal models have shown the importance protective effect of physical activities on chemical tumorigenic insults in the intestine. the role of physical activities in susceptibility to crc has been investigated extensively in epidemiological studies despite the controversial results of these studies. different groups have reported converse correlation, no effect or increased risk of physical activities on crc. considering a very few studies that were taking the effect of leisure time, occupational, and household physical activity on crc, we designed this study to assess these factors on crc in the iranian population. in this case control population - based study, 100 individuals with confirmed diagnosis of crc were recruited to the study in two referral hospitals in isfahan city, iran. we excluded patients with the history of metastatic colon cancer as well as patients with the history of any other chronic disease that could cause limitations in physical and mental activities and potentially interfere with comfortable interview or correct filling of the consent form. among 400 patients that were considered for this study 100 new cases met the inclusion criteria and histopathology confirmed the disease in all participants. in this group, data collection was performed after the diagnosis of each patient. the control group was chosen from companions of other patients (in other clinical sectors) without any history of cancer and limited physical activity. the study was approved by research council of isfahan university of medical sciences (study no : 385342) and written consent form was obtained from all the participants. physical activities of the participants were evaluated using a modified retrospective kriska physical activity questionnaire. this questionnaire is a well - established questionnaire to evaluate past physical history and allows robust analysis of data in retrospective studies. in this questionnaire, 14 leisure physical activities were assessed, including slow easy walking, brisk walking, yoga, swimming, and cycling. we also evaluated the household activities, such as cleaning, changing decoration, and cooking. occupational physical activities were studied considering all physical activities that happen during the work time. to have a reliable assessment of physical activity level we categorized data into four different groups ; number of years, number of months during the past year, number of days in a week, and number of hours in a day. we evaluated the number of each activity in a week by calculating the frequency of that activity per unit of time. different studies have shown that it is difficult to reliably report brisk walking during the day so we evaluated brisk walking separately from other activities. we registered the following data in the questionnaire : demographic data including, age gender, date of birth, current address, marital status, and level of education. we also evaluated other relevant data, such as hormone replacement therapy (starting age, dosage, duration, method of administration), smoking (package / year), alcohol (amount and duration), aspirin usage (regularly at least for 1 year), cholecystectomy, history of cancer, family history of cancer in the first- and second - degree relatives. anthropometric evaluation was conducted in self - report format and it included height (cm), average of weight during the past 5 years (kg) and body mass index (bmi) according to the formula of (weight [kg]/height [m ]). spss statistical software version 15 (2006 by spss inc, chicago, il, inc. the risk of crc was evaluated using multiple binary logistic regression and odds ratio (or) with confidence interval (ci) = 95%. in this case control population - based study, 100 individuals with confirmed diagnosis of crc were recruited to the study in two referral hospitals in isfahan city, iran. we excluded patients with the history of metastatic colon cancer as well as patients with the history of any other chronic disease that could cause limitations in physical and mental activities and potentially interfere with comfortable interview or correct filling of the consent form. among 400 patients that were considered for this study 100 new cases met the inclusion criteria and histopathology confirmed the disease in all participants. in this group, data collection was performed after the diagnosis of each patient. the control group was chosen from companions of other patients (in other clinical sectors) without any history of cancer and limited physical activity. the study was approved by research council of isfahan university of medical sciences (study no : 385342) and written consent form was obtained from all the participants. physical activities of the participants were evaluated using a modified retrospective kriska physical activity questionnaire. this questionnaire is a well - established questionnaire to evaluate past physical history and allows robust analysis of data in retrospective studies. in this questionnaire, 14 leisure physical activities were assessed, including slow easy walking, brisk walking, yoga, swimming, and cycling. we also evaluated the household activities, such as cleaning, changing decoration, and cooking. occupational physical activities were studied considering all physical activities that happen during the work time. to have a reliable assessment of physical activity level we categorized data into four different groups ; number of years, number of months during the past year, number of days in a week, and number of hours in a day. we evaluated the number of each activity in a week by calculating the frequency of that activity per unit of time. different studies have shown that it is difficult to reliably report brisk walking during the day so we evaluated brisk walking separately from other activities. we registered the following data in the questionnaire : demographic data including, age gender, date of birth, current address, marital status, and level of education. we also evaluated other relevant data, such as hormone replacement therapy (starting age, dosage, duration, method of administration), smoking (package / year), alcohol (amount and duration), aspirin usage (regularly at least for 1 year), cholecystectomy, history of cancer, family history of cancer in the first- and second - degree relatives. anthropometric evaluation was conducted in self - report format and it included height (cm), average of weight during the past 5 years (kg) and body mass index (bmi) according to the formula of (weight [kg]/height [m ]). spss statistical software version 15 (2006 by spss inc, chicago, il, inc. the risk of crc was evaluated using multiple binary logistic regression and odds ratio (or) with confidence interval (ci) = 95%. the average of the age was 59.40 11.82 and 55.81 11.96 in case and control group, respectively. we did not observe any significant difference in two groups regarding the age (p = 0.92), gender (p = 0.77), educational level (p = 0.07) and bmi (p = 0.09). the case group showed higher scores for smoking, hormone therapy and in the past 5 years compared to the control [table 1 ]. baseline characteristics of study participants after assessing the age, bmi, smoking, and hormone therapy, next, we evaluated the risk of crc in groups with high leisure physical activity level compared to those with low physical activity and we found 27% reduction in the risk of crc. this is of great importance because we could not detect any significant difference in the risk of crc when considering occupational and house holding physical activities (p = 0.67 and p = 0.73, respectively). the or of the moderate leisure physical activity level in the adjusted model was 0.82 (p = 0.007) and in the nonadjusted model was determined 0.78 (p = 0.009). investigating the bmi in both case and controls did not show a significant difference with the risk of crc [table 2 ]. according to the findings of our study, the protective effect of leisure physical activities in the age groups of 4050 years (or = 0.69, 95% ci : 0.561.9), 5060 (or = 0.74, 95% ci : 0.481.05) and above 60 (or = 0.64, 95% ci : 0.450.52) shows a significant difference although these data in the group of 3040 did not reach significance. our data suggest that the correlation between brisk walking and crc is significant in female versus male (or = 0.76, 95% ci : 0.423.56 and or = 0.79, 95 percent ci : 0.511.56, respectively). our results suggest that there is a significant reverse correlation between crc and physical activity. we observed 27% decrease in the risk of crc in individuals with high level of leisure physical activities. our results are in agreement with the study of slattery., that reported a substantial decrease in the risk of crc and rectal cancer in individuals with high physical activity. there are other studies that suggest an even stronger correlation, for example, in a study conducted in yale university, a 4050% decrease in crc was reported in female and male with high physical activity. cohort studies also show a strong correlation between the risk of crc and leisure physical activities. there are still many aspects of physical activities and their impact on our daily life that needs to be elucidated. we still do not know the mechanism by which physical activity will decrease the risk of crc but resting lifestyle and hyperinsulinemia are among the major causes. like other members of growth factor family, insulin is correlated with tumor growth and anti - apoptotic effects and that may partially explain the higher risk of crc in individuals with increased blood levels of insulin, c - peptide and insulin - like growth factor 1. studies suggest that the increased levels of insulin and having diabetes mellitus are correlated with the risk of proximal, and not distal, crc. reduced exposure of mucosa to carcinogens in diet after exercise, decreased body fat and insulin resistance, improved immune function of t - cells, b - cells and nk - cells, altered interleukin-1 levels, as well as changes in prostaglandin synthesis, modifications in gallbladder acids and cholesterol level as well as gastrointestinal hormones, are among different mechanisms that were suggested to explain the correlation between risk of crc and physical activities. we could not detect any significant correlation between occupational as well as household activities and the risk of crc. although a meta - analysis of 16 cohort studies conducted in 2006 reported a significant decrease in crc risk in individuals with high physical and work activities, but another review in 2007 report no correlation between crc and work activities. it seems that type of study and method of classifying work activities are determining factors that need to be of more attention when analyzing the data. in most of the other studies, housekeeping activities are more reliably measured compared to leisure physical activities because of their routine nature. one of the reasons that may explain why we did not observe any correlation between house holding physical activities and risk of crc is the fact that we studies both colon and rectum cancers together. in the study conducted by larsson, a reverse correlation between housekeeping activities and colon cancer has been reported, although in another study conducted by hou., there was no detectable correlation between rectum cancer and physical activity. some studies have mentioned a strong correlation between obesity and crc in male and to a lesser extent in female. our results do not show any significant correlation between bmi and crc in both genders. we think that recall bias in reporting bmi should be considered in more depth when studying bmi and crc risk in case control studies. in dai., meta - analysis, obesity was reported to have a significant positive effect on crc. the most common physical activity among our participants was walking. according to our finding, brisk other studies have also suggested brisk walking as the most common activity with up to 25% preventive effect on crc when happens more than 40 min / day. one of the limitations of the study was the limited sample size, so the generalizability of the results to the broader community should be done with caution. in addition, numbers of case and controls were equal. another important limitation of this study is a lack of food intake assessment. although due to similarities in the socioeconomic status of both groups, we expected similar dietary habits between the two groups. in some of the previous studies, selection bias and recall bias had affected the data. to avoid this type of biases we conducted this study in a double - blind format. all the interviewers were trained according to protocols and these trainings were repeated during the study to ensure the validity and reliability of the data. in summary, our study shows a reverse correlation between risk of crc and leisure physical activities (especially brisk walking during the day). our study highlights the importance of leisure physical activities and not occupational and housekeeping activities in preventing crc, especially at the age of 40 and above.	background : the prevalence of colorectal cancer (crc) is rapidly increasing in iran. it holds the most prevalent cancer after skin, breast, and gastric cancers among the iranian population. the current study was designed to investigate the effects of leisure time, occupational and household physical activity as well as exercise on the risk of crc in the iranian population.methods:in this population - based case control study, 100 individuals with a recent diagnosis of crc who were eligible for the study were recruited between 2006 and 2008. the control groups were selected from patients companions (excluding first- and second - degree relatives) without past history of cancer or any physical disability. physical activity of the participants was evaluated using a kriska retrospective physical activity questionnaire. the relation between crc and physical activity was assessed via logistic regression model and calculating the odds ratio (or) as well as a confidence interval (ci) of 95%.results : according to the findings, the adjusted or of occupational (or = 0.98, 95%, ci : 0.951.02) and house holding physical activities (or = 1.03, 95% ci : 0.991.08) were not significantly different between the case and control groups for women (p > 0.05). the risk of crc shows a significant reduction in individuals with moderate leisure physical activities compared to those with minimal activities (or = 0.82, ci 95% : 0.730.98).conclusions : the study suggests that the risk of crc will decrease in individuals with higher leisure physical activities (especially with an increase in hours of brisk walking during the day).
pancreatic endocrine tumours are rare tumours, and may produce a variety of hormones including insulin, gastrin, glucagon, pancreatic polypeptide, vasoactive intestinal peptide and somatostatin. the most common pancreatic endocrine tumours are insulinomas and gastrinomas, which are respectively found within the pancreas and the gastrinoma triangle formed by the junction of the cystic and common bile ducts superiorly, the junction of the second and third portions of the duodenum inferiorly, and the junction of the neck and body of the pancreas medially. the tumors may be functional or non - functional, and may be benign or malignant. duodenopancreatic neuroendocrine tumors are rare, although current epidemiological studies worldwide suggest an incidence rate increase. it was assessed the pathological incidence of duodenopancreatic neuroendocrine tumors for 18 years in the netherlands. standardized excerpts from pathological reports of all patients who had a diagnosis of duodenopancreatic neuroendocrine tumors from 1991 until 2009 were collected from the pathologisch anatomisch landelijk geautomatiseerd archief and reviewed. this nationwide network and registry of histopathological and cytopathological data covers 100 percent of the pathological reports in the netherlands. it was identified 905 patients with pancreatic (n=692) or duodenal (n=213) neuroendocrine tumors. the mean annual incidence rates per 1,000,000 persons over 1991 to 2009 were 2.54 for pancreatic and 0.81 for duodenal neuroendocrine tumors. the endocrine pancreas is central in the physiopathology of diabetes mellitus. nutrients and hormones control endocrine pancreatic function and the secretion of insulin and other pancreatic islet hormones. although the pancreas is not usually considered as a target of steroids, increasing evidence indicates that sex steroid hormones modify pancreatic islet function. the biological effects of steroid hormones are transduced by both, classical and non - classical steroid receptors that in turn produce slow genomic and rapid non - genomic responses. in a review, it was focused on the effects of sex steroid hormones on endocrine pancreatic function, with special emphasis in animal studies. to determine if serotonin production by pancreatic endocrine neoplasms is associated with the pancreatic duct stenosis seen in patients with stenosis that is out of proportion to the size of the tumors seen on computed tomographic images an institutional approval was obtained for a study. tissue microarrays constructed from 47 pancreatic endocrine neoplasms from the institutional tissue bank served as controls. only one of the six study patients had a large dominant tumor (4 cm in the pancreatic head). four of the six pancreatic endocrine neoplasms with associated pancreatic duct stricture had prominent stromal fibrosis. serotonin immunoreactivity was present in five (83 %) patients, and this labeling was strong and diffuse in the four patients with prominent fibrosis. by contrast, stromal fibrosis was minimal in the nonimmunoreactive case. only three (6 %) of the 47 control pancreatic endocrine neoplasms were immunoreactive for serotonin. it was concluded that these data suggest that serotonin produced by pancreatic endocrine neoplasms may be associated with local fibrosis and stenosis of the pancreatic duct. clinicians should be aware that small pancreatic endocrine neoplasms can produce pancreatic duct stenosis resulting in ductal dilatation and/or upstream pancreatic atrophy out of proportion to the size of the tumor. it was presented an update on molecular and clinical genetics of solid tumors associated with the various multiple endocrine neoplasias (men) syndromes. men type 1 (men1) describes the association of pituitary, parathyroid, and pancreatic islet cell tumors with a variety of many other lesions. men type 2 (men2) conditions represent at least four different syndromes that associate pheochromocytoma with medullary thyroid carcinoma, hyperparathyroidism, and a number of other manifestations. other pheochromocytoma - associated syndromes include von hippel - lindau disease ; neurofibromatosis 1 ; the recently defined paraganglioma syndromes type 1, 3, and 4 ; carney - stratakis syndrome ; and the carney triad. carney - stratakis syndrome is characterized by the association of paragangliomas and familial gastrointestinal stromal tumors. in the carney triad, patients can manifest gastrointestinal stromal tumors, lung chondroma, paraganglioma, adrenal adenoma and pheochromocytoma, esophageal leiomyoma, and other conditions. the carney complex is yet another form of men that is characterized by skin tumors and pigmented lesions, myxomas, schwannomas, and various endocrine neoplasias. to identify gene expression alterations associated with insulinoma formation and progression in 2 mouse models of multiple endocrine neoplasia type 1 mice were killed at 12 or 16 months, and pancreatic islets were isolated by enzymatic and physical disruption. islets were separated by size representing control, normal, hyperplastic, and adenomous islets. rna was isolated from these islets and profiled on sentrix mouse-6 expression version 1 beadchips. one hundred and one genes that were significantly altered in hyperplastic islets and insulinomas compared with normal islets were identified. of these, 64 gene elements showed reduced messenger rna levels and 37 gene elements had increased gene expression compared with control islets. altered expression of 3 genes, namely, gata6, tspan8, and s100a8, was confirmed by quantitative reverse transcription - polymerase chain reaction, and aberrant levels of tspan8 and lmo2 protein measured by western blot correlated with the changes in messenger rna levels. it was concluded that these results suggest that alterations in gene expression of gata6, tspan8, s100a8, and lmo2 may act via novel pathways that play functionally important roles in men1-associated tumor progression. insulinomas are rare tumors with an estimated incidence of one per 250,000 person - years. most insulinomas are benign with less than 10 percent demonstrating malignant behavior, the vast majority of which occur in adults. a systemic review of the literature revealed only nine cases of malignant insulinomas occurring in children. herein, it was presented a case of metastatic malignant insulinoma in a 12-year - old child. the occurrence of this diagnosis in a child, its unusual pattern of metastases and the challenging management of severe hypoglycemia make this case worth reporting. treatment of hypoglycemia in such patients is difficult and frequently fails to respond to numerous therapeutic agents, requiring continuous dextrose infusion. the authors present the experience with yttirum-90 radioembolization in a patient with metastatic malignant insulinoma who failed to respond to distal pancreatectomy, systemic chemotherapy with capecitabine and everolimus and medical treatment with somatostatin analogues, diazoxide and corticosteroids. treatment with repeated y-90 radioembolization resulted in rapid resolution of hypoglycemic events, allowing discontinuation of dextrose infusion and hospital discharge. however, the effect of y-90 administration seems to be transient and without evidence of tumor shrinkage in imaging studies. octreotide long acting repeatable (lar) is commonly used to control the symptoms of patients with functional neuroendocrine tumors. unfortunately, most patients escape control over time and require higher lar doses or more frequent rescue therapy to remain asymptomatic. previous work has shown that body weight and monthly lar dose will significantly affect circulating plasma octreotide levels in patients undergoing therapy. to determine if other parameters change circulating plasma octreotide levels, it was prospectively studied 82 patients undergoing long - term lar therapy. multivariate analysis demonstrated that the plasma octreotide levels decrease by approximately 3.4 percent for each unit of body mass index (bmi) increase, adjusting for sex and monthly lar dose. plasma octreotide levels for females were approximately 48 percent higher than those for males, adjusting for bmi and monthly lar dose. males are estimated to require 14 mg higher monthly lar doses than females with the same bmi.	pancreatic endocrine tumours are rare tumours, and arise from the types of pancreatic cells that produce hormones. these tumours may or may not secrete hormones themselves and may or may not be cancerous (malignant). functioning tumours secrete a particular hormones which may cause various syndromes. the present article reviews the latest reports on the pancreatic endocrine tumours
a total of 106 participants were recruited by purposive sampling in order to obtain information from subjects with actual experience of handling currently used mmvf materials (table1). they were construction workers in different professions or do - it - yourself consumers aged between 18 and 65 years. forty - four building construction workers were recruited with the help of company safety officers or similar from two large building construction companies (at least 5 were young apprentices). seventeen electricians were recruited from one large building construction company and one small contractor through safety officers and through a representative from the swedish electrical contractors association. eight bricklayers from one large building enterprise and one small contractor were recruited ; one group was mostly spanish - speaking. also, 15 loose wool insulation workers from two small contractors and 8 pipe and ventilation workers from one single ventilation contractor were recruited. fourteen private consumers were recruited : 11 were customers at retail stores who were invited after they had bought insulation materials, and 3 were colleagues or friends of the authors with recent experience of performing mmvf insulation tasks. overview of the study with focus groups and structured interviews concerning skin discomfort caused by man - made vitreous fibres (mmvfs) by profession, work task, and use of different mmvf materials all had experience of working with rock or stone wool (green or grey) and with yellow glass wool. experience with white glass wool and groups with female workers, foremen or other types of personnel are shown in the notes column. company i d : a, peab ab ; b, skanska ; c, jm ab ; d, sprutab ab ; e, ab isolerservice ; f, stersj elektriska ab ; g, universialisolering fredriksson ab. an overview of types of interview, number of participants and tasks is given in table1. 1. some occupational insulation tasks : inner roof insulation (a), cutting a batt for inner room insulation (b), cutting a ventilation insulation batt (c), and installing ventilation pipe insulation (d). interviews (n = 11) were performed to test questions and gather information on working tasks and available fibrous insulation products. in total, 12 focus group sessions were performed during 20092010 : six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33).two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor.two groups of loose wool insulators (n = 15) from two contractors.two groups of electricians (n = 17) employees at a large building construction company and one small contractor. six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33). two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor. two groups of loose wool insulators (two groups of electricians (n = 17) employees at a large building construction company and one small contractor. each focus group interview lasted between 35 and 70 min, and they were conducted at the construction site or in a facility provided by the companies.) first read and reflected separately on the text, and a discussion then followed, where quotes indicating some major opinions were identified and categorized. structured interviews were performed during 20082010 : do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible.pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible. pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. the interviews were audio-recorded.occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. the main questions were developed on the basis of a literature survey, expert consultations, and experience from the pilot structured interview. the following main questions and topics (see questions a, b, and c) were discussed during the focus group interviews, and were highlighted in the structured interviews. as the number of participants in the structured interviews was small, and only covered pipe and ventilation insulators (n = 8) and do - it - yourself consumers (n = 14), as compared with the number of focus group participants (n = 73), who covered different professions, no extensive evaluation of the outcome from the structured interviews was performed. question a : which mmvf material or product in use causes the most skin discomfort ? question b : what task / working position / situation causes the most skin discomfort ? question c : where on the body do you experience discomfort most ? during the focus group interviews, we tried to avoid opinions and discussions concerning working with glass fibre - reinforced wet room boards and wet - area plasterboards. effects caused by inhalation of fibres were also outside the scope of the interviews 25,2022. the study was approved by the regional ethical review board in stockholm, sweden (epn 2009/680 - 31/2), and all participants gave their informed consent. a total of 106 participants were recruited by purposive sampling in order to obtain information from subjects with actual experience of handling currently used mmvf materials (table1). they were construction workers in different professions or do - it - yourself consumers aged between 18 and 65 years. forty - four building construction workers were recruited with the help of company safety officers or similar from two large building construction companies (at least 5 were young apprentices). seventeen electricians were recruited from one large building construction company and one small contractor through safety officers and through a representative from the swedish electrical contractors association. eight bricklayers from one large building enterprise and one small contractor were recruited ; one group was mostly spanish - speaking. also, 15 loose wool insulation workers from two small contractors and 8 pipe and ventilation workers from one single ventilation contractor were recruited. fourteen private consumers were recruited : 11 were customers at retail stores who were invited after they had bought insulation materials, and 3 were colleagues or friends of the authors with recent experience of performing mmvf insulation tasks. overview of the study with focus groups and structured interviews concerning skin discomfort caused by man - made vitreous fibres (mmvfs) by profession, work task, and use of different mmvf materials all had experience of working with rock or stone wool (green or grey) and with yellow glass wool. experience with white glass wool and groups with female workers, foremen or other types of personnel are shown in the notes column. company i d : a, peab ab ; b, skanska ; c, jm ab ; d, sprutab ab ; e, ab isolerservice ; f, stersj elektriska ab ; g, universialisolering fredriksson ab. an overview of types of interview, number of participants and tasks is given in table1. 1. some occupational insulation tasks : inner roof insulation (a), cutting a batt for inner room insulation (b), cutting a ventilation insulation batt (c), and installing ventilation pipe insulation (d). interviews (n = 11) were performed to test questions and gather information on working tasks and available fibrous insulation products. in total, 12 focus group sessions were performed during 20092010 : six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33).two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor.two groups of loose wool insulators (n = 15) from two contractors.two groups of electricians (n = 17) employees at a large building construction company and one small contractor. six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33). two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor. two groups of loose wool insulators (n = 15) from two contractors. two groups of electricians (n = 17) employees at a large building construction company and one small contractor. each focus group interview lasted between 35 and 70 min, and they were conducted at the construction site or in a facility provided by the companies.) first read and reflected separately on the text, and a discussion then followed, where quotes indicating some major opinions were identified and categorized. structured interviews were performed during 20082010 : do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible.pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible. pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. the interviews were audio-recorded.occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. interviews (n = 11) were performed to test questions and gather information on working tasks and available fibrous insulation products. in total, 12 focus group sessions were performed during 20092010 : six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33).two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor.two groups of loose wool insulators (n = 15) from two contractors.two groups of electricians (n = 17) employees at a large building construction company and one small contractor. six groups (one with apprentices only) with building construction workers at two large construction companies (n = 33). two groups of facade insulators (bricklayers) (n = 8) employees at a large building construction company and a small contractor. two groups of loose wool insulators (n = 15) from two contractors. two groups of electricians (n = 17) employees at a large building construction company and one small contractor. each focus group interview lasted between 35 and 70 min, and they were conducted at the construction site or in a facility provided by the companies.) first read and reflected separately on the text, and a discussion then followed, where quotes indicating some major opinions were identified and categorized. structured interviews were performed during 20082010 : do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible.pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. do - it - yourself consumers (n = 14) were interviewed by telephone. only persons who had purchased mmvf products and insulated within the last year were interviewed. the original intention was to perform this as a focus group interview, but, logistically, this was not possible. pipe and ventilation insulators (n = 8) at a medium - sized ventilation contractor were interviewed. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. the interviews were audio-recorded.occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. four teachers at two swedish high schools construction programmes were interviewed about their tuition concerning insulation and the use of mmvfs. occupational hygiene specialists at different construction companies, union representatives, safety officers and personnel working with occupational health care, in total 12 persons, were interviewed. their opinions were not used as a separate input, but they contributed valuable information. the main questions were developed on the basis of a literature survey, expert consultations, and experience from the pilot structured interview. the following main questions and topics (see questions a, b, and c) were discussed during the focus group interviews, and were highlighted in the structured interviews. as the number of participants in the structured interviews was small, and only covered pipe and ventilation insulators (n = 8) and do - it - yourself consumers (n = 14), as compared with the number of focus group participants (n = 73), who covered different professions, no extensive evaluation of the outcome from the structured interviews was performed. question a : which mmvf material or product in use causes the most skin discomfort ? question b : what task / working position / situation causes the most skin discomfort ? question c : where on the body do you experience discomfort most ? during the focus group interviews, we tried to avoid opinions and discussions concerning working with glass fibre - reinforced wet room boards and wet - area plasterboards. effects caused by inhalation of fibres were also outside the scope of the interviews 25,2022. the study was approved by the regional ethical review board in stockholm, sweden (epn 2009/680 - 31/2), and all participants gave their informed consent. the most typical comments and opinions concerning the three main questions in the focus groups are shown in tables24, separately for different professions or tasks. the comments are listed as direct citations (originally in colloquial swedish) as recorded during the focus group sessions. focus group interviews concerning work with man - made vitreous fibres (mmvfs) and skin discomfort ; some of the most typical comments and opinions concerning question a, which mmvf material or product in use causes the most skin discomfort? focus group interviews concerning work with man - made vitreous fibres and skin discomfort ; some of the most typical comments and opinions concerning question b, what task / working position / situation causes the most discomfort? focus group and structured interviews concerning work with man - made vitreous fibres and skin discomfort ; some of the most typical comments and opinions concerning question c, where on the body do you experience discomfort most? the structured interviews with do - it - yourself consumers showed that most informants (11/14) reported some discomfort. one person stated that the yellow fibrous material was most troublesome (question a) ; 1 felt that inner roof insulation was troublesome (question b). the majority of the informants (11 of 14) said that the skin discomfort was felt most on the hands, forearms, and neck (question c), and occasionally in the eyes. one person who reported having had atopic eczema did not report any skin problems related to mmvfs. two of the pipe and ventilation insulators (2/8) reported some skin discomfort, and all of them worked mostly with green / grey fibrous products. some other often discussed topics are shown in tables5 and 6. some selected and typical comments on topics often discussed during focus group or structured interviews on work with man - made vitreous fibres and skin discomfort some more selected and typical comments on topics often discussed during focus group or structured interviews on work with man - made vitreous fibres and skin discomfort this study shows that currently used mmvf products still cause skin discomfort for workers and do - it - yourself consumers. skin irritation caused by mmvf materials the former hazard classification of mmvfs as skin irritants was removed in 2009, but without scientific evidence for the change. the overall aim of the present study was to update and increase our knowledge about the skin effects of mmvf products, focusing on products used in sweden around 2010. in this study using focus groups and structured interviews, we hoped to receive answers to the following questions : which current mmvf material or product in use causes the most skin discomfort?, what task / working position / situation causes the most skin discomfort?, and where on the body do you experience discomfort most? in all, 106 participants contributed with their opinions and experience in focus groups and by structured interviews. the number of focus group participants is quite large as compared with many other focus group studies 2329. participants were building construction workers, bricklayers, loose wool insulators, electricians, pipe and ventilation insulators, high - school teachers, and do - it - yourself consumers. we did not interview workers occupied in disassembling mmvf insulation materials, as this study dealt with current mmvf products. neither did we interview workers at large industrial ventilation companies, workers manufacturing mmvf materials, or people working with other types of glassy (amorphous and vitreous) fibres 24. with regard to the first question, which mmvf material or product in use causes the most skin discomfort?, the most common answer or comment was that yellow glass fibre insulation products caused the most skin discomfort (those available in sweden during 20092010). the loose wool insulators, who seldom used yellow glass fibre wool, considered the green / grey rock wool to cause more skin discomfort than the newer white glass fibre wool. yellow glass fibre insulation products were quite uncommon at the time in the two large construction companies engaged, where more of the grey / green rock wool products were used. yellow glass fibre insulation products were the most frequently displayed products at building materials stores. white glass fibre wool (described as soft as cotton by some of the construction workers) was used by loose wool insulators only. we have not found any scientific report supporting or contradicting this statement. in response to the second question, what task / working position / situation causes the most skin discomfort?, building construction workers and electricians jointly stated that working with the hands above the head (insulation of or electrical work in ceilings or narrow lofts) was the worst scenario, because loose fibrous materials fall down onto the face and body. bricklayers indicated the same, describing the worst situation as lifting the insulation boards up to the next floor or to the frame. loose wool insulators indicated no special worst working situation apart from crawling in attics and in narrow lofts. for the third question, where on the body do you experience discomfort most?, the most common answer was on the neck (inside the collar), wrists, forearms, and face, regardless of profession or work task. this is in agreement with previous studies 7,9,10,14,15. during discussions about skin effects, inhalation discomfort, which was caused by the dusty environment according to the participants,, we asked the participants not to discuss this topic, and we promised to give information about health effects and legislation when the interview was over. focus group interviews have been used for studying the effects of hazardous fibres, for instance in a study aiming to identify important information gaps concerning asbestos exposure assessments 30. in a study in libby, montana, united states, focus group methods were used in a community experiencing a disaster caused by widespread asbestos exposure 31. in both medical research and occupational and public health studies, focus groups have been used frequently to obtain a variety of information from patients or workers 2326,32,33. focus groups have been used in dermatology to obtain data concerning experiences of many conditions, for example pruritus in psoriasis 27, dermatological treatments 28,29,34, and hidradenitis 35. the moderator in the present study (c.m.) had no previous knowledge concerning the technical aspects of the problem ; such knowledge might have helped to improve the questions with an explicit description of the actual working tasks. a technical expert (l.l.) participated, and answered questions about fibre characteristics and legislation during and after the formal part of the interview. could the same answers to the three main questions (a, b, and c) be obtained by some other technique ? the use of simple structured interviews alone would have been possible, but the depth in the answers would have been lost. we believe that the focus group interviews enhanced the reliability of the outcome, particularly for question a (which mmvf material or product in use causes the most skin discomfort?). the interview technique allowed follow - up questions and probing of the information given during the interview, for example when uncertainties were raised about the fibrous material discussed. we also believe that the purposive sampling methods used for recruiting the participants did not influence our findings. another interesting finding obtained from the interviews was that the majority of participants agreed that warm and sweaty situations enhanced skin discomfort independently of tasks. explanations may be the wearing of less clothing and less protective equipment, or the fact that wet / moist skin might increase the deposition of fibres on the skin. the most frequently reported method for removing fibres from the skin after work was to take a shower. most thought that cold water was better than hot water, so that the pores can close and thus stop fibres penetrating ; some thought that hot water opens the pores and lets the fibres out. however, all claimed that showering does not completely remove fibres. accounts of severity and duration of the discomfort ranged from slight itching to sleepless nights. it was not always easy to distinguish opinions and perceptions derived from experiences of currently used materials from those derived from previous exposure to older products. it was quite clear, however, that those with experience of both new and older materials thought that previous mmvf materials caused more severe skin discomfort. occasionally, some new and mmvf - free insulation products were mentioned, indicating that some of these also caused skin discomfort. we did not encourage further discussion on such materials, however, as they were outside the scope of this study. only pipe / ventilation workers and loose wool insulators were normally exposed to different mmvf fibres on a daily basis. the other groups were exposed on separate occasions depending on tasks, for up to a few days a month. electricians and teachers did not themselves work with mmvf materials, but were exposed through others handling them. it is interesting that building construction apprentices performed insulation tasks more often than senior workers, who, we believe, preferred to avoid these tasks. they acknowledged the distribution of the work, but also considered the training exercise to be part of their apprenticeship. protective equipment was generally limited to gloves and long sleeves, depending on room temperature and whether the insulation task was performed indoors or outdoors. the use of eye protectors was compulsory in one company, but this was viewed with some scepticism by some others. others assumed they it would be easy to become accustomed to eye protectors, and saw the benefits of not getting fibres in the eyes. in provisions and general recommendations from the swedish work environment authority on synthetic inorganic fibres ! afs 2004:1 37 ], it is written that with work generating large quantities of dust, protective clothing shall be used. this clothing shall cover sensitive areas of skin, e.g. the neck and forarms. also, breathing protectors shall be used where it is not technically possible to keep the atmospheric concentration of mmvfs at an acceptable level. the fibre products used by participants in the present study were characterized concerning chemical composition and fibre size [there is some indication that coarser fibres of mmvf can cause more severe skin discomfort 15 ]. the products did not differ markedly from what has been reported previously (supporting information doc.s1). yellow glass fibre materials normally had slightly higher fibre diameters (3.26.4 m) than fibres made of rock or stone (1.73.9 m). the new white glass wool used by loose wool insulators had a fibre diameter between 1.9 and 2.3 m, similarly to rock or stone wool. to the best of our knowledge, no data concerning changes in the use of fibrous material during recent decades in sweden have been published. additives may be present in fibrous materials (antistatic agents, anti - mould agents, anti - dust agents, binders, etc.), and their use may have changed over time. however, the possible influence of additives was outside the scope of this study. in 2009 the old risk phrase r38 (irritating to the skin) and the new hazard statement h315 (causes skin irritation) were removed. the decision was not unanimous, and some member states recommended that the classification should be maintained (on the basis of studies published between 1950 and 1980). one reason for the present study was to generate new knowledge, so that classification and labelling can be based on currently used mmvf materials. a high - school construction programme teacher gave the opinion that female students performed the insulation task more accurately and better. he also mentioned that they carried the insulation boards further away from the body than male students, who often carried more boards and were thus in closer contact with them. the authors perceptions from the interviews were that workers and consumers own knowledge on possible skin effects of mmvf fibres was quite low. this often led to speculations and unfounded ideas about risks associated with the materials, as well as notions of mechanisms and treatment. our study shows that mmvf materials still cause skin discomfort for workers and do - it - yourself consumers. on the basis of the outcome of this study, a controlled provocation study with mmvf materials is currently being performed in a human exposure chamber 39,40. we suggest that updated knowledge about skin discomfort and other skin effects resulting from work with mmvf materials should be taken seriously, and should possibly result in a renewal of their classification and labelling as skin irritants. additional supporting information may be found in the online version of this article fibre size and chemical composition of some current insulation mmvf products in sweden.	backgroundman - made vitreous fibres (mmvfs) are used in products for insulation and as reinforcement in materials. contamination of the skin may arise through direct or indirect contact, and from the deposition of airborne fibres. the scientific basis regarding the effects on skin of mmvfs dates from 19701980.objectivesto investigate whether currently used insulation mmvf products still cause skin discomfort.methodsfocus group interviews and structured interviews were performed among workers engaged in insulation tasks and among do - it - yourself consumers with a recent experience of mmvf products.resultsa majority of interviewees experienced skin discomfort when handling mmvf products. complaints caused by traditional (yellow) glass fibre products were more severe than those caused by products of rock or slag wool fibres. the wrists, forearms, neck and face were the locations where the skin was most affected. the situations causing problems varied between occupational tasks, but working with the hands over the head or in narrow spaces were described as the worst situations. building construction apprentices performed insulation tasks more often than senior workers.conclusionsmmvf insulation products do still cause skin discomfort. updated knowledge about people s experiences of work with such products should influence legislation.
the guglielmi detachable coil (gdc) is the first detachable coil approved by the food and drug administration of the united states for treating intracranial aneurysms4,7). although the gdc has been demonstrated as safe and effective for endovascular treatment of intracranial aneurysms, there have been several reports concerning thromboembolic events related to gdc use due to air bubbles and electrothrombosis when it is electrolytically detached. han.4) reported that the electrolytic detachment mechanism of the gdc generated gas bubbles and thrombi from the coil detachment zone and this phenomenon may be a potential cause for thromboembolic complications during treatment of cerebral aneurysms. the target detachable coil (target ; stryker, fremont, target coils have the same electrolytic detachment mechanism as a gdc and we had expected the new advanced coils would generate less bubbles than the conventional gdcs during the detachment. unfortunately, according to our in vitro experiments, the target coils generate larger - sized and more considerable number of air bubbles than the gdcs (fig. recently, lee.8) reported that the air bubbles were larger - sized and generated most frequently in target coil system than other coils through their in vitro experiments. introduction and detachment of the coils may take a share of the procedure after manipulation of microcatheter with microwire. our authors were wondering the air bubbles from the coils matter seriously to patients underwent coil embolization for intracranial aneurysm. however, there was no study for clinical correlation of the air bubbles as thromboembolic source although some authors gave a warning for formation of air bubbles from the electrolytic detachable coils, especially the gdcs or target coils. the purpose of this study was to retrospectively evaluate and compare the incidence of diffusion - weighted imaging positive lesion (dwil)-as practical obtainable finding - between two electrolytic detachable coils (gdc versus target coil) for treating unruptured intracranial aneurysm (uia). from march 2011 to december 2011, we performed endovascular embolization for intracranial aneurysms using one or more target coils for 99 cases in 94 patients. there were 24 men and 70 women (age, 59.310.0 years ; range, 40 - 82 years). eighty - one (81.8%) cases were located in the anterior circulation, including the internal carotid artery, 31 ; posterior communicating artery, 16 ; middle cerebral artery, 14 ; anterior communicating artery, 13 ; anterior cerebral artery, five ; and 18 cases in the posterior circulation, including seven vertebral artery, three posterior inferior cerebellar artery, one anterior inferior cerebellar artery, six basilar artery, and one posterior cerebral artery. the average size of the aneurysms was 6.563.30 mm (range, 2.6 - 24.6 mm). we performed stent - assisted coil embolization in 18 (18.2%) and balloon - assisted in six (6.1%) of 99 cases. the historical control group included 132 consecutive patients, harboring 135 aneurysms, who underwent coil embolization using one or more gdcs during the period from january 2010 to march 2011 before the target coil was launched. inclusion criteria were : 1) uia, 2) one or more gdc or target coils in use with or without other coils, 3) diffusion - weighted image (dwi) examination within 24 hours after coiling, and 4) coiling performed without a balloon or stent. among 222 patients, ninety patients (92 cases) met the inclusion criteria. the patients were subdivided into a gdc - treated group (n=44) and a target - coil treated group (n=48) (fig. saline and contrast medium were heparinized (1000 iu/100 ml), and both a guiding catheter and a microcatheter were placed for a continuous heparinized drip. all patients were administered systemic heparinization (loading dose of 3000 iu followed by 1000 iu / hour) during the procedure. the results of the embolization were classified into complete (no filling of aneurismal rests without a neck remnant), incomplete occlusion (saccular contrast material filling) and neck remnant. all procedure records were reviewed for the number of gdcs or target coils and the total other detached coils. magnetic resonance imaging was performed within 24 hours after the coiling procedure in all cases to check for any silent thromboembolic events, regardless of neurological changes. imaging was performed with 1.5-t system (signa hdxt ; ge medical systems, milwaukee, wi, usa) and a 3.0-t system (magnetom verio ; siemens, erlangen, germany). all mr images were reviewed by two neurointerventionalists (b.- s.k, m.j.k). if dwi abnormalities were detected, their number and location were recorded. aneurysm - related lesions was defined as finding location in the vascular territory downstream from the treated aneurysm, in another cases they were checked as aneurysm - unrelated lesions (i.e., anterior circulation versus posterior circulation, ipsilateral hemisphere territory versus contralateral hemisphere territory except lesion of anterior communicating artery including both territories). the demographic characteristics of the aneurysms, coiling results, and lesions on dwi were compared between the gdc and target coil groups using a contingency table or mean comparison. correlations between multiple dwils (> 10 dots) and distinguished risk factors in the groups were analyzed by logistic regression. we conducted partial correlations analysis to show the statistical differences of continuous variable between dwils and each coil ratio, and adjusted by confound factors. all statistics were performed using spss 12.0 (spss inc., chicago, il, usa), and a p - value 10 dots) and distinguished risk factors in the groups were analyzed by logistic regression. we conducted partial correlations analysis to show the statistical differences of continuous variable between dwils and each coil ratio, and adjusted by confound factors. all statistics were performed using spss 12.0 (spss inc., chicago, il, usa), and a p - value < 0.05 was considered significant. dwi was performed in all 90 patients (92 cases ; two patients underwent coil embolization for aneurysms located on different sites) after the coil embolization. however, all patients with dwil were asymptomatic except for one patient who underwent coil embolization using three gdcs for an unruptured posterior inferior cerebellar artery (pica) aneurysm and suffered headache and dizziness after the procedure. dwils were detected in 31 (70.5%) of 44 cases in the gdc group. the average number of dwils was 5.08.7 (range, 1 - 40) in aneurysm - related territory and 0.81.2 (range, 1 - 4) in aneurysm - unrelated territory. in the target coil group, the number of lesions was 2.15.4 (range, 1 - 32) in aneurysm - related territory and 1.43.1 (range, 1 - 14) in aneurysm - unrelated territory (table 2). the target coil group tended to reveal fewer aneurysm - related dwils, however, it was not significant. no association between multiple dwils with high signal dots over 10 and any of the risk factors (involving group, aneurismal size and occlusion results) was found (table 3). a ratio of the gdcs in total coils was 45.8% (range, 10.0 - 100%) and the target coils were 67.8% (range, 15.4 - 100%) in each the gdc / target coil group. there was no significant correlation between the number of dwils and a ratio of the coils in gdcs or target group (fig. since the endovascular treatment using gdcs was first introduced in 1991, several coil materials have been developed and used for treating intracranial aneurysms. the safety and efficacy of the endovascular technique using coil materials in particular, the coils improved rapidly, which made it possible to improve anatomic and clinical outcomes of endovascular coiling9,11,12,19). although the treatment has world - wide acceptance for intracranial aneurysms, thromboembolic complications still remain as procedure - related main events10,14,17). several studies have shown that thromboembolic events including stroke and transient ischemic attacks occur at a rate of 2.4 - 28%3,10,14). those series defined complications as new focal neurological deficits, mentality changes, or abnormal findings detected on a post - procedural imaging study. pelz.14) reported that thromboembolic events associated with conventional coil embolization using gdcs occurred in 28% of cases, with permanent deficits affecting approximately 5% in their study group. rordorf.16) detected embolic infarctions that were related to the coil placement procedure in eight (61%) of 14 patients, and they were all asymptomatic. soeda.18) reported that dwi showed high signal lesions in 40 (61%) of 66 patients, with 16 patients (40%) eventually developing neurological symptom. in our series, a symptomatic thromboembolism occurred in only one (1.1%) of 90 patients ; however, new high signal lesions on dwi were detected at the aneurysm - related territory in 61.1% (55/90) of patients without neurological symptoms, suggesting silent thromboembolic events. thromboembolic events may be caused by thrombus formation from the delivery system, endosaccular thrombosis, protruding coils into the parent vessel, or other supporting devices for endosaccular stabilization of the coils5). some studies have documented that balloon- or stent - assisted coil embolization increases thromboembolic risk, but this remains controversial1,5). several authors have reported that thromboembolic events are associated with gdc embolization and the use of dwi6,17,18). among the causative factors, rordorf.16) suggested that air bubbles in the fusion solutions may act as emboli, and padolecchia.13) and han.4) investigated the role of electrothrombosis in embolizations performed with gdcs. these studies suggest that thrombi might form during the procedure, as well as immediately after detachment of the coils. the clots that form during the detachment process around the detachment zone or adjacent to the coils may be dislodged during withdrawal of the delivery wire, repositioning of the microcatheter, or repetitive endosaccular introduction of the coil1). the gdc, which is the most commonly used electrolytic detached coil system, has frequent thromboembolic complications due to its detachment system4,13,16). the target coil, which is a new version of the gdc, has been in use since early 2011. several changes and improvements have been implemented over the conventional gdcs. however, according to our in vitro experiment and microscopic examination of target coils, air bubbles were still observed when it is electrolytically detached (fig. 1). in addition, the air bubbles from detach zone of the coils were considerable amount more than the conventional gdcs on the contrary our expectation. in our results, there was no significant correlation between the ratio of gdc and/or target coils and detected high signal dots on dwi (p=0.704). the number of electrolytic detachable coils was not significant for the number of dwil between the two groups. considering these results adjusted by confounding factors (aneurismal size and occlusion results), we suggest that the electrolytic detached coil system may be not a significant factor for thromboembolic events. nevertheless, as electrothrombosis during embolization occurs with gdcs4,13), our results also revealed many cases with dwil. however, the relationship between the electrolytic detachable system and dwil was unclear, and all cases were silent events except one case with mild neurological deterioration. our author reviewed the results in only single gdc (n=5), target (n=17) or other kind (n=6) of coils - using 28 cases in spite of a few cases. there were also no significant correlation between the number of coils and the dwils (4.23.7 ; 1.52.1 ; 0.70.8, p=0.104). no significant difference was observed between the target coil group and the gdc group for aneurysm - related dwil. manufacturers officially announced that the target detachable coil is an advanced model of the gdc coil detachment system. however, target detachable coils still have the electrolytic detachable system and may be not superior to previous generation gdcs for electrothrombosis during conventional coil embolization. as high signal dots in dwi were also detected in the aneurysm - unrelated territory, dwils were not always located in the vascular territory of a parent artery of the aneurysm. lesions can develop from collateral flow through the anterior or posterior communicating artery or from other sites before the coil embolization procedure2,17). the choice of coil material was likely to be influenced by practitioner 's preference at that time during procedure. in addition, we acknowledge that our study is a retrospective analysis with a selection bias because of inclusion of the results in cases used few gdcs or target coils - even if at least the coil was used - to our data. it may be unreasonable to draw exactly the effect of the electrolytic detachable system on the rate of ischemic events on dwi because some other factors (e.g., catheter and coil manipulation, procedural time and experience of practitioner) could affect the environment. therefore, in this study, to exclude the other factors, relatively narrow inclusion criteria (unruptured aneurysm, no supporting devices and simple technique) were applied. moreover, total target detachable coils were used more than one third and the semi - results only involving the gdc or target coils more than fifty percentage were parallel to the overall result. therefore, our results suggest that it may support in a degree the relation between electrolytic detachable coils and dwils. however, for the more significant data coincided with our purpose, prospective study should be performed using unitary detachable coils under unitary environment except other factors. the choice of coil material was likely to be influenced by practitioner 's preference at that time during procedure. in addition, we acknowledge that our study is a retrospective analysis with a selection bias because of inclusion of the results in cases used few gdcs or target coils - even if at least the coil was used - to our data. it may be unreasonable to draw exactly the effect of the electrolytic detachable system on the rate of ischemic events on dwi because some other factors (e.g., catheter and coil manipulation, procedural time and experience of practitioner) could affect the environment. therefore, in this study, to exclude the other factors, relatively narrow inclusion criteria (unruptured aneurysm, no supporting devices and simple technique) were applied. moreover, total target detachable coils were used more than one third and the semi - results only involving the gdc or target coils more than fifty percentage were parallel to the overall result. therefore, our results suggest that it may support in a degree the relation between electrolytic detachable coils and dwils. however, for the more significant data coincided with our purpose, prospective study should be performed using unitary detachable coils under unitary environment except other factors. our study has shown a relatively high prevalence of dwil that occurred after coil embolization for uia using gdcs / target electrolytic detachable coils. no difference in the incidence of dwil was observed between the gdc and the newly developed target coil in a similar environment. our results suggest that electrolytic detachment of the coil may not be a significant contributor to the incidence of dwil during endovascular coil embolization.	objectivethe purpose of this study was to retrospectively evaluate and compare the incidence of diffusion - weighted image (dwi) lesions between the guglielmi detachable coil (gdc) and the target coil for treating unruptured intracranial aneurysm.methodsfrom 2010 to 2011, consecutive 222 patients with an intracranial aneurysm underwent coil embolization. inclusion criterias were : 1) unruptured intracranial aneurysm, 2) one or more gdc or target coils used with or without other coils, 3) dwi examination within 24 hours after coiling, and 4) coiling performed without a balloon or stent.resultsninety patients (92 cases) met the inclusion criteria. dwi lesions were detected in 55 (61.1%) of 90 patients. in the gdc group (n=44), dwi lesions were detected in 31 (70.5%). the average number of dwi lesions was 5.08.7 (meansd ; range, 1 - 40) in aneurysm - related territory. in the target coil group (n=48), dwi lesions were detected in 24 (50.0%). the number of dwi lesion was 2.15.4 (range, 1 - 32) in aneurysm - related territory. there was no significant correlation between a number of coils and dwi lesions. no significant differences were also observed in the number of dwi lesions in each group.conclusionthe gdc and target coils, which have an electrolytic detachable system, showed no differences in the incidence of dwi lesion.
systemic lupus erythematosus (sle, lupus) is a complex inflammatory illness with varied clinical presentations, providing management and assessment challenges to patients and practitioners [1, 2 ]. sle patients live with daily background symptoms and only a few obtain symptom - free periods. a life with lupus usually has an unpredictable pattern of symptom quiescence and periods of exacerbation commonly known as flares. this paper explores lupus flares from the lived experience of 101 australian female sle patients. a novel flare definition, reflecting a chronic illness experience of symptom stability, punctuated by sustained exacerbation over a specified time period, was incorporated. the paper documents findings of patient - perceived flare symptoms, triggers, and management strategies adopted to alleviate symptom exacerbations. the study is part of the initial development phase of a wider cohort study exploring environmental determinants of sle flare events, with data collected retrospectively over a whole year to incorporate potential seasonal influences upon patient wellbeing. study goals were todescribe the patient experience of lupus flares, their features, and attributions of possible triggers and management;compare these flare characteristics between patients meeting american college of rheumatology (acr) classification criteria and those classified as having borderline lupus, falling within the undifferentiated connective tissue disease category as lupus - like disease.treating specialists often use validated tools (e.g., bilag, sledai, selena, and slicc) [68 ] to monitor and objectively assess overall and organ - specific sle activity and damage. however, despite recent consensus definition of lupus flare, differences between clinician and patient determination of flare events occur. these arise when the definition of flare is based solely on biomarker measures and treatment changes, without sufficient recognition of an individuals ' perception of quality of life (qol) impact [1, 1113 ]. flare assessment is also complicated by (i) determination of timing ; (ii) assumptions that clinician review occurs at each flare time - point ; (iii) mistiming of diagnostic assessments ; (iv) reluctance of patients to inform clinicians of symptom changes ; and (v) patient use of self - management strategies. severe flare events are often well supported by investigative confirmation, but determining milder flares with subtle symptom changes still proves difficult. describe the patient experience of lupus flares, their features, and attributions of possible triggers and management ; compare these flare characteristics between patients meeting american college of rheumatology (acr) classification criteria and those classified as having borderline lupus, falling within the undifferentiated connective tissue disease category as lupus - like disease. presentation and definition of a lupus flare from a patient perspective is not readily explored. it is subject to the patient 's perception of the illness impact as a reflection of their health values and priorities. social support and capacity to self - manage, adopting necessary lifestyle changes, are also important considerations. therefore, exploring lupus flares from the patient 's lived experience is an important research area that will increase understanding of lupus health impacts and treatment efficacy [13, 1517 ]. the study was a retrospective analysis of lupus flare characteristics within a large regional area of australia. participant demographic, lifestyle, and medical history was also collected via posted study specific questionnaires. participant sle management and flare history over a 12-month period were explored during a clinic appointment with the medical researcher where study questionnaires were collected and participants were interviewed via a structured interview process to document their perceptions of symptom activity (flare) and management over the previous 12 months. institutional review and approval according to the declaration of helsinki 2008 revision were undertaken and received. sle patients of the autoimmune resource and research centre (arrc) and private immunology clinics in the hunter / central coast regions of nsw, australia, were invited to participate. potential participants were drawn from clinic databases using disease codes sle or lupus as an identifier of potential study recruits. all identified patients were posted a letter of study invitation, along with study information and consent forms. written consent was obtained for all aspects of the study. initial criteria included all identified sle persons aged 1880 years, but, due to low numbers of male respondents, the final study was limited to females. health records of responding patients were reviewed to confirm sle diagnosis and verify self - reported illness onset information, symptoms, disease history characteristics, and management plans, especially pharmaceutical usage. evidence of a positive symptomatic improvement with a pharmaceutical challenge involving either a 3-month regime of hydroxychloroquine or 2 weeks of prednisolone was noted and used to identify a subgroup of borderline sle patients. eligible participants met inclusion criteria and acr sle classification (sle acr 4 +) ; a second comparator group was also included if 3 of the acr criteria plus a positive pharmaceutical challenge were documented within the health record. the comparator group, borderline sle acr 3 + 1, falls within the acr category for undifferentiated connective tissue disease. all participants attended a scheduled appointment to collect study questionnaires and also to document their lived experience of sle symptom exacerbations, features, triggers, and management techniques, as well as perceptions of their flare frequency for the preceding 12 months. this flare assessment was undertaken via valid qualitative techniques with the use of a structured interview method. due to the retrospective nature of the study and the lack of standardized clinical assessment tools documented within health records, the definition was chosen because (i) it was easily understood ; (ii) it contained reference to a chronic illness displaying periods of symptom stability, quiescence, and exacerbation ; (iii) it recognised symptom background levels ; and (iv) it best allowed exploration of the patient experience. flare was defined as : the appearance of a new clinical sign / symptom or the clinical worsening of a previous sign / symptom that had been stable for at least the previous 30 days and which persisted for a minimum of 24 hours.the flare interview was conducted by the same medical researcher without clinician involvement. each interview was in a verbatim structured format commencing with reading of the definition and a scripted clarification example. fifteen questions exploring the patients ' flare experience followed, without participant response prompting or discussion. all responses were concurrently documented by the researcher to allow free communication flow and to reduce recall bias. structured interview format and question proforma used in the study have been provided as supplementary information available online at http://dx.doi.org/10.1155/2014/816729. the appearance of a new clinical sign / symptom or the clinical worsening of a previous sign / symptom that had been stable for at least the previous 30 days and which persisted for a minimum of 24 hours. calculation of total counts of flare days and collection of data related to other perceived flare risk factors have been outlined in a previous paper. data were analysed with the use of inductive thematic analysis to ensure complete data richness. separate analysis was performed for symptoms experienced in flares, identified triggers to flares, and management strategies for alleviating flare symptoms. to examine differences between assigned sle groups, flare groups, and independent variables, continuous variables were analysed via one - way anova, and categorical variables were analysed via fisher 's exact test of independence. all significant p values (0.05) were noted, with analysis performed using stata v11.0 (statacorp lp, college station, texas, usa). documented evidence of 4 out of 11 acr criteria was confirmed in 83 participants, and 21 participants displayed 3 out of 11 acr criteria plus a positive pharmacological challenge. demographic data for separate and combined groups of the remaining 101 participants are shown in table 1. differences between groups were found for medical review periods with the borderline sle reporting shorter periods. group also had a larger proportion of participants not exceeding educational training past 15 years of age. higher proportions of some acr criteria were found in the sle group : malar rash (71.3%, p = 0.01), photosensitivity (53.8%, p = 0.01), oral and nasal ulcers (36.3%, p = 0.00), and renal (46.3%, p = 0.03) and haematological disorders (48.8%, p = 0.02). immune therapy medication (itm) use was similar for each group ; however, vitamin d supplementation was less in the borderline sle group. combined participant group mean age was 48 (12.9) years with mean disease duration of 7.7 (6.7) years. illness comorbidity was reported in 65.4%, with 61.4% of participants reporting more than one autoimmune illness. there was a high representation of participants reaching advanced or vocational level and above (57.5%) educational levels. a minority (18.75%) reported a ses of below australian average. flare frequency information and reported symptoms along with relevant interview questions are presented in table 2. self - reported individual flare events ranged from 0 to 52 flares (mean 6.8). total flare days ranged from 0 to 240 days (mean 29.9 38.8) with 2 participants reporting major adverse renal events resulting in prolonged hospitalization. a majority of participants (63, 62.4%) reported a decreased flare frequency since implementation of medical management and a further 24 (23.8%) spontaneously created a 3rd response category, remained the same. overall, general health improvement was recorded for 51 (50%) participants and deterioration was recorded for in 29 (28.4%). despite having a nonflaring year, 3 (25%) of the 12 nonflaring participants current pharmacological immune therapy medications (itm) for the cohort varied and included methotrexate, plaquenil, prednisolone, imuran, intravenous immunoglobulin, dapsone, and cellcept. it is of note that 8 (67%) of the 12 nonflaring participants did not currently take steroidal based medications and, of these, 1 was not on any itm. in comparison, 42 (47%) flare group participants did not take steroids and 15 (17%) were not on any itm. the broad spectrum of individual clinical presentation is reflected by the variety of symptom exacerbation descriptors reported. in total 376 symptom exacerbations were reported with a mean of 3.8 (2.2 : range 010) different symptoms for each participant. the 376 symptoms were thematically grouped into 29 separate codes with the 4 most frequent flare symptoms being joint and muscle pain (73.3%), fatigue (65.4%), skin rash (29.7%), and headaches (23.8%). stress and anxiety, which are often linked as known flare triggers [3, 11, 22 ], were reported as flare symptoms along with depression and mood disturbance (3%). no significant difference between sle groups was found for flare counts, frequency, general health vas scores, or flare symptoms. the unpredictable nature of symptom exacerbation and flares can impact on long term health and quality of life. this can lead to participants ' awareness of subtle changes in symptoms and drawing links between flare triggers and changes in daily activities and agents. within our cohort, 29 general trigger themes were identified from a total of 292 individual triggers. these are presented in table 3. a majority of participants (96%) identified triggers with a mean of 3 (1.5, range : 06) different triggers identified by individual participants regardless of flare group. a small proportion of participants (4%) that reported flare events did not identify any trigger to these events. the results were consistent with frequently cited triggers including stress (54, 53.5%), becoming run down (47, 37.6%), uv exposure (37, 36.6%), and infection (16, 15.8%), as well as triggers relating to physical responses to temperature, work, and sleep deprivation. environmental influences on symptoms, in particular temperature sensations, included themes of overheating (20, 19.8%), weather / changes in temperature (20, 19.8%), and cold temperature interestingly, exposure to strong chemicals, specifically, bleach and pesticides were reported by 8 (7.9%). triggers associated with working and being upset are well - established associations in sle which were also recorded within our cohort. the theme of work was represented by impact from workloads, deadlines, difficult bosses, hard jobs, and work hours (early and long). physiological responses to changes in medications were reported in 17% of the nonflare group and only 6% of the flare group as an exacerbation - contributing factor. to examine possible correlations between identified triggers, reported trigger themes and symptom exacerbations of the 89 flare group participants were cross - referenced in a matrix format, presented as table 4. this matrix, whilst not documenting single reports of symptoms, does clearly show that flare symptoms of joint and/or muscle pain, fatigue, rash (malar, photosensitive and other), and headache were almost universally linked to the 15 most frequently reported trigger themes. the symptom of swelling (representing swollen finger, swollen hands or feet, swollen joints, and fluid retention) also featured as common cross - links to many triggers but was not linked to weather / sudden temperature changes or cold temperatures. predictably, these 2 triggers along with doing too much and infection were cross - linked to symptoms in participants with known raynaud 's phenomenon comorbidity. it is also of interest that 4 of the top 6 triggers identified in this study accord with established and known sle flare triggers of stress, overexertion, uv exposure, and postinfective episodes. in addition to these our study also identified new potential environmental triggers to symptom exacerbation : sudden weather changes and overheating. this suggests that thermoregulatory dysfunction in people with sle could have a role in disease activity and symptom changes. participant responses, tabulated within table 5, show that 50% of participants change their steroidal medications to manage exacerbations, with 14% making these changes without medical advice. one participant, despite not taking steroids as part of her regular itm, reported self - administration of steroids when flaring. i 've been living with this for a long time and i know what works quickly and how to do it.participants did not readily notify their doctors of flare events. only 25% notified their general practitioner and a lesser proportion (19%) notified their specialist ; a further 14% clarified their response as when i see them and 6% only if bad. participant comments noted different reasons for not accessing medical help, such as follows:i ca n't get an appointment when i 'm flaring, so i do all i can first, moving things around and hope it helps. comments also reflected a degree of resignation about their illness and the perception of a lack of empathy and understanding received from their medical supports:i used to, but now they just look at you and say the numbers look fine, but i still feel like crap.i only tell him when i see him, do n't want to bother him. sometimes the gp helps, but they do n't understand really.a total of 219 management strategies were reported and grouped into 12 separate themes. no differences were found between groups apart from the theme of diet modification for flare groups (p = 0.03), derived from small numbers of participants. modifications specified concentrated on reducing processed foods, in particular breads or gluten containing foods, and increasing fresh fruits and vegetables. diet due to hot and spicy foods being perceived as a flare trigger. i 've been living with this for a long time and i know what works quickly and how to do it. i ca n't get an appointment when i 'm flaring, so i do all i can first, moving things around and hope it helps. i used to, but now they just look at you and say the numbers look fine, but i still feel like crap. i only tell him when i see him, do n't want to bother him. changes in medications incorporating itm as well as analgesics, anti - inflammatory medications, and topical ointments were employed in 67.3% of participants. time out, take it easy, and rest was also reported in 66.3% of participants. one participant comment suggested the need to self - monitor and change behaviour:i try to keep on top of things ; i 'm learning to be kinder to myself, saying no. it gets easier to know when it 's happening ; it 's just a feeling i get to slow down. but it still frustrates me.the strategy of pacing and planning rest periods was adopted by 11% of our cohort and was often reported in conjunction with relaxation (9%) and complementary therapy techniques including meditation. therapies reported were a mix of exercise related activities, tai chi, pilates, and yoga ; homeopathic practitioner treatments (acupuncture, massage, and chiropractor) ; and a holistic soft tissue interestingly, the use of herbs, probiotics, tonics, and homeopathy medications such as olive leaf, glucosamine, and fish oils, whilst reported as routine use in background questionnaires, was not reported as specific flare management strategies. i try to keep on top of things ; i 'm learning to be kinder to myself, saying no. it gets easier to know when it 's happening ; it 's just a feeling i get to slow down. changing daily behaviour including reducing workloads, avoiding computers, and changing environments were reported by 17% of participants. of these, strategies to reduce the amount of outdoor hours and uv light, as well as staying within a warm or cool environment, were reported most frequently. the use of simple everyday aids as a first - line treatment to alleviate symptoms was reported in 16% of participants as a management strategy. aids specified included heating devices (wheat bags, gloves, heater, and electric blankets) and also hot or cold showers / baths with oils / minerals. this study was designed to improve our understanding of sle flare symptoms, triggers, and management from the patient 's viewpoint. the majority of people living with sle experience a daily symptom background level despite medical management. heightened perception of symptom intensity can occur frequently ; therefore the flare definition used specified that the variation needed to be sustained over a 24-hour minimum. patient interpretation of flare events is based on a number of factors including overall illness severity, personal insight, and perceived degree of symptom impact. some patients report flare only when illness exacerbations are persistent, not explained by their activities, and require management help. eighty - eight percent of our cohort reported 1 or more flare events (mean 6.8) ; 2 participants reported weekly flare events ; 56 (63%) of the flare group reported fewer than 5 flare events for the study year. monthly flare events lasting for an average of 3.4 days were reported by 14 participants. of interest, 11 of these, a prospective norwegian study found an average monthly flare rate of 2.5. the specific timing and matching of symptoms to separate events fitzgerald and grossman suggest that the assessment of flare timing and frequency is particularly difficult in observational cohorts with irregular follow - up : flares are variably coded as single or multiple events. acknowledging this, flare frequencies reported in our study could reflect (i) a mixture of multiple single symptom events ; (ii) a single continuous event with multiple different symptom exacerbations ; or (iii) a single continuous event with the same symptom exacerbation resurfacing due to inadequate resolution. of these, 5 (42%) had a complex symptom spectrum with 6 or more acr criteria. all nonflaring participants reported flare triggers and also management strategies used in the past to alleviate flare events. this indicates a level of illness understanding that allows optimisation of self - management strategies preventing and reducing flare events. a small number commented that they had used steroids for management of exacerbations in the past, indicating that their pharmaceutical regime had altered over time. patient flare experiences differed in symptom presentation, triggers, and management strategies across individuals, but not between sle groups. cohort reported flare symptoms of joint and muscle pain, fatigue, rash, headaches, fevers, joint swelling, and cognitive clouding are consistent with other published literatures as being most frequently reported [3, 11, 24 ], although our rates did differ slightly, possibly indicating factors unique to the australian environment. particular differences included reduced reports of headache (23.8%) and cognitive impairment (11.9%) [17, 25 ] ; also, joint and muscle pain (73.3%) was more frequent than fatigue (65.4%) [3, 11 ]. gastrointestinal symptoms (13.9%) and shortness of breath (9.9%), not reported in other studies, were also frequent in this australian cohort. the flare behaviour similarities between borderline and classic lupus patients is noteworthy and is consistent with the proposal that lupus represents a spectrum of disease rather than a dichotomous state of lupus being present or absent. differentiation between symptoms of normal illness fluctuations, treatment side effects, and symptom exacerbations classified as flares can be difficult for both clinicians and patients. differences can lie in the level of importance placed on symptoms without sufficient regard to qol impacts [1, 1113 ]. this potential discrepancy between physician and patient flare definitions was demonstrated within this study by infrequent patient reports of some symptoms (ulcers, alopecia, seizures, and serositis) used within clinician assessment tools and acr classification. the unpredictable nature of symptom fluctuations can impact greatly on an individual 's qol resulting in heightened awareness of symptom changes and recognition of suspected causal triggers [16, 24 ]. confirming causal links is difficult and is not often undertaken ; however, the role environmental triggers play in lupus development and flare has been described in the literature, with flare triggers of stress, overexertion, uv exposure, and infection being most often discussed [2628 ]. additional evidence also supports a role for oestrogens, drugs (e.g., hydralazine), and vitamin d as immune modulators in sle [29, 30 ]. our study supported established understandings of flare triggers with stress, overexertion, uv exposure, and postinfective episodes being most readily identified as potential triggers. the cohort identified these as the first, second, third, and sixth most frequent triggers along with several other perceived triggers. the identification of weather changes (19.8%) and physiological responses to temperature changes such as overheating (19.8%) and cold temperatures (9.9%) are of great interest in light of predictions of climate change and weather instability. dysfunction of the thermoregulatory system with either vascular or autonomic origins has been reported in sle and other connective tissue disorders studies [31, 32 ]. theories of causation vary and have included treatment / pharmacological driven responses, ischemia due to vasculitis, response to circulating complement - fixing autoantibodies directed against sympathetic and parasympathetic nervous structures, and proinflammatory cytokine activity. however, more directed studies are required to investigate and establish lupus flare and temperature change associations. the trigger - symptom matrix (table 4) consistently linked frequently reported symptoms to reported flare triggers, independent of individual patient attributions. not surprisingly, symptoms of fatigue and pain were cross - linked for all combinations examined including newly identified triggers associated with temperature alteration. it is of interest that potential associations between excessive proinflammatory cytokines have also been found for symptoms of fatigue, depression, and overheating in breast cancer patients prompting thoughts of similar connection possibilities in people with sle. the matrix development was undertaken to aid in the identification and validation of trigger and symptom associations and was sensitive enough to successfully link a known symptom trigger (cold temperature) to raynaud 's symptoms in patients known to have this comorbid illness. this process could be further developed in larger multinational studies with reference to other comorbidities and also symptom spectrums of sle patients allowing development of personalized self - management advice for day - to - day living. trigger discussions within published studies have also identified activities associated with work [20, 35, 36 ] and household chemicals [3739 ] as being important considerations. these despite not being named within the top triggers were identified by our participants (work 8.9%, chemicals 7.9%) and will be the focus of future research. the majority of participants, independent of flare group, identified perceived triggers (95%) and adopted flare management strategies (98%). coping skill development along with understanding symptom manifestations in many chronic illnesses is reported to increase patient resilience and self - efficacy. this was also evident in our cohort with participants reporting that they seek out medical help once all self - management strategies have been exhausted. the reluctance of flaring participants, to both inform and involve treating clinicians, was based primarily on past experience and highlights the importance our participants placed on self - management. concerns about costs, waiting times, travel distance, perceived lack of empathy, and understanding of symptom impact were expressed by our participants as in other studies [4043 ]. self - management strategies relied heavily on increasing rest periods, use of aids, and the addition of pain and anti - inflammatory medications prior to adjustment of itm. participants used retained steroid supplies and self - adjustment to alleviate symptoms and reduce need for medical interventions. this often occurred without adherence to staged dosage decline and without knowledge of medical risks. exploration of published literature suggests that self - adjustment of medications is not unique to this study cohort [15, 44, 45 ]. usage was more common in patients of a younger age and higher level of education and those dissatisfied with medical care. participants within our study often used alternative therapies, with increased usage during flares. relaxation (8.9%) and the complementary therapies (9.9%) reported were a mixture of massage, bowen therapy, acupuncture, and herbs. flare attribution to a build - up of toxins was suggested by a few participants as a reason for flares events. therapies were used as both a detox facilitator and symptom alleviator along with diet modifications such as adopting a fresh food or gluten - free diet. overall, participants reported that they did whatever they could to stay as well as possible. the fact that symptom flares still occurred despite all these itm and lifestyle changes created distress through a constant reminder of disease. over 62.4% of our participants reported a decreased flare frequency since medical management had commenced. despite this reduction only 50% reported a general health improvement. this demonstrates that there is a considerable retained disease burden associated with living with lupus. retrospective studies have inherent limitations associated with participant recall bias ; however they allow in - depth exploration of the lived lupus experience within a flexible and cost - effective study design. consequently, they are useful in exploring hypotheses allowing refinement of focus for future research. due to their flexibility the results can often be more generalized ; however, results of our study should be taken in the context of a homogeneous female caucasian population. the study design incorporated an audit process to validate and standardise sle classification and also allowed cross - correlation of self - reported medical information, therefore reducing potential recall and misclassification bias within methods. individual participants may have varying interpretations of what constitutes a flare. to reduce participant misinterpretation, the approach provided guidance to defining fluctuating symptoms as part of daily illness changes and sustained exacerbations more representative of flare events. the use of a single structured interview without response exploration may have narrowed results, although participants freely volunteered comments and response clarifications. importantly, this approach was supported by (i) convergent results found between this study and other sle populations as well as other autoimmune illnesses and (ii) internal consistency using the trigger - symptom matrix. this paper provides insight into the patient experience of sle flare particularly perceived symptoms, triggers, and management strategies. in addition to known flare triggers of stress, infection, and uv light, the study identified new and important potential flare triggers. the finding of patient associations between their flares and changes in temperature and weather is of particular concern in light of growing predictions of climate change. triggers in relation to daily activities as part of working or home based cleaning (chemical exposure) raises potential for identifying specific triggers. further research focusing on these activities may provide evidence for extension of current management guides for lupus and flare prevention. in summary, we found that, each year, the average sle patient suffers 30 days of symptom flares with around 7 discrete episodes, which are often self - managed without medical input. many expressed barriers and reluctance to include medical support in their flare management, highlighting the need for improvements in patient / clinician interactions and the need for developing shared health management plans.	individuals living with lupus commonly experience daily backgrounds of symptoms managed to acceptable tolerance levels to prevent organ damage. despite management, exacerbation periods (flares) still occur. varied clinical presentations and unpredictable symptom exacerbation patterns provide management and assessment challenges. patient perceptions of symptoms vary with perceived impact, lifestyles, available support, and self - management capacity. therefore, to increase our understanding of lupus ' health impacts and management, it was important to explore lupus flare characteristics from the patient viewpoint. lupus flares in 101 australian female patients were retrospectively explored with the use of a novel flare definition. qualitative methods were used to explore patient - perceived flare symptoms, triggers, and management strategies adopted to alleviate symptom exacerbations. a mean of 29.9 flare days, with 6.8 discrete flares, was experienced. the study confirmed that patients perceive stress, infection, and uv light as flare triggers and identified new potential triggers of temperature and weather changes, work, and chemical exposure from home cleaning. the majority of flares were self - managed with patients making considered management choices without medical input. barriers to seeking medical support included appointment timings and past negative experiences reflecting incongruence between clinician and patient views of symptom impact, assessment, and ultimately flare occurrence.
visceral leishmaniasis (vl) is the vector - borne disease and has great importance in public health due to the fatality among children. although there is an increasing interest to vl but there is not enough approach for control measures. there are various epidemiological features of vl which indicating the needs for defining different control measures (who 1990). kala - azar is a mediterranean - form in iran which the leishmania infantum isolated from vl patients (mohebali. 2005), the sand flies species from subgenera larroussius and paraphlebotomus acts as vectors (azizi. 2006) and the dogs and wild canids are considered as principal reservoirs (edrissian. asymptomic infected dogs are suspected as the main reservoir host for sand flies to establish the transmission cycle to humans (moshfeh. the confirmed endemic foci of vl are distributed in different provinces and districts of iran including : ardebil province, east azerbaijan province (kaleibar, ahar, azarshahr districts), fars province (ghir - karzin, jahrom) semnan province, bushehr province (poshtkooh), qom province, kerman province (baft) and alborz (savejbolagh district) (mohebali 2013). moreover, the sporadic cases are reported from different parts of iran annually. the isolated parasite from animal reservoir was identified as l. infantum lon49 using biochemical assay (isoenzyme) in ardabil and azarbaijan provinces. the isolated parasite is exactly the same strain which previously isolated from human cases, so it can certainly be considered that the canine family is the most important reservoirs for humans (mohebali., the interruption of the transmission cycle seems to be the most effective (ramezani., the common control strategies between humans and carnivores vl (zvl) due to l. infantum is limited to early diagnosis and treatment with expensive and potentially toxic pentavalent antimonial drugs (killick - kendrick. treatment of infected dogs with meglumine antimobiate (glucantime) may be led to decrease of sensitivity of l. infantum to these drugs (gramiccia. there is no record for using of flumethrin on dogs as intervention method against control of sand flies in vl foci around the world. continuous efforts for the control of leishmaniasis and the applicability of these studies will be needed more than ever in the new and the old world. as surely as the vectors could be spread and transmitted the parasite from dogs to man (braga. 1998). at the present, the methods that can prevent the dogs against visceral leishmaniasis could not be prevented spreading of infected sandflies (reithinger. prophylactic soaps, shampoos and sprays with base of pyrethroids have been used on dogs with different success around the world (ashford 1989). yet, any protective measure which could protected completely the dogs against vl, is not introduced (christopher 1996). in the endemic area of the vl foci, it is recommended that all seropositive dogs are eliminated from infected areas. in southern europe the prevalence rate of vl is about 40 % but the elimination of dogs has not been accepted in these communities. in the most endemic areas, the health policy administration system is based on the elimination of seropositive dogs (dietze. 1998), but due to resource and financial limitations and failure of availability of necessary supplies are not be realized with suitable methods (david. 1998). in europe, the chemical treatment of dogs using antimonial components have been used for dog treatment, but this method is not recommended due to drug resistant of the parasites among dogs. the drug therapy of vl among dogs may led to temporary treatment of infected dogs (gramiccia. 1992, alessandro. 1997) but this drugs do not prevent recrudesce of vl. important notice that the use of these drugs does not stop the infection of sand flies that feed on infected dogs (alvar. application of residual insecticides is advisable for the control of endophagic sandflies in human dwelling, stables, poultries (marcondes and nascimento 1995, jin. 2004) but are not applicable for the exophagic sandflies which include most of vl vectors that their resting places is outdoor (jin. the present study was carried out in order to evaluate the pour - on formulation of flumethrin on owned dogs in meshkinshahr district during the activity of sandflies and effects of this method was assessed based on blood - feeding and mortality of exposed sand flies treated dogs. totally 118 dogs of the same race and gender were selected. due to higher age (over one year) 3 dogs the blood samples was prepared on filter paper in order to detect the positive cases of vl among selected dogs using direct agglutination test (dat) which was done in laboratory of medical parasitology, school of public health, tehran university of medical sciences, iran. before application of flumethrin, the parazyquantel and albendazole was administered to the dogs in order to protect the research team and it is recommended to the family to bury the dogs feces up to 48 hours in a sanitary manner. the location of experiments in mazrae - khalaf village was selected in front of the house where the cattle stables as well as the nest of sheep dog placed adjacent to it. the bed net size of 2 2 2 meters was installed and a sheet was spread on top of dogs for collection of knockdown sand flies. the live sand flies were collected using suction tube from indoor and outdoor early morning as well as evening and transferred to the laboratory and fed on 5 % sucrose solution during three successive days. the sugar pad was removed 12 hours before test. at the dusk (19:30 pm) which is synchronize with activity of sand flies, first the control dogs and then the treated dogs were anesthetized with xylazine and transferred to the inside of each net. after this stage, 150 live sand flies were released inside each bed net and the environmental condition including temperature and humidity was recorded. subsequently the exposed sand flies were transferred to the paper caps covered with net according to physiological conditions eg blood - fed or unfed. the blood feeding condition was observed immediately after 2 hours after experiments. before pour - on application of flumethrin, titer of the serum in dogs was determinted indicating negative. for determination of flumethrin effect on vector, two indicators were considered in relation to blood feeding and viability of exposed sand flies compared to control group. the blood - feeding index (bfi) of sand flies were 345 out of 620 (55.6%) and 105 out of 614 (17.1%) from 20 up to 80 days post - treatment in control and treated groups (table 1). the bfi was varied from 56.5 to 53.5 % and from 12.3 to 25 % respectively in control and treatment groups (fig. the difference between bfi in control and treated groups was significant (p < 0.0001). blood - feeding index (bfi) of sand flies after application of pour on flumethrin on dogs using bed net trap, meshkinshahr, ardabil province, 2013 blood - feeding index (bfi) of sand flies after application of pour on flumethrin in dogs using bed net traps, meshkinshahr, ardabil province, 2013 application of pour - on flumethrin during the peak of blood - feeding activity of sand flies were assessed at four interval times post - treatment (fig. the ibf of sand flies on treated dogs was 75.0 to 87.7 % and on untreated dogs was 46.541.3 % respectively. the ibf of sand flies was stasistically differenent between treated and control dogs (p < 0.0001). inhibition of blood - feeding rate (ibf) of sand flies after application of pour - on flumethrin on dogs, in bed net trap, meshkinshahr, ardabil province, 2013 the mortality rate of unfed and blood - fed sand flies was studied. the total number of dead blood - fed sand flies was 115 out of 620 in treated dogs and 100 out of 614 respectively in control and treated groups. the variation of mortality rate in the dead blood - fed sandflies during four interval times was 2532 % and 1538 % respectively in control and treated groups (table 2, fig. mortality rate of blood - fed and unfed sand flies after application pour - on flumethrin on dogs compared to control group using bed net traps, meshkinshahr, ardabil province, 2013 mortality rate of sand flies after application pour - on flumethrin on dogs using bed net traps, meshkinshahr, ardabil province, 2013 the mortality rate of dead unfed sand flies was varied from 5 to 51 % and 94.8 to 100 % respectively in control and treated groups. the difference in total mortality rate in control and treated sand flies was significant (p < 0.001). the combined effect of inhibition of blood feeding and mortality rate of sand flies exposed to treated dogs compared with control group. the combined effect was varied between 94.8 to 100 % and 19.5 to 53.3 % respectively in control and treated groups. the difference of combined effect between four interval times of observation was not significant either treated neither group nor control except for 20 days in control group (p < 0.001). before pour - on application of flumethrin, titer of the serum in dogs was determinted indicating negative. for determination of flumethrin effect on vector, two indicators were considered in relation to blood feeding and viability of exposed sand flies compared to control group. the blood - feeding index (bfi) of sand flies were 345 out of 620 (55.6%) and 105 out of 614 (17.1%) from 20 up to 80 days post - treatment in control and treated groups (table 1). the bfi was varied from 56.5 to 53.5 % and from 12.3 to 25 % respectively in control and treatment groups (fig. the difference between bfi in control and treated groups was significant (p < 0.0001). blood - feeding index (bfi) of sand flies after application of pour on flumethrin on dogs using bed net trap, meshkinshahr, ardabil province, 2013 blood - feeding index (bfi) of sand flies after application of pour on flumethrin in dogs using bed net traps, meshkinshahr, ardabil province, 2013 application of pour - on flumethrin during the peak of blood - feeding activity of sand flies were assessed at four interval times post - treatment (fig. the ibf of sand flies on treated dogs was 75.0 to 87.7 % and on untreated dogs was 46.541.3 % respectively. the ibf of sand flies was stasistically differenent between treated and control dogs (p < 0.0001). inhibition of blood - feeding rate (ibf) of sand flies after application of pour - on flumethrin on dogs, in bed net trap, meshkinshahr, ardabil province, 2013 the total number of dead blood - fed sand flies was 115 out of 620 in treated dogs and 100 out of 614 respectively in control and treated groups. the variation of mortality rate in the dead blood - fed sandflies during four interval times was 2532 % and 1538 % respectively in control and treated groups (table 2, fig. mortality rate of blood - fed and unfed sand flies after application pour - on flumethrin on dogs compared to control group using bed net traps, meshkinshahr, ardabil province, 2013 mortality rate of sand flies after application pour - on flumethrin on dogs using bed net traps, meshkinshahr, ardabil province, 2013 the mortality rate of dead unfed sand flies was varied from 5 to 51 % and 94.8 to 100 % respectively in control and treated groups. the difference in total mortality rate in control and treated sand flies was significant (p < 0.001). the combined effect of inhibition of blood feeding and mortality rate of sand flies exposed to treated dogs compared with control group. the combined effect was varied between 94.8 to 100 % and 19.5 to 53.3 % respectively in control and treated groups. the difference of combined effect between four interval times of observation was not significant either treated neither group nor control except for 20 days in control group (p < 0.001). major are the responsible for the transmission of the disease (nadim. 1997, 2000, 2005, 2012, azizi. according to the census of iranian center for control of disease, during the 1988 to 2005, the total number of recorded cases of vl in the country was 2056 cases which 624 of them (30%) related to ardabil province (mohebali 2013). in a seroepidomiological study to determine the prevalence of vl between the years of 2002 to 2005, the more positive cases related to the northwest, especially ardabil province (mohebali., the control approach of vl due to l. infantum is reserved to early detection and treatment of the patients with expensive and potentially toxic of pentavalent antimonial drugs. in some countries, where the disease is endemic situation, the main control strategies has been established for indoor residual spraying as well as reservoir population control (infected dog culling). in brazil, in spite of residual spraying of indoor places and annually elimination of the dogs, the trend of zvl has been increased over the past 20 years (marcelino. methods for the elimination of dogs was questionable due to availability of facilities as well as social acceptance (reithinger. treatment of leishmania - infected dogs with anti - leishmania drugs was not effective due to recrudescence of vl (up to 74.0%) (mohebali. 2002). while waiting for a vaccine against leishmania, the new ways switch to interrupt the vl transmission, by passing the dogs from livestock deltamethrin impregnated bath (david. 1994, guanghua. 1994) as well as topical application of pyrethroid lotions. epidemiological effects of topical application of insecticide on dogs, depends on not only reducing the number of sand flies blood - fed on dog, but also survival rate of sand flies infected with leishmania (reithinger. the main way for the control of vl is protection human and dosmetic reservoir from bite of sand flies (poli. 1997). despite the nature of vl epidemiology which the special species of sand flies are responsible for transmission of the disease from infected dogs to others including human. nevertheless due to specific behavior of the vectors, control of vl different methods including vaccinations, treatment of dogs, separation of infected dogs has been used with different results in the vl foci around the world (ashford. in the recent decade, the application of deltamethrin impregnated collars has been successfully used with high social acceptance as prevention measure for the dogs in different countries. the latter method has the repellency and killing effects against sand flies and so protected the dogs against vl) ramezani. the feeding rate of sand flies on treated dogs was 3.3 folds lower than control. the inhibition of feeding rate on treated dogs was 1.8 fold higher than controls respectively. the difference between total mortality of treated and control groups was highly significant (p= 0.0001). in this study, the immediate mortality rate of sand flies was 5 fold more than control group during 2 hours exposure time. the delayed mortality rate of sand flies was known as a best indicator for the evaluation. the delayed mortality rate in blood - fed sand flies was 94.8 % compared to 27.5 % in control group. it seems the age of dogs is an important factor for pour - on application of flumethrin. it also appears that inhibition of blood feeding or combined effects of flumethrin is prior to killing effect. according to the results, this method could be a good alternative for control of sand flies of vl with high social acceptance by dog owners.	background : visceral leishmaniasis (vl) is one of the most important parasitic zoonotic diseases in the world. domestic dogs are the main domestic reservoirs of vl in endemic foci of iran. various methods, including vaccination, treatment of dogs, detection and removal of infected dogs have different results around the world. general policy on control of canine visceral leishmaniasis is protection of them from sand fly bites. the aim of this study was evaluation of pour - on application of flumethrin on dogs against blood - feeding and mortality of field - caught sand flies.methods:once every 20 days from may untill september 2013, the treated and control dogs were exposed with field caught sandflies for 2 hours under bed net traps. after the exposure time, both alive and dead sand flies were transferred in netted cups to the laboratory. the mortality rate of them was assessed after 24 hours. the blood - fed or unfed conditions were determined 2 hours after exposure to the dogs under stereomicroscope.results:the blood feeding index was varied from 12.0 to 25.0 % and 53.0 to 58.0 % for treated and control dogs respectively (p < 0.0001). the blood feeding inhibition was 75.087.0 % and 41.046.0 % for the control and treated dogs (p < 0.0001), respectively.the total mortality rate was 94.0100 % and 19.058.0 % respectively for the treated and control groups (p < 0.001).conclustion : application of pour - on flumethrin on dogs caused 90100 % mortality until 2.5 month and inhibited the blood - feeding of sand flies.
although the etiology of ibd is unknown, many investigators have probed whether a pathogenic cause can be attributed to onset of disease, either in full or in part.5, 6, 7 although no pathogen has been shown consistently to be associated with ibd, a subset of cd patients do show increased prevalence of a unique enteropathogenic strain of the b2 phylotype e coli termed aiec.2, 8, 9 ileal biopsy specimens from cd and uc patients have shown that aiec are associated primarily with cd ; however, more recent studies have shown an equal prevalence of aiec strains in uc as well as cd, suggesting that this pathobiont may have a greater association with ibd than first thought.10, 11, 12 although lf82 is the most referenced aiec, it is only one of many aiec strains that were found in ibd patients.2, 13 it has been remarkably difficult to identify a specific genetic or molecular marker of aiec, thus in vitro assays are used to validate aiec. however, there may be some genetic signatures that differ between the b2 aiec phylotype compared with other aiec phylotypes. one potential aiec candidate gene is the novel aiec serine protease vat - aiec, which promotes expansion and adherence of aiec to intestinal epithelial cells (iecs). expression of this gene was approximately 3-fold higher in cd - aiec isolates compared with non - aiec isolates from cd patients or healthy subjects. it therefore could be considered a selective rather than an exclusive genetic marker for aiec. aiec are distinct from other strains of e coli because they show nonclassic virulence factors of adherence and invasion (ie, lack of a type iii secretion system). notably, aiec pathogenesis involves survival and replication in iecs and macrophages in vivo and in vitro that further exacerbated barrier dysfunction, this topic is discussed later (figure 1).9, 13 martinez - medina and garcia - gil published an excellent comprehensive review on the definition, characteristics, and molecular basis of aiec pathogenicity. the intestine is lined by a single layer of epithelial cells that are interconnected by transmembrane proteins to create physical connections with one another and to form a selectively permeable barrier. this leaky barrier serves multiple functions that are essential for intestinal homeostasis : generation of ion solute concentration gradients, absorption of nutrients, antimicrobial peptide secretion (reviewed by mccole and barrett), and protection of the host from toxins and pathogens (reviewed by peterson and artis). disruption of barrier function can lead to loss of microbiome diversity and promotion of aiec expansion and pathogenesis.1, 6 iecs express molecular sensors that sample the luminal content for bacterial antigens, and in some cases the bacteria itself, and translate this information to host immune cells. this process prevents the host immune system from a robust proinflammatory response against commensal bacteria while maintaining the ability to protect against pathogens. the first identified ibd - associated gene, cytoplasmic nucleotide - binding oligomerization domain 2 (nod2/card15), a nod - like receptor family member, serves as a sensor to bacterial peptidoglycans (bps) (reviewed by philpott). binding of bps to nod2 results in the activation of nuclear factorb (nf-b) and autophagolysis, and the appearance of nod2 at the site of bacterial endocytosis in epithelial cells drives autophagolysis. mutations in nod2 result in an increased immune response to bacteria and bacterial antigens that is accompanied by impaired autophagolysis of aiec and subsequently survival of aiec in immune cells.23, 24 conversely, overexpression of nod2 and other genes involved in autophagy (eg, by activation of the eukaryotic translation initiation factor 2 alpha kinase 4-eukaryotic translation initiation factor 2 alpha - activating transcription factor 4 [eif2ak4-eif2a - atf4 ] pathway), results in an increased autophagy response to aiec and a reduction in aiec survival in macrophages.25, 26, 27 thus, functional autophagolysis in both intestinal epithelial cells and macrophages is required for aiec degradation and prevention of aiec - induced barrier dysfunction. it is clear that proinflammatory cytokine signaling plays a major role in altered intestinal permeability and this may be associated with aiec pathogenesis. loss of negative regulation of proinflammatory cytokines (eg, interferon- [ifn- ] and tumor necrosis factor- [tnf- ]), and, conversely, overstimulation of the proinflammatory response, plays a role in altered intestinal permeability and this may be associated with aiec expansion and pathogenesis.22, 29, 30, 31 a key mediator of signaling by many cytokines, nf-b has been shown to regulate the development and response of the immune system, inflammation, and cancer. defective nf-b activation can result in a defective immune cell response to bps (driven by interleukin [il]8) and bacterial lipopolysaccharides, and defective autophagy.33, 34, 35 taken together, dysfunctional regulation of proinflammatory cytokines may be associated with an inappropriate response to aiec and may play a role in the promotion of aiec expansion and pathogenesis. with respect to the clinical intersection between altered expression of genes in ibd and genes involved in aiec expansion, patients who are at risk of developing cd also overexpress carcinoembryonic antigen related cell - adhesion molecule 6 (ceacam6), placing them at a higher risk for aiec adherence and invasion via interaction of bacterial type 1 pili and long polar fimbriae (lpf) to host ceacam6. ceacam6, normally expressed on the apical membrane of human epithelial cells from the large intestine, is regulated by the proinflammatory cytokines ifn- and tnf-.36, 38 studies using isolated human peyer s patches or mouse ileal biopsy specimens containing peyer s patches showed that bacterial pili specifically targeted peyer s patch m - cells (discussed later) expressing ceacam6 ; however, lpf - independent aiec invasion and adherence also has been shown in other types of intestinal epithelial cells.36, 39 nevertheless, aiec can up - regulate ceacam6 indirectly via activation of proinflammatory cytokines, thus leading to increased expression of ceacam6 and further binding and internalization of aiec. carcinoembryonic antigen human bacterial artifical chromosome 10 (ceabac10) transgenic mice that overexpress human ceacams show no clinical evidence of colitis ; however, these same mice infected with aiec show a barrier defect. it would be expected, therefore, that patients with nod2 mutations and/or increased expression of ileal ceacam6 are more at risk to develop ibd. in support of this statement, ceacam6 and the pore - forming tj protein claudin-2, isolated from ileal biopsy specimens of cd patients, are not only co - localized, but expression levels are both increased compared with control subjects, thereby promoting a favorable environment for aiec adherence and invasion and further exacerbation of barrier dysfunction, ultimately leading to a vicious cycle of proinflammatory cytokine release and increased barrier permeability. interestingly, although ceacam6 is overexpressed in ileal mucosa of ibd patients, the protein is not normally expressed in ileal mucosa. the mechanism of aiec internalization involves macropinocytosis and vacuolization of the bacteria into iecs and macrophages.9, 42, 43, 44 aiec have been found in late endosomes of intestinal epithelial cells, as evident by co - localization with the lysosomal marker lysosomal - associated membrane protein-1 (lamp1).24, 44 similarly, mycobacterium avium subspecies paratuberculosis has been localized in endosomes of intestinal epithelial cells. the process of internalization into endosomes also occurs during reorganization of tjs and is discussed later. aiec - containing endosomes mature into phagolysosomes, which then can exit iecs, as discussed later. alternatively, aiec replication is permitted in autophagolysis - deficient, aiec - containing phagosome / endosome / lysosomes, leading to loss of organelle membrane integrity, release of the bacteria into the cytoplasm, and basolateral exit of the bacteria from the iec (figure 1).46, 47 aiec translocation through the epithelial barrier is dependent on type 1 pili and binding of lpf to glycoprotein 2 (gp2) to aiec disruption of epithelial junctions microfold cells (m - cells).39, 43, 48 m - cells are required for the development of host immunity, are involved in bacterial sampling,50, 51, 52, 53 and can serve as an alternative gateway for aiec39, 54 into and through the epithelium (reviewed by pravda). the aiec - containing endosome can mature into a phagosome and exit basolaterally from the iec, where it can be processed by macrophages (figure 1).42, 44 after basolateral exit from epithelial cells, aiec exposure to lamina propria immune cells induces a proinflammatory response and a tnf-dependent increase in m - cell development, thereby further permitting the adherence and invasion of aiec and exacerbation of barrier permeability. aiec invasion elicits a proinflammatory response by triggering secretion of proinflammatory cytokines from iecs, resulting in recruitment of immune cells,13, 57 and, subsequently, nf-b signaling, which allows survival of aiec in macrophages. in summary, cytokines play a key role in the pathogenesis of aiec and aiec - induced epithelial barrier dysfunction. in addition to the unknown etiology of ibd, it is uncertain whether inflammation precedes epithelial barrier dysfunction or if barrier dysfunction results in chronic inflammation such as that observed in ibd. however, some evidence points to an underlying barrier defect that renders individuals susceptible to disease. first - degree relatives of cd patients show asymptomatic barrier dysfunction associated with subclinical immune activation, indicating that a barrier defect may precede or is at least an early event in the disease.59, 60 in addition, altered permeability across the intestinal epithelium is a predictor of relapse in human cd ; whereas increased permeability precedes inflammation in rodent models, canine models, and human disease,59, 64, 65, 66 and has been shown to be the underlying mechanism of ibd in these patients. pathologic stimuli, such as aiec, in part influence epithelial barrier integrity by altering the network of proteins that regulate barrier permeability known as the intercellular apical junctional complex (ajc). the ajc, comprised of several different proteins with distinct responsibilities, directs the paracellular movement of ions and solutes and thus is responsible to maintain appropriate epithelial barrier permeability. the structural units of the ajc responsible for regulating barrier integrity and permeability are adherens junctions (aj) and apical tjs. ajs and tjs are associated with scaffolding proteins that cooperate with the cytoskeleton of the cell providing the ajc with a dynamic means of regulation. dysregulation of tj proteins is observed in ibd patients (table 1).68, 69, 70, 71 that the ajc is required for barrier integrity has led to the hypothesis that defects in the ajc may play a role in ibd pathogenesis. alteration of the ajc can occur in response to exposure to aiec and other enteropathogenic e coli.71, 72, 73, 74 aiec have been shown to decrease transepithelial electrical resistance of iecs owing to disruption of the ajc.47, 67 the molecular interaction of aiec with iecs induces an inflammatory response leading to the overproduction of proinflammatory cytokines, with ifn- and tnf- as the major effectors, and this is a key step in the pathogenesis of ibd. ifn- and tnf- both have been shown to increase gut permeability by acting on tj proteins and tj strand complexity,76, 77 which then further exacerbates barrier dysfunction. aiec - dependent, cytokine - induced alterations in aj and tj proteins is discussed later. as previously discussed, an increase in expression of both ceacam6 and claudin-2 is associated with increased barrier permeability. interestingly, targeting of adherens junction proteins, occludin, and claudin 3/4 and other tight junction proteins by helicobacter pylori, vibrio cholera, and enteropathogenic e coli, respectively, has been shown previously,78, 79 supporting the possibility that aiec directly bind to tj proteins. it has been shown that aiec results in increased claudin-2 expression in mice and human beings, and because aiec pathogenesis is not dependent solely on interaction of ceacam6 with aiec, it is possible that claudin-2 could serve as a gateway for aiec entry into iecs while co - internalization of tj proteins and aiec may promote aiec invasion and increased barrier permeability. whether aiec are able to bind directly to tj proteins has yet to be shown experimentally. distinct from the functions of adherens junctions, tjs serve as a paracellular gateway for passive fluid movement and solute flux and limit the passive movement of proteins and lipids, thereby generating a concentration gradient as well as playing a role in polarization of epithelia. the conglomeration of proteins that make up the tjs include the claudin family of transmembrane proteins, junctional adhesion molecule - a (jam - a), occludin, and zonula occludens members 13 (others not included in this article are reviewed elsewhere) (figure 1). claudin-2 is a prominent member of the claudin family because it forms a cation - selective pore that permits paracellular sodium and water flux.80, 81 overexpression of claudin-2 results in an increase in epithelial barrier permeability both in vitro and in vivo.82, 83, 84, 85 il13 and tnf- induce a barrier defect in uc by up - regulating claudin-2 to facilitate the pore pathway, although il6 also has been shown to increase claudin-2 expression in intestinal epithelial cells.86, 87 that epithelial barrier leakiness is related inversely to tj strand complexity supports the association of increased levels of claudin-2 with increased barrier permeability in ibd.70, 88 conversely, a decrease in claudin-2 expression results in a tighter epithelial barrier. adherens junctions are primarily responsible for cell cell recognition as well as initiating and maintaining cell cell contact and polarization. the major aj protein is e - cadherin, a transmembrane protein associated with the catenin family of cytoplasmic proteins (figure 1). e - cadherin plays a critical role in adhesion of cells and is required for the formation of tjs. e - cadherin single - nucleotide polymorphisms and dysregulation of e - cadherin are associated with ibd (reviewed by mccole), supporting a role for aj defects in ibd - associated barrier dysfunction. furthermore, aiec, in addition to other intestinal pathogens, are able to displace e - cadherin in vitro,47, 93 potentially providing another mechanism of invasion and barrier disruption. jam - a is another psd95-dlga - zonula occludens-1 homology domain (pdz)-domain containing integral protein localized to tjs. jam - a shows pdz - domain dependent interactions with the scaffolding protein zonula occludens 1 (zo-1) and cell polarity partitioning defective protein-3. expression of jam - a was shown to be decreased together with zo-1 in ibd, and loss of jam - a results in increased epithelial barrier permeability.101, 102 it is unknown whether aiec directly results in the down - regulation of jam - a or if it is a consequence of down - regulation of zo-1 ; however, aiec may regulate jam - a indirectly via the release of inflammatory cytokines from infected macrophages. nevertheless, it is possible that aiec, in addition to altering zo-1, may alter jam - a and partitioning defective protein-3dependent targeting of tj proteins to the membrane, resulting in disrupted tj complex integrity and altered cell polarity, further promoting ibd pathogenesis. occludin, a 65-kilodalton integral transmembrane protein, is involved in the stability and regulation of tjs. although down - regulation of occludin is observed in cd and uc patients, the uc - associated proinflammatory cytokine il13 alone was not able to abolish occludin protein levels in a colorectal cancer cell line (ht-29). this discrepancy was attributed to a dilution of the number of cells isolated from the inflamed tissue of uc patients. nevertheless, it has been shown from in vitro work that occludin plays a key role in promoting barrier function and is redistributed away from tight junctions after enterohemorrhagic e coli infection of human colonoid monolayers.17, 74 further work to elucidate the in vivo role of occludin is needed because mice lacking occludin do not show defects in tj morphology despite histologic abnormalities in some tissues. proinflammatory cytokine - dependent phosphorylation of myosin light chain (mlc) by myosin light chain kinase (mlck) or rho - associated kinase results in rapid reorganization of the ajc.67, 83, 105 although it remains to be confirmed, aiec could affect occludin distribution indirectly through bacteria - dependent induction of proinflammatory cytokines from immune cells, activation of mlck or rho - associated kinase, and subsequent redistribution of occludin. zo is a pdz homology domain containing plaque protein involved in scaffolding of tj proteins to the cytoskeleton. zo-1, -2, and -3 contain pdz domains that facilitate anchorage of tj proteins such as claudins, occludin, and jam - a to the cytoplasm and thereby play a key role in tj formation and regulation. tnf- induces an increase in tj permeability via nf-b dependent down - regulation of zo-1 protein and mlck - dependent95, 96, 109, 110, 111 redistribution of tj proteins in an apoptosis - independent manner (reviewed by bruewer). similarly, aiec disrupt tj composition by altering zo-1, resulting in increased appearance of gaps between cells.47, 93 conversely, probiotic and commensal bacteria promote up - regulation of zo proteins and strengthening of the epithelial barrier (reviewed by ulluwishewa), potentially supporting the notion that when probiotic bacteria outcompete pathogens, this not only will return the balance of the microbiota, but also enable restitution of the tj complex. in the absence of bacteria, regulation of the ajc is mediated by the interaction of the cytoskeleton with the proteins of the ajc or by cytokines. ifn- induces macropinocytosis of occludin, jam - a, and claudin-1 via myosin 2dependent vacuolarization of the apical membrane into early endosomes, resulting in an increase in barrier permeability. that aiec also are internalized by macropinocytosis illustrates a direct disruption of the ajc by the bacteria and establishes an additional virulence factor for the invasion of aiec. data to date clearly support the notion that ibd pathogenesis is dependent on dysregulation of multiple factors including disruption of the intestinal barrier and immune dysregulation, and is associated with profound alterations in gut microbial communities. altered intestinal homeostasis drives expansion of aiec and results in compromised barrier function. aiec are able to invade, survive, and replicate within host cells, leading to exacerbation of barrier dysfunction by disruption of tj proteins, and ultimately contributing to ibd pathogenesis. if aiec does indeed play an important role in ibd pathogenesis it will be vital to better understand unresolved questions regarding its appearance in disease. specifically : (1) what are the host genetic and environmental factors that promote aiec expansion in some patients but not all ; (2) how is regional variation in aiec expansion determined ; and (3) can therapeutic interventions (probiotics, prebiotics, small molecules) specifically neutralize or modify aiec pathogenic behavior ? aiec are capable of compromising intestinal epithelial barrier properties both directly and indirectly to enhance their pathogenic impact. therefore, in addition to the earlier - described approaches, it will be of key interest to determine if strategies designed to enhance the epithelial barrier can minimize the pathogenic impact of aiec and its putative contribution to ibd.	pathobiont expansion, such as that of adherent - invasive escherichia coli (aiec), is an emerging factor associated with inflammatory bowel disease. the intestinal epithelial barrier is the first line of defense against these pathogens. inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of aiec. aiec in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier. one critical element of the epithelial barrier is the tight junction. increasing evidence suggests that aiec may selectively target protein components of tight junctions, leading to increased barrier permeability. this may represent one mechanism by which aiec could contribute to the development of inflammatory bowel disease. this review article discusses potential mechanisms by which aiec can disrupt epithelial tight junction function and intestinal barrier function.
humans are confronted with a number of microorganisms, viruses, and parasites that require continued surveillance and defense against [1, 2 ]. polymorphonuclear leukocytes (pmns), monocytes, and tissue macrophages are important cells in the innate immune response to microorganisms and parasites [1, 3 ]. all derived from the same myeloid progenitor cell in the bone marrow. blood monocytes are an intermediate stage of development which then further differentiates in tissues to various macrophage populations. tissue macrophages, as well as, other cells of the innate immune system, are critical for these surveillance and defense activities. upon stimulation by microbial particles or other endogenous factors such as cytokines, macrophages can de novo synthesize and release a large variety of cytokines (il-1, il-6, il-8, il-10, il-12, tnf, ifn, ifn, mcp - l, mcp-3, mif, m - csf, g - csf, gm - csf, mip - l, mip-2, lif, osm, and tgf-). some cytokines can stimulate the production of cytokines by macrophages (il-3, gm - csf, ifn), while others can suppress it (il-4, il-10, il-13, tgf). in addition, in many organs, tissue macrophages serve as important regulatory cells, through their synthesis and secretion of a spectrum of cytokines and growth factors [57 ]. toll - like receptors (tlrs) of mammalian innate immune defense systems play a crucial role in the initiation of adaptive immune responses to bacteria invasion [811 ]. a critical feature of macrophages required for specific recognition and response includes the expression of optimum levels of different tlrs [811 ]. upon the interaction with specific pathogen - associated molecular patterns (pamps), tlrs trigger the signal transduction cascades that lead to cellular responses including the engulfment of invasive microbes, induction of inflammatory cytokines, and generation of ros to kill the invasive pathogens. several mammalian tlrs genes have been identified, and at least ten human tlrs have been cloned. tlr1, predominantly distributed in monocytes / macrophages and pmns, is implicated with trl2 in the recognition of the native configuration of mycobacterial lipoprotein and several other lipopeptides [10, 12 ]. tlr4 and cd14, on the other hand, are required for the recognition of the gram - negative bacteria - derived lipopolysaccharide (lps). during sepsis, the binding of lps to extracellular domain of tlr4 receptors on phagocytic cells, macrophages, and neutrophils elicits activation of the tlr4/irak-2/nf-b complex to signal production and release of proinflammatory mediators (cytokines) phagocytic cells. although tlrs play an important role in the adequate response of immune cells, the deregulation of their expression and function causes defective immune responses [1416 ] and the occurrence of chronic diseases [1719 ]. to understand the molecular mechanisms underlying macrophage differentiation, investigators have turned to human myeloid cell lines such as hl-60, u937, thp-1, and ml-1 [2023 ]. in vitro, these myeloid cell lines continuously proliferate in suspension culture and can be induced to differentiate into macrophages by 12-o - tetradecanoylphorbol-13-acetate (tpa) or 1,25-dihydroxyvitamin d3. these cell lines have been widely used as cell models for studying the molecular and cellular aspects of myeloid differentiation. ml-1 cells were originally isolated from a patient with acute myeloblastic leukemia [25, 26 ]. these cells readily differentiate into macrophages in response to a low concentration of tpa (0.3 ng / ml or 0.5 nm) [25, 26 ]. in this process, the differentiation signal induced by tpa is mediated via a redox - regulated extracellular receptor kinase (erk1/2). differentiated ml-1 cells demonstrate a number of biochemical and functional endpoint indicators of macrophage differentiation including : nonspecific esterase (nse) phagocytosis, plasma membrane nadph oxidase and accompanying superoxide generation, and cell surface markers [2831 ]. in the present study, we have characterized the temporal upregulation of tlr1, tlr2, tlr4, and irak2 expression during the differentiation of ml-1 cells leading to increased responsiveness to lps and lta. our data also show that the increased expression of tlr1 and tlr4 induced by tpa is at least in part mediated through activation of erks. lipoteichoic acid (lta) (major constituent of the gram - positive bacteria cell wall) and lipopolysaccharide (lps) (escherichia coli) were purchased from sigma - aldrich. the human cytokines il-1 alpha, il-1 beta, il-6, il-8, il-10, il-12(p40), gmcsf, ifn - gamma, tnf- and ip-10 cytokine multiplex immunoassay reagents were obtained from upstate biotechnology, lake placid, ny, usa. the alexa fluor 488-labeled mouse anti - human abs against tlr1, tlr4, cd14, and human myeloid leukemia ml-1 cells, originally isolated from a patient with acute myeloblastic leukemia, were kindly provided by dr. ruth craig, dartmouth school of medicine, nh. all cell culture media and supplements were obtained from invitrogen (carlsband, ca). cells were cultured in rpmi 1640 medium supplemented with 10% heat - inactivated fetal bovine serum, 2 mm l - glutamine, antibiotics (50 u / ml penicillin and 50 g / ml streptomycin), and maintained at 37c in a humidified 5% co2 atmosphere as previously described [25, 26, 32 ]. the differentiation of ml-1 cells to macrophages entails a 6-day protocol : three days exposure to tpa (5 nm) and followed by three days of culture in the absence of tpa. both the concentration of tpa used and the time of its presence are critical for optimal macrophage differentiation, particularly mitochondrial maturation. louis, mo) was dissolved in dmso to obtain a 100 m stock solution and further diluted before use. for all experiments, cells were cultured at an initial density of 5 10 cells / ml. for negative controls, the cells were incubated in the absence of tpa, in medium containing an equivalent 1% (v / v) dmso. ml-1 cells were grown at density of 10 cells / ml in a 150 ml dish and treated with vehicle (dmso) or 5 nm tpa (n = 3). the purified rna was subjected to affymetrix oligonucleotide microarray analysis using human genome u133 2.0 plus array chip. six replicates, including three controls and three tpa treated ml-1 cells samples, were examined. the levels of tlr1, tlr4, and cd14 were determined using facs - scan analysis. cells (1 10) resuspended in pbs containing 0.1% sodium azide and 5% fbs were incubated on ice for 30 min and then incubated with (alexa - fluor 488) conjugated anti - tlr1, tlr4, or cd14 antibodies for 1 hour. the cells were washed twice, fixed in 2% formaldehyde in pbs, and analyzed by facs - scan analysis. negative controls were stained with isotype - matched (alexa - fluor 488)-conjugated igg and compensation was adjusted using the single - stained cell samples. the fluorescence intensities were determined using cellquest software (becton dickinson, bedford, ma). selected panel of inflammatory mediators (il-1 alpha, il-1 beta, il-6, il-8, il-10, il-12(p40), gmcsf, ifn - gamma, tnf - a, and ip-10) in cell culture medium were analyzed following stimulation of ml-1 cells or differentiated ml-1 cells (dml-1) with lps or lta for 16 hours, using cytokine multiplex immunoassay reagents (upstate biotechnology, lake placid, new york, united states) analyzed by luminex 100 (luminex corporation, austin, texas, united states). the generation of cellular ros, in particularextracellular h2o2, was assessed using horseradish peroxidase - dependent luminol - derived chemiluminescence(cl) as previously described. one million cells were incubated with luminol (10 m) and horseradish peroxidase (10 g / ml) in complete pbs, 500 l (pbs with 0.5 mm mgcl2, 0.7 mm cacl2, 0.1% glucose). cellular cl was measured continuously in a berthold lb9505 luminometer (pforzheim, germany) for 1.5 hours. in our previous studies, we have demonstrated that human myeloid ml-1 cells differentiate to macrophages upon stimulation with the phorbol ester tpa. to further confirm that tpa - treated ml-1 cells exhibit macrophage characteristics, cd14 gene expression, a key macrophage marker, was determined at the mrna and protein level. examination of the microarray analysis (figure 1(a)), and flow cytometry analysis (figure 1(b)) revealed a strong induction of cd14 gene expression upon stimulation with tpa and in a time - dependent fashion. toll - like receptors (tlrs) of mammalian innate immune defense systems play a crucial role in the initiation of adaptive immune responses to bacteria invasion [811 ]. accordingly, we assessed the temporal expression of different tlr gene products in response to tpa - induced macrophage differentiation of ml-1 cells. analysis of microarray data revealed significant increases in the mrna levels of tlr1, tlr2, tlr4, and tlr8 expression also that of irak-2 in ml1 cells compared to control (dmso - treated cells) (figure 2). interestingly, only the gene expression of the tlr members indicated above were changed in response to tpa. as shown in figure 2, the mrna for each of the tlrs showed different expression patterns, with tlr4 showing maximum mrna as early as 6 hours of tpa addition, after which it decreased. interestingly, irak-2 mrna exhibited the same temporal pattern as tlr4 (figure 2(b)). on the other hand, the mrna for tlr2 and tlr8 was apparent at 6 days of differentiation. flow cytometry analysis confirmed enhanced tlr1 and tlr4 protein expression by ml-1cells (figure 3(a)). immunocytochemistry data revealed a basal level of expression of tlr4 but not tlr1 in the undifferentiated ml-1 cells (figure 3(b)), which increased with macrophage differentiation. in our previous studies [23, 27, 35 ], we reported that the mitogen - activated protein kinase (mapk) cascade plays a crucial role in the initiation of the macrophage differentiation signal induced by tpa. using u0126, a mek inhibitor, we examined whether mapk - derived differentiation signal was required for tlr-1 and tlr-4 expression - induced by tpa in ml-1 cells. pretreatment with 10 m u0126 effectively blocked both tlr1 and tlr4 protein expression induced by tpa in ml-1 cells (figure 4). since ml-1 cells already expressed a basal level of tlr4 protein, the observed increase in lps and lta response may be due in part to the upregulation of irak-2 gene expression in response to tpa. together, these observations suggested that tlr1, tlr2 tlr4, and cd14 and irak-2 gene expression is induced in ml-1 cells upon stimulation with tpa. this observation is supported by a previous report that tpa induces tlr2 expression in u937 cells. to determine whether tpa - differentiated ml-1 cells exhibit macrophage - like responses following stimulation with bacterial particles lps and lta, cytokine multiplex immunoassay was used to assess the levels of selected cytokine production and release. incubation with lta (10 g / ml) and lps (100 ng / ml) resulted in robust production of various cytokines by the differentiated ml-1cells (figure 5). for example, lps either induced il-1 alpha and beta, il-6, tnf alpha and ip-10, or to a much greater extent than lta. the presence of il-10, il-12p40, and interferon gamma was similar in response to lps and lta. these observations support our previous findings that tpa treatment induces ml-1 cell differentiation to functional macrophages. to determine whether tpa - differentiated ml-1 cells exhibit a change in ros levels in response to lps and lta, luminol - derived cl was used. horseradish peroxidase - dependent luminol - derived cl indicates the presence of extracellular hydrogen peroxide. as shown in figure 6, ml-1 cells exhibit a significant luminol - derived cl, indicative of the presence of extracellular hydrogen peroxide. we have previously determined that in unstimulated macrophages that this basal ros is mitochondrial in origin. the differentiated ml-1 cells exhibited a delayed but a more sustained production of hydrogen peroxide in the presence of lps or lta. addition of dpi (10 m), a potent inhibitor of nad(p)h - dependent enzymes, rapidly suppressed the luminol - derived cl suggesting a role of nadph oxidase and/or mitochondria complex i in the observed ros production by ml-1 cells (figure 6). the basal unstimulated ros production by differentiated ml-1 cells was also blocked by dpi, as previously shown (figure 6). interestingly, the sustained, increased ros with lps or lta treatment was not apparent until 3040 minutes after their addition. in this regard, ros from nadph oxidase in alveolar macrophages is apparent almost immediately after tpa addition to activate pkc. macrophages are key members of the innate immune system and are known to play crucial roles in initiating and maintaining the immune response [38, 39 ]. our immunocytochemistry data show a basal expression of human myeloid ml-1 cells differentiate into macrophage - like cells when incubated with tpa. we have demonstrated that the tpa - induced differentiation signal is mediated via a redox - mediated activation of the extracellular - regulated kinase (erk1/2), a family member of mitogen - activated protein kinases, mapks, to signal cell growth arrest and cell attachment during the initiation of the differentiation process [23, 27 ]. in the present study, we investigated the mechanism by which tpa treatment may lead to increase in ml-1 cell response to stimulation by bacterial particle lps and lta. we have shown that tpa treatments result in the upregulation of tlr1, tlr2, tlr4, and cd14 in ml-1 cells, which lead in part to the potentiation of their responses to lps and lta. this observation correlates with a previous study by shuto. in 2007, which has suggested the existence of a direct correlation between the levels of tlr2 proteins expression and the cellular response to tlr2 ligands in differentiated hl-60 cells. has also shown an induction of tlr2 gene expression in response to tpa in u937 cells. our immunocytochemistry data show a basal expression of tlr4 but not tlr1 in undifferentiated ml-1 cells. however, upon tpa treatment, both tlr1 and tlr4 expressions were upregulated not only at the mrna but also at the protein level. an interesting finding is that interleukin-1 receptor - associated kinases (irak-2) mrna were significantly upregulated in response to tpa treatment in ml-1 cells. the induction of irak-2 expression in ml-1 cells correlates with the observed increase responsiveness to tlr2 and tlr4 ligands (lps and lta). irak-2 has been implicated to participate in multiple toll - like receptor signaling pathways that lead to nf-b activation and inflammatory cytokine production [41, 42 ]. irak-2 loss - of - function mutants or knock - down of irak-2 expression by small interfering rna suppresses tlr3, tlr4, and tlr8 signaling to nf-b in human cell lines, and importantly, tlr4-mediated chemokine production in primary human cells [41, 42 ]. together, these observations strongly suggest that tpa treatment not only induces tlr1, tlr2, and tlr4 genes expression, but also that of the irak-2 in ml-1 cells. irak-2 participates in multiple toll - like receptor signaling pathways that lead to nf-b via activation and inflammatory cytokine production. the regulation of irak-2 gene expression is known to play a role in the control mechanism of inflammatory responses. since ml-1 cells express basal tlr4 level, the upregulation of irak-2 gene expression in response to tpa appears to be at least in part a determinant factor in the increased nfb - mediated cytokines production induced by lps and lta. tlrs expression on macrophages is essential for recognition and response to bacterial particle [811, 43 ]. as such, impaired expression of tlrs for example, macrophages from tlr1 mice and tlr2 mice were shown to be hyporesponsive to borrelia burgdorferi outer - surface lipoprotein (ospa) vaccination. therefore, defects in the tlr1/2 signaling pathway may account for human hyporesponsiveness to ospa vaccination. interestingly, recent studies have proposed that the overexpression of tlr4 may play roles in the occurrence of inflammatory diseases [1719 ]. our suggestion that tlr1 and tlr4 expression required mapk activation is based on (i) our previous observations that tpa induces a ros - mediated erk1/2 activation in ml-1 cells [23, 27 ] and (ii) the tlr1 and tlr4 protein expression in ml-1 induced by tpa was blocked by u0126, an agent that is known to effectively suppress mek activity. this observation is consistent with previous reports by jang. that demonstrated that tpa - induced tlr2 expression in u937 cells ros generation in response to bacterial lps and lta is crucial for pathogen killing by immune cells [8, 44 ]. however, sustained production of ros during immune responses and sepsis can cause damage to macromolecules, cell death and tissue injury [45, 46 ]. in tpa - differentiated ml-1 cells we have identified two sources of ros, nad(p)h oxidase, and the mitochondrial electron transport chain. here, we demonstrated that tpa - differentiated ml-1 cells exhibit a prolonged generation of ros upon stimulation with lps and lta. our conclusion that the mitochondrial electron transport chain and/or nadph oxidase may be the source(s) of this ros is based on the observation that dpi (10 m), an agent that we have shown effective in inhibiting both nadph oxidase and mitochondria complex iin ml-1 cells, immediately and completely suppressed the prolonged ros generation induced by lps and lta. while dpi inhibition does not allow us to conclude the source of ros elicited by lps and lta, there are several studies that have demonstrated interactions between tlr and mitochondria. for example, a recent study has shown that a subset of toll - like receptors (tlr1, tlr2, and tlr4) promote recruitment of mitochondria to macrophage phagosomes and induce the generation of mitochondrial ros. these results implicate mros as an important component of antibacterial responses and establish mitochondria as hubs for innate immune signaling. similarly, toll - like receptor 4 mediates ros - mediated mitochondrial dna damage and biogenic responses in the liver of mice exposed to heat - inactivated e. coli. collectively, the data presented in this study demonstrate that tpa - induced differentiation of ml-1 cells to macrophages is accompanied by the expression of members of the tlr / irak-2/nf-b complex. this leads to a robust downstream tlr / irak-2/nfb - mediated cytokine production and an increase in sustained cellular oxidative metabolism and ros upon cellular interaction with bacterial lps and lta.	12-o - tetradecanoylphorbol 13-acetate (tpa) induces the differentiation of human myeloid ml-1 cells to macrophages. in the current study, the expression, responsiveness, and regulation of toll - like receptors (tlrs) in tpa - induced ml-1-derived macrophages were investigated. we have found that tpa - induced differentiation of ml-1 cells was accompanied by the upregulation of tlr1, tlr2, tlr4, and cd14 expression at both the mrna and protein levels. interestingly, tlr1 and tlr4 protein expression on ml-1 cells could be blocked by pretreatment with u0126, suggesting the role of an erk1/2-induced differentiation signal in this process. in addition, the expression of irak-2, a key member of the tlr / irak-2/nf-b - dependent signaling cascade was also induced in response to tpa. accordingly, we demonstrated an increased cellular release of inflammatory cytokines (tnf- and various interleukins) upon stimulation with lps and lta ligands for tlr4 and tlr2, respectively. furthermore, using luminol - dependent chemiluminescence, addition of lps and lta induces a sustained dpi - inhibitable generation of reactive oxygen species (ros) by the differentiated ml-1 cells. together, these data suggest that the increase in the responsiveness of tpa - treated ml-1 cells to lps and lta occurs in response to the upregulation of their respective receptors as well as an induction of the irak-2 gene expression.
nonalcoholic fatty liver disease (nafl) is a term applied to the accumulation of fat in the liver in the absence of alcohol consumption. nafl refers to a wide spectrum of liver damage ranging from simple steatosis to non - alcoholic steatohepatitis (nash) to advanced fibrosis and finally, cirrhosis. the clinical significance of nafl is derived mostly from its high prevalence in the general population and in its possible progression to liver failure. obesity, type 2 diabetes mellitus and hyperlipidemia are the major risk factors associated with nafl. a bulk of evidence suggests that insulin resistance plays a major role in the pathogenesis of nafl and nash. however with meticulous questioning, more than half would mention fatigue, right upper quadrant pain and occasional discomfort, although the significance of these symptoms is uncertainmost cases initially come to medical attention by the incidental finding of increased liver echogenicity during ultrasonography performed for other reasons. ultrasonography could be used as a non - invasive predictor of liver histology in both moderate and severe steatosis, and advanced fibrosis with acceptable sensitivity and specificity. nevertheless, gradual and sustained weight reduction and exercise are most frequently recommended for overweight nafl patients. weight reduction can not only improve symptoms, but also the risk of progression of nafl to cirrhosis. in the present study we compare the effect of a weight reduction diet on liver ultrasonographic findings in overweight nafl patients. to our knowledge, only one study has thus far researched weight reduction in nafl adults. from july 2003 to december 2005 all patients incidentally found to have fatty liver by ultrasonography who referred to radiology and gastroenterology clinics were included. fasting blood sugar (fbs), and liver transaminase were checked for all patients. waist circumference was measured using a measuring tap placed in a horizontal plane around the abdomen at the level of the iliac creast. patients with body mass index (bmi) above 25 and of the ages 16 to 60 were included in the study. patients not consenting to the study or not able to refer for regular follow - up were also excluded. the degree of steatosis was graded by ultrasonography from 0 to 3 according to table 2. the diet contained 50 percent carbohydrates, 30 percent fat and 20 percent protein given in three meals with a deficit of 500 calories planned to ideally reduce weight by 2 kg per month. patients were followed monthly by a nutritionist. at the end of month three, anthropometric and ultrasonographic measures before and after dieting were compared using student s paired t - test by spss version 14. the study protocol was approved by the institutional review board and ethics committee of the digestive disease research center (ddrc) of tehran university of medical sciences (tums). in this study, 23 patients (13 females, 10 males) were enrolled. the decrease in weight, bmi, waist circumference and hip circumference were statistically significant. the ultrasonographic grade decreased in all patients ; eight had a two grade reduction and 15 patients reduced one grade (table 4). the decrease in ultrasonographic grade had a significant correlation with decrease in weight and bmi, but not with changes in waist and hip circumferences (table 5). the amount of decrease in weight and bmi was significantly greater in patients whose ultrasonographic grades improved by two scores when compared to patients who only had a one score improvement. men had a significantly greater weight loss (p<0.01), but changes in ultrasonographic grade, bmi, waist circumference and hip circumference were not significantly different among the two sexes. the treatment of nafl is controversial and even the necessity of treatment is doubted when there is no indication of liver damage. even in nash, where liver damage is well documented, there is no universally accepted treatment. when treatment is instituted, evaluation of treatment response is usually through transaminase levels or liver histology. in nafl patients, where the transaminase levels are normal to begin with, histology is the only guide to treatment. but liver biopsy is a fairly invasive procedure and is difficult to justify in subjects with normal liver transaminase levels where the prognosis is generally considered to be very good. in such cases another non - invasive method, such as ultrasonography, could be a good substitute. in this study we observed a significant improvement in ultrasonographic features of nafl by a low calorie diet. unfortunately, we did not have histologic confirmation but we believe it is safe to assume that improvement in histology parallels improvement in ultrasonography. ultrasonography has an acceptable sensitivity and specificity in evaluating liver steatosis and provides a non - invasive predictor of liver histology and degree of steatosis in nafl patients. based on the current study, we suggest ultrasonography as a guide to weight loss. we also observed that patients with a higher initial ultrasonographic grade had greater improvement. it can be concluded that more severe cases will respond better to weight loss. in our study, the diet was designed with a modest calorie deficit aiming at a maximum weight loss of 2 kg per month. the gradual weight loss is important as rapid and severe weight loss, starvation, or even total parenteral nutrition, could result in or perpetuate fatty liver disease 3 - 5. although slightly more rapid weight loss might also be safe, a modest diet would result in better patient compliance. as mentioned above, the necessity of treatment for non - complicated nafl with no indication of liver injury is in doubt. but considering the safety and other obvious benefits of gradual weight loss, we believe it should be offered to all overweight cases of nafl. we conclude that weight loss is effective in improving the ultrasonographinc grade of steatosis and ultrasonography may be used as a non - invasive indicator of improvement of liver steatosis in patients with nafl during weight reduction.	background non - alcoholic fatty liver (nafl) includes a spectrum of diseases ranging from simple hepatic steatosis to nonalcoholic steatohepatitis (nash) and cirrhosis. nafl is typically seen in association with obesity, diabetes and hypertriglyceridaemia. in order to seek the role of diet therapy in treatment of nafl, we compared the ultrasonographic findings of patients with fatty liver disease before and after standard diet therapy. methods twenty - three overweight or obese subjects with incidental fatty liver discovered during ultrasonography were included. subjects underwent 3 months of diet therapy, and anthropometric data including weight, height, bmi, waist circumference, and hip circumference were measured. ultrasonographic findings were graded from 0 to 3. changes in ultrasonographic findings and anthropometric data were studied. results after three months of dieting, the ultrasonographic grade of all patients decreased by one or two grades. fifteen patients decreased one grade while 8 others decreased by 2 grades. we observed a significant correlation between the decrease in ultrasonographic grade and the decrease in weight and bmi. conclusion our study indicates that standard diet therapy could be used as an effective treatment for nafl patients.
study of prescriptions is considered as part of drug utilization, which encompasses the study of marketing, distribution and prescription of various drugs in society, along with evaluation of its medical, social, and economic consequences. the basic aim of studying drug utilization is to encourage and facilitate rational use of medications. the importance of psychotropic medications in the management of various psychiatric disorders, almost all treatment guidelines formulated by various scientific organizations provide information about how to use psychotropic agents in the management of a particular disorder. however, it is often discussed that many patients receive irrational prescriptions, which either do not provide any benefit to the patients or actually harm them. accordingly, understanding the prescriptions can partly provide us information about the type of care received by patients. these studies have evaluated the national prescription patterns and have additionally looked at the prescription patterns in general practice, specialist care, emergency services, and extreme of ages. studies have also looked at the prescription patterns specific to various psychiatric disorders such as depression, bipolar disorder, and psychosis. data are also available in the form of prescription patterns for antidepressants, mood stabilizers and antipsychotics in patients with mental disorders. some of the authors have also looked at the influence of demographic factors like gender on the prescription patterns. in recent times, many studies have evaluated the prescription patterns in various asian countries too, in which data from a few centers in india has also been included. some of the studies from india have also evaluated the prescription patterns of patients with various psychiatric disorders, but these studies have included small number of patients and have been mostly limited to one center only. it is often difficult to compare these studies because of the different time frames in which these were conducted. in recent times, a study was published with a large sample size covering patients with different diagnoses, but this study too included patients from one center only. only one multicentric study has evaluated the prescription patterns, but it limited itself to use of antidepressants in patients with depression. this study was conducted under the aegis of indian psychiatric society (ips), and it showed escitalopram was the most commonly prescribed antidepressant, and selective serotonin reuptake inhibitors (ssris) were the most commonly prescribed class of antidepressants. polypharmacy in the form of use of combination of two antidepressants in the same patient was infrequent and benzodiazepines were used as a co - prescription in a significantly higher proportion of patients. most studies from india do not provide data with regard to the dosages used. in this background, this multicentric study funded by the ips aimed to assess the first prescription handed over to the psychiatrically ill patients on their contact with a psychiatrist. the study was approved by the ips ethical review board and/or by the local institutional ethics committees. initially, 22 centers showed their willingness to participate, but finally 11 centers participated. of the centers which participated, maximum numbers of sites were from north india (chandigarh, ludhiana, mullana, rohtak, and udaipur) and there were 2 centers each from central india (agra and lucknow) and western india (ahmedabad and vadodara). there was one center each from eastern part of india (guwahati) and southern india (chennai). among the participating centers, 1 center was in the centrally funded institute (chandigarh), 5 centers were in the state government run medical colleges (agra, ahmadabad, guwahati, lucknow and rohtak), 4 centers were in privately run medical colleges (chennai, mullana, udaipur and vadodara) and 1 privately run clinic (ludhiana). this study followed a cross - sectional design and the prescriptions handed over to the patients were assessed only once at the time of intake into the study. the inclusion criteria for the study was age more than equal to 15 years and the patient must have fulfilled the criteria of one of the mental disorders as per icd-10, assessed by using mini international neuropsychiatric interview. current level of functioning was assessed on the global assessment of functioning (gaf) scale. all patients fulfilling the selection criteria were approached and explained about the purpose of the study. written informed consent was obtained from the patients or their primary caregivers before they were included in the study. descriptive analysis was carried out using mean and standard deviation with range for continuous variables and frequency and percentages for ordinal or nominal variables. the main reason for exclusion of cases was having included patients with only neurological disorders (for example, epilepsy). distribution of patients across different centers as shown in table 2, majority (89.4%) of the patients were in the age range of 20 - 65 years with a mean age of the study sample being 37.28 (sd : 13.53) years. there was nearly equal distribution of patients of either gender, those from nuclear versus nonnuclear families, employed versus unemployed and urban versus rural locality. about two - third of the patients had family income of less than indian rupees 7322 and 71.8% were married. sociodemographic profile of study sample (n=4480) as shown in table 3, more than half of the study sample had the diagnosis of affective disorder, majority being of unipolar depressive disorders (n = 1957) and remaining having a diagnosis of bipolar disorder (n = 474). nearly one - fifth of patients had diagnosis of a psychotic disorder, with schizophrenia (n = 592) being the most common diagnosis in this category followed by unspecified nonorganic psychosis (n = 81), acute and transient psychosis (n = 74), schizoaffective disorder (n = 39) and persistent delusional disorder (n = 33). another one - fifth of the patients had neurotic, stress - related and somatoform disorders, with other anxiety disorders (n = 425) being the most common followed by obsessive compulsive disorder (n = 240). other small groups of neurotic, stress - related and somatoform disorders included dissociative disorders (n = 84), phobic anxiety disorder (n = 54), reaction to severe stress, adjustment disorders (n = 44) and other neurotic disorders (n = 22). diagnostic distribution of the study sample most of the patients (87.5%) were assigned one psychiatric diagnosis ; a few patients (12.2%) had 2 diagnoses, while only occasional patients (0.4%) were diagnosed to have three psychiatric disorders. about one - tenth (n = 451 ; 10.1%) had comorbid neurological diagnosis, comorbid physical illness related to cardiovascular system (i.e. hypertension) was assigned to 4.1% patients (n = 201) and very few patients (n = 98 ; 2.2%) were diagnosed to have an endocrine disorder (i.e. diabetes mellitus or hypothyroidism). the mean gaf scale score for the study sample was 56.73. among the various psychotropics, about one - eighth of the patients were prescribed one of the classical mood stabilizers (i.e. lithium carbonate, valproate, carbamazepine, ox - carbamazepine, and lamotrigine). prescription patterns of the study sample in terms of diagnostic groups, 95.5% of patients with unipolar depression were prescribed one antidepressant, 95.1% of patients with psychosis were prescribed at least one antipsychotic medication, 78.7% of bipolar patients received one antipsychotic medication, and 74.7% of bipolar patients received one of the mood stabilizers. in terms of polypharmacy, that is., prescription of more than one medication from the same group (example, two antidepressants), highest level of polypharmacy was seen for antidepressants (8%), followed by antipsychotics (5.6%). very few patients were prescribed pregabalin as anxiolytic (0.8%) and still fewer (0.3%) were on various cognitive enhancers. in addition, nearly one - fifth of the prescriptions included medications other than psychotropics, in the form of nutritional supplements, trihexyphenidyl, propranolol, antidiabetic and antihypertensive medications. as is evident from table 5, escitalopram was the most commonly prescribed antidepressant, followed by sertraline and amitriptyline. in total, selective serotonin reuptake inhibitors (ssris) formed 71.4% of the total prescriptions. one - fifth (19.2%) of the antidepressant prescriptions were those of tricyclic antidepressants (tcas) (amitriptyline, imipramine, clomipramine, and dothiepin). details of the mean and median doses along with most commonly used doses for each antidepressant are shown in table 5. prescription pattern for antidepressants among the antipsychotics, olanzapine (42.9%) was the most commonly prescribed antipsychotic medication, followed by risperidone (26.7%) and amisulpride (11.6%). details of the mean and median doses along with most commonly used doses for each antipsychotic are shown in table 6. prescription pattern for antipsychotics among the patients prescribed mood stabilizers, about two - third (65.8%) were prescribed valproate, and 22.3% were prescribed lithium. prescription pattern for mood stabilizers among the benzodiazepines, clonazepam formed 64% of all the benzodiazepine prescriptions, and it was followed by lorazepam and diazepam. prescription pattern for benzodiazepines and other sedatives as shown in table 9, olanzapine was the most commonly prescribed antipsychotic followed by risperidone in patients with psychosis and bipolar disorder. risperidone was the most common antipsychotic in the unipolar affective disorders and amisulpride was the most common antipsychotic in the anxiety disorder group. however, it is important to note that most of the patients with unipolar affective disorders and anxiety disorders were not on any antipsychotic medications. escitalopram was the most commonly prescribed antidepressant in patients with affective disorders and anxiety disorders. however, in patients with psychosis, fluoxetine was prescribed more commonly than escitalopram. as is evident from table 9, two - third of patients with psychotic disorder were prescribed benzodiazepines and about 70% of patients with affective disorders and anxiety disorders were prescribed benzodiazepine. in all the diagnostic groups, clonazepam was the most commonly prescribed benzodiazepine, with about half of the patients with unipolar affective disorders and anxiety disorders receiving clonazepam. prescription pattern as per diagnostic categories in terms of diagnosis and polypharmacy, the rate of antidepressant polypharmacy in unipolar depression group was 13.64%, 0.63% in bipolar depression, 10% in neurotic, stress - related and somatoform disorders and 0.35% of patients with psychotic disorders received more than one antidepressant. in terms of antipsychotic polypharmacy, 20.9% of patients with psychotic disorders received more than one antipsychotic medications, and the antipsychotic polypharmacy rate was 0.61% for unipolar depression, 12.65% for bipolar depression and 0.4% for neurotic, stress - related and somatoform disorders. mood stabilizer polypharmacy in bipolar depression was 10.33%, in unipolar depression was 0.11%, in anxiety disorders was 0.2% and that for psychotic disorders was 0.1%. benzodiazepine polypharmacy in unipolar depression group was 1%, in bipolar depression was 0.6%, in anxiety disorders were 1.3% and that for psychotic disorders was 1.1%. for various major medications, few differences were seen across different treatment centers as shown in table 10. among the antidepressants, escitalopram was the most common antidepressant across 7 out of 11 centers and second most common in 3 centers. in terms of antipsychotics, olanzapine was the most commonly prescribed antipsychotic across 6 out of 11 centers and second most common across rest of the centers. among mood stabilizers, valproate was more often prescribed in 8 out of 11 centers and in terms of benzodiazepines, clonazepam was the most common benzodiazepine prescribed in 6 out of the 11 centers. most commonly prescribed medications across different centers in terms of polypharmacy, there were significant differences across various centers. for antidepressants, polypharmacy for mood stabilizers varied from 0% to 27.3% and for benzodiazepines varied from 0% to 8%. as shown in table 2, majority (89.4%) of the patients were in the age range of 20 - 65 years with a mean age of the study sample being 37.28 (sd : 13.53) years. there was nearly equal distribution of patients of either gender, those from nuclear versus nonnuclear families, employed versus unemployed and urban versus rural locality. about two - third of the patients had family income of less than indian rupees 7322 and 71.8% were married. as shown in table 3, more than half of the study sample had the diagnosis of affective disorder, majority being of unipolar depressive disorders (n = 1957) and remaining having a diagnosis of bipolar disorder (n = 474). nearly one - fifth of patients had diagnosis of a psychotic disorder, with schizophrenia (n = 592) being the most common diagnosis in this category followed by unspecified nonorganic psychosis (n = 81), acute and transient psychosis (n = 74), schizoaffective disorder (n = 39) and persistent delusional disorder (n = 33). another one - fifth of the patients had neurotic, stress - related and somatoform disorders, with other anxiety disorders (n = 425) being the most common followed by obsessive compulsive disorder (n = 240). other small groups of neurotic, stress - related and somatoform disorders included dissociative disorders (n = 84), phobic anxiety disorder (n = 54), reaction to severe stress, adjustment disorders (n = 44) and other neurotic disorders (n = 22). diagnostic distribution of the study sample most of the patients (87.5%) were assigned one psychiatric diagnosis ; a few patients (12.2%) had 2 diagnoses, while only occasional patients (0.4%) were diagnosed to have three psychiatric disorders. about one - tenth (n = 451 ; 10.1%) had comorbid neurological diagnosis, comorbid physical illness related to cardiovascular system (i.e. hypertension) was assigned to 4.1% patients (n = 201) and very few patients (n = 98 ; 2.2%) were diagnosed to have an endocrine disorder (i.e. diabetes mellitus or hypothyroidism). about one - eighth of the patients were prescribed one of the classical mood stabilizers (i.e. lithium carbonate, valproate, carbamazepine, ox - carbamazepine, and lamotrigine). prescription patterns of the study sample in terms of diagnostic groups, 95.5% of patients with unipolar depression were prescribed one antidepressant, 95.1% of patients with psychosis were prescribed at least one antipsychotic medication, 78.7% of bipolar patients received one antipsychotic medication, and 74.7% of bipolar patients received one of the mood stabilizers. in terms of polypharmacy, that is., prescription of more than one medication from the same group (example, two antidepressants), highest level of polypharmacy was seen for antidepressants (8%), followed by antipsychotics (5.6%). very few patients were prescribed pregabalin as anxiolytic (0.8%) and still fewer (0.3%) were on various cognitive enhancers. the mean number of medications prescribed for a patient was 2.51. in addition, nearly one - fifth of the prescriptions included medications other than psychotropics, in the form of nutritional supplements, trihexyphenidyl, propranolol, antidiabetic and antihypertensive medications. as is evident from table 5, escitalopram was the most commonly prescribed antidepressant, followed by sertraline and amitriptyline. in total, selective serotonin reuptake inhibitors (ssris) formed 71.4% of the total prescriptions. one - fifth (19.2%) of the antidepressant prescriptions were those of tricyclic antidepressants (tcas) (amitriptyline, imipramine, clomipramine, and dothiepin). details of the mean and median doses along with most commonly used doses for each antidepressant are shown in table 5. among the antipsychotics, olanzapine (42.9%) was the most commonly prescribed antipsychotic medication, followed by risperidone (26.7%) and amisulpride (11.6%). details of the mean and median doses along with most commonly used doses for each antipsychotic are shown in table 6. prescription pattern for antipsychotics among the patients prescribed mood stabilizers, about two - third (65.8%) were prescribed valproate, and 22.3% were prescribed lithium. the other details of prescriptions among the benzodiazepines, clonazepam formed 64% of all the benzodiazepine prescriptions, and it was followed by lorazepam and diazepam. as shown in table 9, olanzapine was the most commonly prescribed antipsychotic followed by risperidone in patients with psychosis and bipolar disorder. risperidone was the most common antipsychotic in the unipolar affective disorders and amisulpride was the most common antipsychotic in the anxiety disorder group. however, it is important to note that most of the patients with unipolar affective disorders and anxiety disorders were not on any antipsychotic medications. escitalopram was the most commonly prescribed antidepressant in patients with affective disorders and anxiety disorders. however, in patients with psychosis, fluoxetine was prescribed more commonly than escitalopram. as is evident from table 9, two - third of patients with psychotic disorder were prescribed benzodiazepines and about 70% of patients with affective disorders and anxiety disorders were prescribed benzodiazepine. in all the diagnostic groups, clonazepam was the most commonly prescribed benzodiazepine, with about half of the patients with unipolar affective disorders and anxiety disorders receiving clonazepam. prescription pattern as per diagnostic categories in terms of diagnosis and polypharmacy, the rate of antidepressant polypharmacy in unipolar depression group was 13.64%, 0.63% in bipolar depression, 10% in neurotic, stress - related and somatoform disorders and 0.35% of patients with psychotic disorders received more than one antidepressant. in terms of antipsychotic polypharmacy, 20.9% of patients with psychotic disorders received more than one antipsychotic medications, and the antipsychotic polypharmacy rate was 0.61% for unipolar depression, 12.65% for bipolar depression and 0.4% for neurotic, stress - related and somatoform disorders. mood stabilizer polypharmacy in bipolar depression was 10.33%, in unipolar depression was 0.11%, in anxiety disorders was 0.2% and that for psychotic disorders was 0.1%. benzodiazepine polypharmacy in unipolar depression group was 1%, in bipolar depression was 0.6%, in anxiety disorders were 1.3% and that for psychotic disorders was 1.1%. for various major medications, few differences were seen across different treatment centers as shown in table 10. among the antidepressants, escitalopram was the most common antidepressant across 7 out of 11 centers and second most common in 3 centers. in terms of antipsychotics, olanzapine was the most commonly prescribed antipsychotic across 6 out of 11 centers and second most common across rest of the centers. among mood stabilizers, valproate was more often prescribed in 8 out of 11 centers and in terms of benzodiazepines, clonazepam was the most common benzodiazepine prescribed in 6 out of the 11 centers. most commonly prescribed medications across different centers in terms of polypharmacy, there were significant differences across various centers. for antidepressants, polypharmacy for mood stabilizers varied from 0% to 27.3% and for benzodiazepines varied from 0% to 8%. this is the first multicentric study from india, which has attempted to evaluate the prescription pattern of various psychotropic agents across multiple diagnostic groups. this study included 4480 patients aged 15 or more years spread across 11 centers in india. the main focus of the study was prescriptions given to consecutive patients contacting the specific mental health services for the first time, irrespective of their past treatment history. this study shows that the escitalopram is the most commonly prescribed antidepressant, followed by sertraline and amitriptyline. as a class when we compare these findings with other studies from india, findings concur with some of the studies, but differ from that reported from other centers and from those reported more than a decade ago. when we compare the findings of this study with reports from the west, certain similarities are again noted. a multicountry study from europe, which evaluated the prescription of antidepressants to patients with first episode depression or those suffering from recurrent depressive disorder and requiring an antidepressant for a new episode reported that about two - third (63.3%) of patients were prescribed an ssri, whereas snri formed 13.6% of the total antidepressant prescriptions. a multicountry survey from asia also reported that together ssris and snris form 77% of all antidepressant prescriptions. in this study, tcas formed one - sixth (16.8%) of the total prescriptions, with amitriptyline being the most commonly prescribed antidepressant from this class. thus, tcas are still considered an important option in the management of depression and this finding is similar to the findings noted by grover. in their study from chandigarh. however, when compared with older studies over the years there is a reduction in the prescriptions of tcas. in various diagnostic groups, escitalopram again emerged as the most commonly prescribed antidepressant medication in patients with unipolar depression, in patients with bipolar disorder and those with neurotic, stress - related and somatoform disorders and it emerged as the second most commonly prescribed antidepressant in those with psychotic disorders. these findings suggest that, probably diagnosis is not the only factor that influences the choice of antidepressants, and there could be many other factors such as clinicians experience, patient profile, side - effect profile of medication, etc. there is wide variation in the concomitant antidepressant prescription in patients with schizophrenia as reported in the literature. some of the studies from asian countries report similar prescription rates for antidepressants in patients with schizophrenia as seen in this study. however, a study from malaysia reported that majority of patients with schizophrenia received antidepressants. the rate of antidepressant prescriptions in this study are much lower than that reported in the studies from west, which reported antidepressant prescription rates of 37.4 - 40% in patients with schizophrenia. it is important to note that the lower antidepressant prescription rate in patients of schizophrenia could have been influenced by the fact that the present study focused only on the first prescriptions. it is quite likely that most patients with psychotic disorders might have presented in the acute psychotic phase, when clinicians would prefer to use antipsychotics only. other reason for lower rate of antidepressant prescriptions in patients with psychosis in the present study could be the recent evidence that suggests that some of the atypical antipsychotics also influence mood. in the present study, this prescription rate of antidepressants in patients with bipolar disorder is similar to that reported in previous studies from india. the commonly prescribed antidepressants in bipolar disorder group in this study included escitalopram, fluoxetine and sertraline, which is also very akin to the previous study, which also reported escitalopram and sertraline to be the commonly prescribed antidepressants in patients with bipolar disorder. however, this rate of antidepressant prescription in patients with bipolar disorder is much less than that reported from some of the western countries. the lower prescription of antidepressants in this group could be a reflection of concern for manic / hypomanic switch. in psychotic patients, olanzapine was the most commonly prescribed antipsychotic medication, followed by risperidone, amisulpride, and quetiapine. typical antipsychotics formed about 18% of all antipsychotic prescriptions, and the rest were all atypical antipsychotics. this prescription pattern is very similar to that reported in previous studies from india as well as in the survey conducted on indian psychiatrists. when we compare the findings with those reported from western countries and other countries from asia, certain similarities are evident. recent studies from various part of the world suggest increase in the prescription of atypical antipsychotics contributing to about 76.9 - 77.2% of all antipsychotic prescriptions. however, a study from malaysia reported haloperidol to be the most commonly used oral antipsychotic medication and atypical antipsychotics formed only 32% of all the prescriptions. all these findings suggest that many factors like availability of particular medications in a country, availability in the dispensary, cost, etc., could influence the prescription patterns, and there is a need to evaluate the role of these factors. nearly 75% received monotherapy (i.e. only one antipsychotic medication), 20.9% patients received more than one antipsychotic medications, and 4.9% did not receive any antipsychotic. this rate of monotherapy, nonprescription of antipsychotics and polypharmacy in patients with psychosis is comparable to that reported from other countries. the proportion of patients receiving antipsychotic polypharmacy in this study was much less than 51.6%, reported in a multicountry study from asia. focused on inpatients whereas the present study evaluated the prescriptions at the first contact. a study from finland assessed antipsychotic polypharmacy in outpatients with diagnosis of schizophrenia and reported that 46.2% of patients received polypharmacy ; however, it is important to note that the study defined polypharmacy as concurrent use of 2 antipsychotics for the duration of at least 2 months. risperidone, amisulpride and olanzapine were the most commonly prescribed antipsychotics in this diagnostic group. this antipsychotic prescription rate in unipolar depression group is about half than that reported in some of the studies from the west, but is similar to the 8.6% prescription rate of second generation antipsychotics in patients with nonpsychotic depression. when we compared the antipsychotic prescription rate in patients of unipolar depression with previous studies from india, the rates were slightly higher than one of the earlier studies (6% vs. 10% in the current study), but less than that reported in other studies. these variations could be attributed to the differences in the clinical profile of the patients studied. more than three - fourth of the patients with bipolar disorder received antipsychotics ; olanzapine, risperidone, and quetiapine being the most commonly prescribed antipsychotic in this diagnostic group. this rate is much higher than that reported in previous studies from other countries and india. however, the preference of atypical antipsychotics in this group is similar to the findings from other countries and is possibly a reflection of the emerging evidence that atypical antipsychotic also have mood stabilizing properties. about one - eighth of the patients with neurotic, stress - related and somatoform disorders received antipsychotics ; amisulpride, aripiprazole, olanzapine, and risperidone were the most commonly prescribed antipsychotics in this diagnostic group. the rate of antipsychotic prescription in patients with neurotic, stress - related and somatoform disorders is much higher than that reported in a previous study from india, but lower than that reported in a study from the west in which antipsychotic prescription rates in patients with anxiety disorders was 10.6% during the years 1996 - 1999 and it increased to 21.3% during the years 2004 - 2007. in the present study, nearly three - fourth of the patients with bipolar disorder were prescribed one of the classical mood stabilizers, with valproate being prescribed much more than other mood stabilizers. existing literature is somewhat contradictory with respect to mood stabilizer prescriptions in patients with bipolar disorder with some studies reporting 72 - 82% the prescription rate of classical mood stabilizers in bipolar patients. many studies from the west have reported more use of lithium compared to valproate contrary to the findings of the present study. conversely, preference for valproate over lithium is also supported by some of the studies and also by the data, which suggests that over the years valproate is increasingly preferred compared to lithium. earlier studies from india too suggest contradictory trends ; one study reported higher use of valproate, while the other reported preference for lithium. these differences could be due to different samples studied i.e., one study focused on follow - up patients and the other evaluated the first prescription of patients. higher preference of valproate in the first prescriptions of patients with bipolar disorder could be due to the evidence for higher efficacy of valproate in treatment of acute mania. other reasons of preference for valproate could be the ease in increasing its doses, and relatively lesser number of investigations required both prior to starting of valproate and thereafter. very few patients (4%) in this study with psychotic disorders received concurrent mood stabilizers studies for various other countries suggest that. prescription of mood stabilizers was found in 20.4% of patients with schizophrenia admitted to inpatient units in various countries of asia. the mood stabilizer prescription rate in patients with psychosis as noted in this study is higher than that reported by a previous study from india. it is difficult to account for such differences in the absence of the study of factors determining the drug - prescription. in the unipolar depression (7%) and neurotic, stress - related and somatoform disorders (3.3%) groups although few patients received mood stabilizers yet this prescription rate was significantly higher than that reported by grover. as discussed earlier, these higher prescription rates could be due to variation in prescription patterns across different study centers. more than two - third of patients with any of the diagnostic group were prescribed a benzodiazepine, with clonazepam being the most commonly prescribed agent in all the diagnostic groups. lorazepam was the second most commonly prescribed benzodiazepine in the psychotic and bipolar disorder group ; diazepam was the second most commonly prescribed benzodiazepine in the unipolar depression group ; etizolam was the second most commonly prescribed benzodiazepine in the neurotic, stress - related and somatoform disorders group. these benzodiazepine prescription rates are similar to those of the earlier study from a center in north india, but are less than that reported for depression and bipolar disorder in studies from lucknow. the benzodiazepine prescription rates across different disorders observed in this study was much higher than that reported in studies from the west. when one looks at the doses of various medications, it is evident that lower doses are more preferred by the psychiatrists in india for most of the antidepressants, antipsychotics, mood stabilizers and benzodiazepines. the commonly used doses for most of the psychotropics in majority of the patients are in the usual recommended doses as given in the various treatment guidelines. this is contradictory to many studies from the west, which report use of megadoses of various medications in some of the patients. when we compare the doses with that reported in the survey of psychiatrists from india, the doses in this study were on the lower side. a possible reason for the same could be that in the present study we had evaluated the first prescription only. a study from the asian countries reported that about 18% of patient with schizophrenia were prescribed high doses of antipsychotic (i.e. more than 1000 mg / day of chlorpromazine equivalent). the proportion of patients receiving doses of antipsychotics on the higher side was much lower in the present study. rate of antidepressant polypharmacy (i.e. concurrent use of two antidepressants) in the index study was 8% for the whole study sample, this is about half the rate reported in the previous ips multicentric study which looked at the prescription of antidepressants in patients with depression. antidepressant polypharmacy in patients with depression in the present study was comparable with that in the previously reported study. the rate of antidepressant monotherapy in this study is significantly higher than that reported in a study from japan ; however, it is important to note that the study from japan followed a longitudinal study design and the patients were evaluated over a period of time. it is quite possible that while carrying out the longitudinal assessments, it becomes more evident that certain patients do not respond to a particular antidepressant and resultantly require combination / augmentation with another agent. in terms of antipsychotic polypharmacy, one - fifth of patients with psychotic disorders received more than one antipsychotic medication concurrently, and the antipsychotic polypharmacy rate was 0.61% for unipolar depression, 12.65% for bipolar depression and 0.4% for neurotic, stress - related and somatoform disorders. these rates are much higher than that reported in a previous study from india for all the diagnostic groups and that reporting prescription patterns in patients with bipolar disorder. the overall rate of use of antipsychotic combinations across different age groups in this study was less than that reported in some of the studies from the west. mood stabilizer polypharmacy in patients with bipolar disorder was 10.33%, less than that reported in studies from the west. in terms of variation across different centers, escitalopram emerged as the most commonly used antidepressant in 7 out of the 11 participating centers and was ranked as the second most commonly used antidepressant in other 3 centers. therefore, it can be concluded that the results of the present study were not influenced by the prescription patterns of one particular center alone. of the 11 centers, olanzapine and risperidone emerged in 8 centers as the two most commonly prescribed antipsychotics. in other 3 centers, again olanzapine was among the two most commonly used antipsychotic. for mood stabilizers, valproate emerged as the most commonly prescribed mood stabilizer across 8 different centers and second most commonly prescribed mood stabilizer in 2 out the remaining 3 centers. this suggests a preference of valproate over other mood stabilizers in management of acute symptoms of bipolar disorder. clonazepam emerged as the most commonly prescribed benzodiazepine across 7 of the 11 centers and second most commonly prescribed benzodiazepine in 3 out of the remaining 4 centers. however, there were certain differences in the rate of polypharmacy across different treatment centers, with some centers virtually reporting no same class polypharmacy. the study was limited to assessment of the first prescription handed over to the patients, and this does not reflect the prescriptions received by patients in various stages of their illness. the study was also limited by the cross - sectional assessment and was not able to provide information about the duration of prescriptions. further, the study did not evaluate the relationship of prescriptions with particular symptoms per se and was limited to the presence or absence of a particular diagnosis. the study also did not focus on assessment of side - effects experienced by the patients, nor did it evaluate the factors associated with the prescriber which can influence the prescription patterns. this study also does not provide information about the tolerability of various doses used and can not be generalized to inpatients, who are often more severely ill and require higher doses of medications. the study also did not look at the relationship of prescription patterns and drugs available free of cost in the dispensary of the hospital. similarly, although the study focused on first prescriptions, it must be understood that all the patients were not necessarily seeking treatment for the first time or were drug - nave. it is quite possible that a proportion of patients would have been on treatment prior to contacting the particular study site. in future multicentric studies must evaluate the relationship of prescriptions with various clinical parameters such as age of onset, duration of symptoms, past history of treatment and type of symptoms, past history of side - effects, etc., in addition, the studies must attempt to evaluate the prescriptions over a period of time to study the duration of use of each medication, and the side - effects experienced, in order to reach a better conclusion in terms of polypharmacy and particularly the use of drugs like benzodiazepines. this study suggests that there is little difference in prescription patterns of various psychotropics across different treatment centers, irrespective of the treatment setting, that is., central institute, government medical college or private practice, except for the prevalence of polypharmacy. olanzapine and risperidone are the most commonly prescribed antipsychotics, valproate and lithium are the most commonly prescribed mood stabilizers and clonazepam and lorazepam are the most commonly prescribed benzodiazepines.	background : there is a lack of national level data from india on prescription of psychotropics by psychiatrists.aim and objective : this study aimed to assess the first prescription handed over to the psychiatrically ill patients whenever they contact a psychiatrist.materials and methods : data were collected across 11 centers. psychiatric diagnosis was made as per the international classification of diseases classification of mental and behavioural disorders 10th edition criteria based on mini international neuropsychiatric interview, and the data of psychotropic prescriptions was collected.results:study included 4480 patients, slightly more than half of the subjects were of male (54.8%) and most of the participants were married (71.8%). half of the participants were from the urban background, and about half (46.9%) were educated up to or beyond high school. the most common diagnostic category was that of affective disorders (54.3%), followed by neurotic, stress - related and somatoform disorders (22.2%) and psychotic disorders (19.1%). other diagnostic categories formed a very small proportion of the study participants. among the antidepressants, most commonly prescribed antidepressant included escitalopram followed by sertraline. escitalopram was the most common antidepressant across 7 out of 11 centers and second most common in three centers. among the antipsychotics, the most commonly prescribed antipsychotic was olanzapine followed by risperidone. olanzapine was the most commonly prescribed antipsychotic across 6 out of 11 centers and second most common antipsychotic across rest of the centers. among the mood stabilizers valproate was prescribed more often, and it was the most commonly prescribed mood stabilizer in 8 out of 11 centers. clonazepam was prescribed as anxiolytic about 5 times more commonly than lorazepam. clonazepam was the most common benzodiazepine prescribed in 6 out of the 11 centers. rate of polypharmacy was low.conclusion:escitalopram is the most commonly prescribed antidepressant, olanzapine is the most commonly prescribed antipsychotic and clonazepam is most commonly prescribed benzodiazepine. there are very few variations in prescription patterns across various centers.
isotretinoin is a vitamin a synthetic analogue that has been widely used by dermatologists in the treatment of acne. among the many adverse effects of isotretinoin, eye dryness, corneal and retinal abnormalities, myalgia, true myopathy, psychiatric problems, and several autoimmune disorders have been described [2, 3, 4, 5, 6, 7, 8 ]. a literature review has revealed very few case reports of autoimmune disorders associated with isotretinoin intake. isotretinoin - induced inflammatory bowel disease (ibd) was the most common of these autoimmune disorders. some of the patients developed ibd after discontinuation of isotretinoin, while some developed ibd during the intake of isotretinoin. there is only one report of an association between isotretinoin intake and immune - mediated diabetes mellitus. in this case, it was concluded that latent autoimmune diabetes mellitus could be clinically revealed after isotretinoin use. guillain - barr syndrome, which is an autoimmune disease of the nervous system, has been reported in two patients taking oral isotretinoin, and both cases received intravenous immunoglobulin. to the best of our knowledge, this is the first reported case of isotretinoin - induced autoimmune thyroiditis and ocular myasthenia gravis (omg) resulting from use of isotretinoin. a 19-year - old caucasian male was referred to our clinic, with a three - week history of diplopia and variable bilateral ptosis (fig. 1 and fig. usa) at 1 mg / kg / day for severe acne disease, for the previous six months. no use of other medications or occurrence of any stressful events was recorded in his recent history. he first experienced a sudden onset of right eye ptosis, which recovered spontaneously within two weeks. one week later, he noted left eye ptosis that was resolved within one week. two weeks later, the right eye ptosis recurred and was accompanied by sudden onset of diplopia. the ophthalmic, general medical and family histories for neuromuscular or autoimmune disorders were negative. physical examination showed moderate periorbital edema and limitations of up- and down - gaze in the left eye. thyroid function tests revealed free t4 of 3.08 ng / dl (normal range 0.931.7 ng / dl), free t3 of 13.85 pg / ml (normal range 2.04.4 pg / ml) and thyroid - stimulating hormone (tsh) < 0.005 mu / ml (normal range 0.274.2 iu / ml). the anti - thyroglobulin antibody level was normal, whereas tsh receptor antibody (trab) [40 u / l (normal < 15 u / l) ] and anti - thyroid - peroxidase antibody levels were elevated [492.2 iu / ml (normal 034 iu / ml) ]. the thyroid ultrasound showed moderately heterogeneous, reduced echogenicity, which was consistent with thyroiditis. propylthiouracil (propycil), at 200 mg / day, was prescribed by the endocrinologist. the acetylcholine receptor antibody test, tensilon test, and single fiber electromyography were normal. computerized tomography of the chest showed no evidence of thymomas. in accordance with the clinical characteristics, oral pyridostigmine (mestinon, valeant pharmaceuticals, aliso viejo, calif., usa) at 180 mg / day was initiated. at the second - month follow - up, usa), at 2.5 units, was injected into the right inferior rectus muscle. after one month, variable ptosis recurred and diplopia persisted. in order to exclude myopathies ragged red fibers were seen in the samples, which was consistent with a mitochondrial cytopathy (mc). pyridostigmine was stopped and oral co - enzyme q10 (co - enzyme q10, life time, meridian, idaho, usa) at 30 mg / day was initiated. cardiac and neuromuscular disorders may be associated with mc, but were not detected in the patient. no clinical improvement was seen within one month ; therefore, co - enzyme q10 was discontinued. oral pyridostigmine at 360 mg / day and oral prednisolone (deltacortril, pfizer, new york, n.y., usa) at 40 mg / day were initiated to treat the concomitant autoimmune thyroiditis and omg. regarding the patient presented in this case report, there is a possible relationship between isotretinoin intake and concomitant autoimmune thyroiditis and omg. the clinical symptoms did not reverse upon completion of the acne treatment, since the autoimmune reactions had already been triggered. it is reported that isotretinoin has immunomodulating effects that may induce some autoimmune diseases such as crohn 's disease, immune - mediated diabetes, and guillain - barr syndrome [5, 6, 7, 8, 9 ]. genetic predisposition and a variety of environmental factors drugs are one of the important environmental factors, which may be the trigger factor for the development of autoimmune diseases in genetically predisposed people. it has been shown that isotretinoin is involved in induction of apoptosis, activation of t cells and b cells [9, 11, 12 ]. in our case, it is unclear how isotretinoin caused the development of autoimmune thyroiditis and omg, but it is possible to suggest that isotretinoin triggered autoimmunity through its immunomodulating effects [9, 11, 12 ]. most screening tests for mitochondrial cytopathy are not sensitive, and in this case report, the observed ragged red fibers probably led to a false positive diagnosis. the clinical and laboratory findings and the response to combined pyridostigmine and steroid treatment were consistent with concomitant autoimmune thyroiditis and omg. the laboratory and thyroid ultrasound findings and moderate periorbital edema suggested thyroid eye disease (ted). graves disease was the most likely etiology for the current ted, because tsh receptor antibody was elevated in addition to hormone levels, indicating hyperthyroidism. periorbital swelling, eyelid retraction, proptosis, and restrictive and congestive ocular myopathy are common signs of ted. in the present case, diplopia, due to involvement of extraocular muscles, was a persistent finding and did not resolve until combined high - dose oral pyridostigmine and prednisolone was initiated. however, normal orbital mri and negative forced duction tests did not support a diagnosis of graves ocular myopathy. however, ptosis, which is not a feature of ted, was one of the presenting signs in the present case. the spontaneous recovery of the ptosis at the second - month follow - up and the following recurrences of the ptosis were most likely due to the fluctuating nature of omg. however, it has been reported that these tests may be negative in some omg cases. in the current case, the variable ptosis, the limitations of eye movements, and response to pyridostigmine were used to determine the diagnosis of omg. steroids may be effective in both ted and omg, supporting concomitant diagnosis of both diseases. although oral steroid and pyridostigmine relieved the symptoms, thyroid hormone levels must be checked during the follow - up. although a clinical diagnosis may be confirmed by laboratory testing, clinical findings of fluctuating and fatigable weakness leading to ptosis and diplopia are the most important elements of diagnosis. in the present case, the emphasis was solely on ted, because of negative test results for omg. variable ptosis and involvement of the extraocular muscles without any enlarged muscles, as observed by orbital mri, supported the diagnosis of concomitant autoimmune thyroiditis and omg. patients with acne should be assessed for autoimmune disorders or any predisposition to their development prior to treatment. clinicians prescribing isotretinoin should be aware of the possible association between isotretinoin intake and concomitant autoimmune thyroiditis and omg. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal.	introductionthere are many adverse effects that have been described for isotretinoin. to the best of our knowledge, this is the first report of a possible association of oral isotretinoin intake with autoimmune thyroiditis and ocular myasthenia gravis (omg).case presentationa 19-year - old caucasian male, who had used oral isotretinoin for severe acne disease for the previous six months, was referred to our clinic. he had a three - week history of diplopia and variable bilateral ptosis. physical examination showed moderate periorbital edema and limitations of up- and down - gaze in the left eye. laboratory findings and thyroid ultrasound were consistent with autoimmune thyroiditis. antithyroid therapy did not relieve the clinical symptoms. concomitant omg was suspected. variable ptosis and a positive response to oral prednisolone of 40 mg / day and pyridostigmine of 360 mg / day supported the diagnosis of concomitant autoimmune thyroiditis and omg.conclusionautoimmune disorders may be triggered by oral isotretinoin treatment. clinicians prescribing isotretinoin should be aware of the possible association between isotretinoin intake and concomitant autoimmune thyroiditis and omg.
a 40-year - old colombian man living in central colombia, bogota, was referred to local hospital for turbid white urine that had appeared 2 months earlier. milky. the phenomenon was intermittent : the patient would pass discoloured urine during 4 or 5 days without complaints of fever, burning or urgency. thereafter, the urine was clear or pink. this anxious man had sought medical attention in various facilities, and despite the fact that no urinary tract infection was found by urine cytology and cultures, he had been repeatedly treated by urinary antibiotics that did not produce any effect on his albinuria. causes of turbid white urine at first visit to our nephrology unit, the patient kept describing his urine as milky. we verified that his description was appropriate by collecting urine in various occasions. in fact, analysing the urine revealed a massive proteinuria of 5 g / l, microhaematuria (1520 red cells per ml) and leucocyturia (1015 leucocytes / ml).. a closer examination of urinary cytology showed that the white cell population consisted of abundant lymphocytes. further investigations revealed a normal renal function, with a creatinine clearance in the order of 105 ml / min. proteinuria was profuse (6.7 g/24 h), but serum albumin concentration was normal (43 g / l), and so were his serum lipids (total cholesterol : 4.9 mmol / l, triglycerides 1.9 mmol / l). no oedema, no hypoalbuminaemia, no hyperlipidaemia and fasting coincided with a clearing of the urine so it could not be a nephrotic syndrome. the rest of the laboratory work up was also non - contributive : leucocytes 5700/mm, (neutrophils 64%, lymphocytes 23%), haemoglobin 16.5 g / dl, haematocrit 48.2%, platelets 285.000/mm. a cystoscopy that found white urine in the bladder (thus excluding malingering) did not disclose tumoural or infectious lesions. the patient observed that the hospital diet that he found meagre also coincided with a clearing of the urine. this remark prompted us to ask the department of nutrition to give him fat meals. with a lipid - rich diet urine turned white and clotty. with a fat - free diet it was found that milky urine was demulsified with chloroform and sudan dye made it turn red. a new cystoscopy was made with previous administration of a fat - rich diet and disclosed a punctiform hole in the posterior wall of the bladder with a leak of white fluid (figure 3). iodopovidone closed the fistula and the patient was discharged with instructions to avoid fat foods. cystoscopic view of white urine flowing from a hole in the bladder wall (arrow). its differential diagnosis includes phosphates, calcium and urates that in excess give the urine a turbid, white or cloudy appearance, although the amounts of these minerals are rarely high enough to produce albinuria. the urine ph plays a role in provoking a turbid discolouration of the urine : claude bernard in his doctoral thesis presented in paris in 1843 explained that rabbits, a species feeding on vegetal, excrete alkaline turbid urine that turns clear by adding acid to it. when thinking of mineral crystal deposits, some clues are given by the urine ph : phosphaturia is related to alkaline urine and sediment analysis shows identifiable crystals. when chyluria is centrifuged, it remains white, which permits to differentiate it from mineral deposits. other substances that can induce albinuria are pus in severe urinary tract infections, or caseous material in urinary tuberculosis (tb). once such causes have been ruled out, the diagnosis of chyluria is likely to be appropriate, but this has no reason to be in non - endemic regions of filariasis infection, which was the case in our patient who lives in central colombia. chyluria implies an abnormal communication between lymphatics and the urinary tract, mediated by any obstruction of intestinal lymph drainage resulting in lymphatic vessel dilatation and rupture into the urinary tract. this parasitic disease is frequent in geographic areas between latitudes 40 north and 30 south. about 10% of the population in these countries suffer from filariasis, and one - tenth of the affected patients present with albinuria caused by urinary - lymphatic fistulae. nonetheless, chyluria can be related to other rare non - parasitic diseases such as fungal infections, congenital abnormalities of the lymphatic vessels, malignancy, trauma and pregnancy. white urine emission alternates with macrohaematuria and is sometimes accompanied with renal colic produced by chyle clots. proteinuria is explained by the high concentration of albumin and fibrinogen in chyle (3060 g / l), and leucocyturia consists of lymphocytes. a lipid - rich meal is rapidly followed by albinuria, whereas a fat - free the urine to turn clear. firstly, it shows that chyluria may occur in any part of the world, and that european readers may come across a case of albinuria in a patient who never lived in an endemic parasitic region where w. bancrofti infection is common. secondly, diagnosing chyluria may simply be achieved by dietary manoeuvres, alternating a lipid - rich diet followed by a fat - free diet. however, albinuria and chyluria may be caused by congenital or acquired aetiologies of lymphatic fistulae. when fortune smiles, as it was in our case, the urologist may observe white urine running from a punctiform hole and treat albinuria by sclerotherapy or povidone injection into the fistula. however, a fat - poor diet must be pursued : the fistula might reopen, and besides, a patient whose bmi is 30 can not but benefit from it.	turbid white urine albinuria is defined as a urine discoloration described as milky or cloudy. one of the most frequent causes of turbid white urine is chyluria complicating filariasis (table 1). the extant causes of albinuria are non parasitic and rare. amongst their aetiologies stand excessive mineral sediment excretion such as calciuria and phosphaturia, massive pyuria and fungal infections, and rarely congenital malformations of the lymphatic vessels. malingering is also possible, in patients adding milk to their urine. we observed a case of albinuria in which the diagnostic work up led to diagnosing an exceptional cause of chyluria in a patient living in a region of colombia where filariasis is not endemic.
stress granules (sgs) are large cytoplasmic rna - protein assemblies that form during cellular stress, and play a pivotal role in the integrated stress response (kedersha., 2013). following translational arrest, polysomes disassemble leading to a sudden increase in free mrna levels in the cytoplasm. it is believed that this free mrna subsequently nucleates the formation of sgs by binding of a large set of rna - binding proteins (rbps) (jain., 2016). interestingly, several of these sg proteins have been linked to the neurodegenerative disorders amyotrophic lateral sclerosis (als) and frontotemporal lobar degeneration (ftld), in which neuronal cytoplasmic fibrillar aggregates of rbps, e.g., tdp-43 and fus, appear (boeynaems. it is thought that these inclusion bodies play a key role in the disease pathogenesis (ramaswami., 2013). moreover, mutations in several of these sg proteins are also the genetic cause of the disease in a subset of familial cases (renton., 2014). hence, sgs have been suggested as a stepping stone towards pathological aggregation in als and ftld (aguzzi and altmeyer, 2016, king., 2012, based on previous observations from other rna granules and nucleoli (brangwynne., 2009, brangwynne., 2011), recent studies have shown that the physical basis of sg formation is liquid - liquid phase separation (llps) (burke., 2015, conicella., 2016, lin., 2015,, 2015, murakami., 2015, patel., 2015). depending on in vitro conditions, recombinant tdp-43, fus, and other hnrnps spontaneously demix from an aqueous solution and form liquid - like protein - rich droplets. it was shown that the intrinsically disordered prion - like domains (prlds) of these proteins by themselves are sufficient in inducing llps. interestingly, these liquid - like droplets can mature over time to more fibrillar states, and this process is accelerated by disease - causing mutations (conicella., 2016, molliex., 2015, murakami., 2015, patel., 2015). other reports have shown that these prlds can also form reversible hydrogels, which have a high sheet content (kato., 2012). it was recently suggested that cellular sgs consist of a stable core surrounded by a liquid - like shell, and atp - dependent remodeling seems necessary for maintaining sg fluidity (jain., 2016, kedersha., 2016). these data point at the importance of a tight control of sg dynamics in preventing pathological aggregation. as mentioned above, prlds can undergo llps or form hydrogels in vitro (burke., 2015, kato., 2012, lin., 2015,, 2015, murakami., 2015, patel., 2015). this type of low complexity domain (lcd) shows a strong sequence similarity to yeast prions in that they are enriched in polar uncharged amino acids (usually glutamine / asparagine) and glycine (king., 2012). degenerate tyrosine - containing repeats in the prlds of fus and other hnrnps, which can mediate hydrophobic and interactions, seem to be crucial in promoting hydrogel formation (kato., 2012). the llps of these prlds indeed is inhibited by aliphatic alcohols, which interfere with hydrophobic interactions (molliex., phase separation based on hydrophobic interactions is also likely to underlie nuclear speckle and nuclear pore mesh formation (frey., 2006, moreover, we find that pathogenic arginine - rich dipeptide repeats in c9orf72 als / ftld also undergo llps in vitro and in cells, hereby uncovering a potential new toxic pathway in the disease. sg proteins also contain other domains that might be non - redundant in phase separation. besides canonical rna - binding domains, e.g., rrm, these proteins are also enriched in disordered arginine - rich sequences, e.g., rgg boxes (aguzzi and altmeyer, 2016) (figure 1a), which can bind rna or poly(adp - ribose) and seed llps or aggregation of prlds (altmeyer., 2015, burke., 2015, lin., 2015, molliex., 2015, patel., 2015, schwartz., 2013). while globular rrms have a clear rna - binding function, these disordered arginine - rich motifs are more promiscuous and also serve as protein binding modules (dormann., 2012, thandapani., 2013). given the strong conservation of both disorder and arginine content in rbps (varadi., 2015), we hypothesize that arginine - rich domains could play a role in llps in physiological and pathological processes. in fact, 40 amino - acid - long arg / gly - rich peptides from the rgg boxes of fus, hnrnpa1, and fmrp spontaneously phase separate in the presence of a molecular crowder (figures 1b1d, s1a, and s1b). this emerging basic mechanism of llps puts particular emphasis on recent observations from c9orf72 hexanucleotide repeat expansions, which are the most common genetic cause of als and ftld (dejesus - hernandez. a potential toxic mechanism is the generation of five species of dipeptide repeats (dprs) through aberrant translation of the repeat rna (ash. several groups have shown that two of them, namely, glycine - arginine (gr) and proline - arginine (pr) repeats, are highly toxic (boeynaems., 2016a, freibaum., 2015, jovii., 2015, we wondered whether these peptides exhibit a behavior similar to the rgg peptides in vitro. synthetic gr20 and pr20, but not gp20, strongly phase separated following the addition of a molecular crowder (figures 1c and 1d). interestingly, the extent of phase separation for all examined peptides is significantly correlated with arginine content (figures 1c and s2a), which indicates that arginines are likely to be the mediators of phase separation for these peptides. to further investigate such arginine - mediated phase separations, we used the pr dpr as a model. as shown for other proteins (molliex., 2015, nott., 2015, patel., 2015), phase separation of pr20 is temperature dependent and reversible (figures 2a, 2b, and s1a s1c). pr20 droplets are highly circular (figure 2c), suggesting that they minimize surface tension. when we applied shear stress to these droplets, we indeed observed plastic deformation with minimization of their surface area after relaxation (figure 2d). there was also a conservation of volume after the fusion of two droplets (figure 2e). all these observations indicate that pr20 droplets behave as a liquid. besides temperature, llps of pr was also sensitive to protein concentration, molecular crowding, and the length of the peptide (figures 3a and 3b). in agreement with the polar nature of the peptide, we found that llps was not perturbed by an aliphatic alcohol, but was hindered by increasing the salt concentration (figures 3c, 3d, and s1d). additionally, the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ans) showed maximum intensity at 495 nm and was reduced following llps of pr20 (figure s1f) (hawe., 2008). these observations argue against hydrophobic interactions and indicate the importance of electrostatic forces via arginines in llps. phase separation of like - charged inorganic polyelectrolytes has been reported before, and due to charge repulsion, this process is only conceivable in the presence of counteranions in solution (brangwynne. we reasoned that phosphate ions in our buffer could be a likely candidate in stabilizing pr - pr interactions. in accordance, when we incubated pr in pure water with the molecular crowder and added different salts, the extent of phase separation depended on the charge of the anions (figure 3e). indeed, addition of polyu rna promoted llps in a dose - dependent manner even without molecular crowders being present (figures 3f, 4a, and s1e) ; and this was also the case for other arginine - rich peptides (figure s2a). similar to the peg - induced llps, pr20-rna droplets were highly circular (figure 4a). they also continuously increased in size over time, as measured by dynamic light scattering (dls) (figure 4b), mimicking observations for cellular sgs (wheeler., 2016). due to its polyvalent nature, polyu was much more effective than phosphate and crowding agent peg in promoting llps. besides purely electrostatic interactions, arginines can also engage in interactions with aromatic side chains (brangwynne., 2015). in support of the contribution of this mechanism, incubating pr20 with poly - tyrosine induced clustering of the insoluble poly - tyrosine polymers (figures 3 g and s1h). disordered linear binding motifs often undergo induced folding following ligand binding (tompa, 2016), but they can also remain predominantly disordered when bound in so - called fuzzy complexes (tompa and fuxreiter, 2008) and when phase separated, as already observed in the case of fus lcd droplets (burke., 2015). cd spectroscopy clearly illustrated the random coil nature of the dispersed pr20 peptide, and this was also the case for other arginine - rich peptides (figures s2b however, after titration with polyu rna, the pr20 cd spectrum was lost, i.e., there is no signal for the peptide within droplets (figure s2 g). therefore, we applied two other approaches to monitor the structural state of pr20 in droplets, both supporting its largely disordered (fuzzy) state. fluorescence anisotropy of peptide tagged with a fluorophore (figure s2h) is very low (0.02), and even after the addition of polyu rna it only increases to about 0.2, far from the value measured in the highly viscous glycerol (0.38), the latter being close to the theoretical upper limit (0.4) for an immobile fluorophore. global hydrogen - deuterium exchange (hdx) experiments (figures s2i and s2j) show full accessibility of the free peptide, which is compatible with its disordered state. following the addition of polyu rna, accessibility drops only slightly (to 80% of its initial value), indicating that the peptide is still largely accessible, and suggesting that it stays disordered after llps. to check whether the pr - rna droplets could have any internal fibrillar structure, we performed cryo - transmission electron microscopy (tem), and could not discern any visible structures in the droplets (figure 4c). further, we performed fluorescence recovery after photobleaching (frap) analyses with the fluorescently tagged pr20 (figures 4d and 4e), and found in full - droplet bleaching experiments that demixed pr readily interchanges with pr in the dispersed phase (figure 4d). aging of the pr - rna droplets had no effect on intradroplet fluorescence recovery (figure 4e). this result is in contrast to findings for prld llps, which undergo a liquid - to - solid transition likely through sheet formation (conicella., 2016, we took soluble hela cell lysate, to which we added increasing concentrations of pr30. the pr30 peptide spontaneously demixed, and could be separated from the solution by gentle centrifugation. the resultant pellet was subsequently washed and analyzed by gel electrophoresis (figure 5a). unexpectedly, this pellet was largely resistant to washing steps, suggesting that pr induced the precipitation of different cellular proteins to the insoluble fraction. the demixing of pr was also largely independent of the presence of rna in the lysate, as addition of rnase only had a modest effect on the extent of protein precipitation (figure s3). to see whether weak interactions could still be involved in the process, we performed mild crosslinking with paraformaldehyde as recently used to identify both the stable core and liquid - like shell of cellular sgs (jain., 2016). the pr fraction seemed to be enriched for a specific set of proteins as was evident from the band pattern. mass spectroscopy (ms) analysis on both crosslinked and uncrosslinked samples identified 874 proteins that were detected and quantified in both conditions. we performed gene ontology (go) enrichment analyses to identify overrepresented biological processes and cellular compartments in our hit list. significantly overrepresented terms centered on rna and protein metabolism (go biological process ; figure 5b) and different cellular liquid - like compartments (go cellular compartment ; figure s3). indeed, when comparing the protein content of the pr30 precipitate with those of stress granules and nucleoli, we found a strong overlap with both cellular liquid compartments (figure 5c), while this was not the case for an unrelated organelle (figure s4). the pr30 precipitate was also enriched for metastable aggregation - prone proteins (figure 5d). given that 60% of identified proteins have been implicated in rna metabolism (figure s4), we evaluated overrepresentation of rna binding domains. these included globular rrm or dead / h box domains and disordered rgg boxes (figure 5e). besides rgg boxes, r motifs were also highly abundant (figure 5f), once more illustrating the importance of arginine - rich binding modules in llps. lastly, we found that the pr30 precipitate is enriched for disordered (figure 5 g) and aggregation - prone supersaturated proteins (figure 5h) (ciryam., 2013). quantitative analysis revealed that 190 proteins were more abundant after crosslinking (figure s5), which suggests that weak interactions between these proteins and the pr30-induced insoluble fraction could be stabilized by our mild crosslinking protocol. interestingly, this set of cross - linking - sensitive proteins was enriched for different protein families involved in stress granule biology, such as hnrnps (molliex., 2015) and aminoacyl transferases (david., 2011) although both 40s and 60s ribosomal proteins were highly abundant in our protein set identified by ms, only 40s, but not 60s, constituents were overrepresented in the crosslinking - sensitive fraction. cellular sgs also contain 40s, but exclude 60s subunits (kedersha., our in vitro cell - free findings, we overexpressed a codon - optimized pr100 construct in hela cells (figure 6a). as previously reported, pr100 predominantly localized to the nucleolus (kwon., 2014, tao., 2015, wen., 2014, yamakawa., 2015). yet, the evidence whether arginine - rich dprs affect sgs has remained ambiguous (tao. we found cytoplasmic pr100 granules positive for sg markers after overexpression of pr100 (figure 6a), and this process was dose and length dependent (figure s6). pr100 expression was more efficient at inducing sgs than pa100 expression, showing that these sgs do not just originate from the transfection protocol (figure 6b). pr100-induced sgs were also responsive to known sg modifying compounds, i.e., arsenite and puromycin treatment enhanced sg formation, whereas cyclohexamide reduced their number (figure 6c). by using a mutant mef cell line carrying a non - phosphorylatable form of eif2, we found that pr100 requires the integrated stress response for sg induction (figure 6d). also the presence of g3bp1/2 seemed to be required for this, based on the use of knockout cell lines (figure 6e) (kedersha. when studying these pr100-induced sgs in more detail, we observed that they are slightly less dynamic than arsenite - induced sgs, based on frap analysis of a g3bp1-gfp fusion protein (figure 6f). moreover, the protein content of pr100 sgs was strongly altered compared to arsenite sgs. while there was no difference in the sg enrichment of a general sg marker (yb-1), both a late sg marker (ddx6) and als - related proteins carrying a prld (ataxin-2 and tdp-43) were significantly enriched in pr100 sgs compared to arsenite sgs (figure 6 g). the current hypothesis for the formation of aggregates in als / ftld patients is a liquid - to - solid phase transition or perturbed clearance of sgs. although not observed in a living system so far, in vitro droplets of fus and hnrnps have been shown to mature to a solid state over the course of hours (molliex., 2015, patel., 2015). this likely corresponds to a transition of their prlds from a disordered fuzzy state to a rigid sheet structure (burke., 2015, molliex., 2015, patel., 2015). since we found that (1) pr repeats could engage in interactions with tyrosines, (2) pr induced the precipitation of several of these proteins in vitro, and (3) such proteins were enriched in pr100 sgs, we wondered whether pr could affect the dynamics and maturation of prld droplets. addition of pr30, but not gp30, to fus lcd droplets interfered with their spontaneous fusion dynamics (figure 7a). additionally, fus lcd phase separation was also enhanced by addition of pr30 (figures 7a and 7b). in agreement with the reduced dynamics of the droplets, kinetic thioflavin - t fluorescence analysis of fus lcd droplets supplemented with pr30, indicated an increase in sheet content over time (figures 7c and s7). in this study, we have investigated whether highly repetitive and disordered arginine - rich peptides generated from expanded c9orf72 repeats in als / ftld could play a role in llps. this phenomenon is of intense interest since it could explain the biogenesis and physical underpinnings of sgs, which are the prime suspect of seeding for pathological protein aggregation in als and ftld (king., 2012, ramaswami., 2013). until now, llps of prld containing rbps has been mostly attributed to hydrophobic interactions. however, arginine - rich domains are highly enriched in cellular liquid - like compartments, such as stress granules and the nucleolus, and both protein disorder and arginine - content are highly conserved in rbps (varadi., 2015). this conservation suggests that these arginine - rich domains must be under selective pressure, highlighting their functionality and suggesting that a similar gain - of - function may underlie the pathological role of c9orf72 dprs in als. in fact, rgg boxes have been shown to affect prld aggregation and llps by nucleating on rna or poly(adp - ribose) (altmeyer., 2015, lin., 2015,, 2015, patel., 2015, schwartz., 2013). we hence hypothesized that arginine - rich dprs could play a more direct role in phase separation through their disorder (zhang., indeed, we found that arginine - rich dprs derived from the c9orf72 repeat expansion undergo llps, a process mimicked by short synthetic peptides corresponding to the rgg boxes of three sg proteins, which also spontaneously phase separated in vitro. this phase transition was directly correlated with the arginine content of the peptides. as shown previously for inorganic polymers, such like - charged phase separation was dependent on counterions (tiraferri., 2015). recently, two other studies have also reported a similar behavior for artificial arginine - rich peptides (aumiller and keating, 2016) or peptides derived from nucleolar proteins (mitrea., 2016). intriguingly, when we added cellular proteins to pr, this resulted in the formation of an insoluble pellet, rather than liquid droplets. however, crosslinking experiments showed that specific weak protein - protein interactions did occur in our test tube experiment. this is consistent with recent reports indicating that cellular sgs likely consist of a stable core surrounded by a liquid shell, rather than a homogeneous liquid droplet (jain. the pr30 interactome using ms showed that pr was able to interact with a large, yet specific, set of proteins enriched for rna - binding domains, arginine - rich motifs and protein disorder. numerous hits were known constituents of endogenous liquid organelles, and, interestingly, several have been directly implicated in als / ftld. hence, we suggest that dprs may play a role in disease by upsetting the internal functional balance of membraneless organelles (brangwynne., 2015). the data of our in vitro precipitation experiment are in agreement with three recent studies showing that intracellular pr and gr peptides can bind with a large set of proteins involved in rna- and sg metabolism (lee., 2016, lin., 2016, lopez - gonzalez., 2016 when overexpressed in cells, pr was indeed able to seed sg assembly, and this sg induction was dependent on phosphorylation of eif2 and the presence of g3bp proteins. although sg induction by pr was shown recently by others (lee., 2016, yamakawa., 2015), the mechanism through which this occurred remained unknown and of intense interest from a therapeutic perspective. in this light, eif2 modulation has already been shown to ameliorate toxicity in different tdp-43 als models (kim., 2014). g3bp proteins have been shown to be essential in eif2-controlled sg assembly (kedersha., 2016), and the g3bp rgg domain plays a crucial role in this process, once more highlighting the importance of arginine - rich domains in sg metabolism. pr - induced sgs displayed reduced dynamics and were enriched for als - related proteins. moreover, pr was also able to accelerate the liquid - to - solid maturation of prld droplets, a process reminiscent of pathological aggregation (conicella., 2016, molliex. in line with our findings, two other reports also recently found that arginine - rich dprs can affect sgs and other higher order assemblies in similar ways (lee. sgs are generally considered to be the stepping stone toward pathological aggregation of rbps in disease. identifying the mechanisms behind altered sg dynamics and a deeper appreciation of their biology, hence, will provide us with invaluable clues to better understand the etiology of disease. disease mutations are known to target several sg proteins or proteins involved in sg dynamics (ramaswami., 2013). however, the reason why wild - type proteins mislocalize to the cytoplasm and aggregate in c9orf72 cases remained elusive. recently, we and others have shown that nucleocytoplasmic transport defects caused by arginine - rich dprs could explain the mislocalization of these rbps (boeynaems., 2016a, boeynaems., 2016b, freibaum., 2015, jovii., 2015). by focusing now on the biophysical behavior of arginine - rich dprs, we and others have found that they could also be directly implicated in the cytoplasmic aggregation of rbps. these data suggest that dprs could initiate a pathogenic cascade in c9orf72 als / ftld, by targeting the nuclear transport - stress granule axis (boeynaems. reagent or resourcesourceidentifierantibodiesanti - flag, mousesigma - aldrichf3165 ; rrid : ab_259529anti - flag, rabbitcell signaling#2368santi - g3bp1, mouseabcamab56574 ; rrid : ab_941699anti - ddx6, rabbitabcamab40684 ; rrid : ab_941268anti - yb1, rabbitabcamab76149 ; rrid : ab_2219276anti - tdp-43, rabbitproteintech12892 - 1-ap ; rrid : ab_2200505anti - ataxin-2, mousebd biosciences611378 ; rrid : ab_398900anti - tia1, goatsanta cruzsc-1751 ; rrid : ab_2201433bacterial and virus strainse. coli, bl21 star (de3)thermo scientificc601003chemicals, peptides, and recombinant proteinspr20this papern / apr30this papern / agr30this papern / agp30this papern / afus rggthis papern / ahnrnpa1 rggthis papern / afmrp rggthis papern / afus lcdr., 20151,6-hexanediolsigma - aldrich240117polyu rnasigma - aldrichp9528poly - tyrosinesigma - aldrichp1800peg300sigma - aldrich81160anssigma - aldricha1028complete protease inhibitor cocktailsigma - aldrich11697498001arsenitesigma - aldrichs7400lipofectamine 3000thermo scientificl3000015nucbluethermo scientificr37605prolong gold antifadethermo scientificp36934critical commercial assaysalexa fluor 488 protein labeling kitthermo scientifica10235alexa fluor 568 protein labeling kitthermo scientifica10238deposited datamas spec datathis paperpride : pxd005509experimental models : cell lineshelaatccccl-2mef eif2a ssdr., 2015plasmid : pr100-flag - mcherrythis studyn / a further information and requests for reagents may be directed to and will be fulfilled by the lead contact, ludo van den bosch ([email protected]). paul taylor (st. jude, usa) (figley., 2014). u2os g3bp1/2 ko cell line, and eif2 ss and aa mefs were a kind gift of dr. paul anderson (harvard, usa) (emara., 2012, kedersha., u2os and hela cells (atcc) were cultured in high glucose dmem (invitrogen) supplemented with 10% fetal bovine serum (greiner), 4 mm glutamax (invitrogen), penicillin (100 u / ml), streptomycin (100 g / ml) and non - essencial amino acids (1%). flag - tagged dpr50 expression constructs were synthesized by genscript (piscataway, usa). fus lc domain was expressed in e. coli and purified as described previously (burke., 2015). hnrnpa2 lc domain (residues 190 - 341) was expressed with a tev - cleavable n - terminal hexahistidine tag, purified from the inclusion body via histrap (ge healthcare) in urea containing buffers, concentrated, diluted into native buffer for tev cleavage of hexahistidine tag, resolublized by addition of solid urea, separated from his - tagged tev protease and cleaved his - tag by histrap, and concentrated to 1 - 2 mm in 8 m urea 20 mm mes ph 5.5. peptides were diluted to the indicated concentrations in 100 mm k2hpo4/kh2po4 buffer at ph 7, unless otherwise indicated. peg300, polyu rna or poly - l - tyrosine (sigma - aldrich) were added at the indicated concentrations. for fluorescence microscopy and frap analyses, pr - alexa 568 and polyu30-alexa 488 were spiked in at 200 nm and 100 nm respectively. fus lc droplets were generated by diluting the stock solution to the desired concentration in 50 mm mes buffer at ph 5, with the indicated nacl concentrations. hnrnpa2 lc droplets were generated by diluting the stock solution to the desired concentration in 20 mm napi buffer with 50 mm nacl at ph 7.5. od600 of 60l samples was measured using truview microcuvettes in a smartspec plus spectrophotometer (bio - rad). turbidity of 100l of fus and hnrnpa2 lc droplets was measured on a spectra max m5 microplate reader (molecular devices). fus lc droplets were generated by diluting the stock solution to 250m in 50m mes buffer at ph 5, 200m nacl. fluorescent droplets were incubated in plastic cell counter slides (bio - rad) at room temperature. every two hours three arbitrary fields were imaged on a zeiss lsm 510 meta nlo confocal microscope. tht was added to 100l pr30 + lc droplet mixtures at a concentration of 20m. fluorescence intensity over time was followed using a spectra max m5 microplate reader (molecular devices). lag time was estimated as the time point of a local minimum in the curve. cd spectra of the peptides analyzed in this study were recorded in a jasco 715 spectropolarimeter equipped with a ptc 423s peltier element. peptides were loaded into a 0.2 mm quartz cuvette at 30m concentration (50 mm k2hpo4/kh2po4 buffer at ph 7), and spectra were recorded with 50nm / min scan speed at 25c with 9 acquisitions averaged on the fly. spectra were background corrected with the buffer spectrum (50 mm k2hpo4/kh2po4 buffer at ph 7), and converted into molar ellipticity. pr droplet (250m pr20 + 0.04g/l polyu rna) sample (3.5 l) was applied to a 300 mesh lacey quantifoil grid and incubated for 30 s. next, excess buffer was removed by blotting the grids for 3 s using a whatman 1 filter paper and the sample was snap frozen by plunging in liquid ethane at a temperature of 180c and stored in liquid nitrogen until visualization. next, the samples were transferred to a gatan 914 cryoholder and imaged at low dose conditions at 177c, using a jeol jem1400 tem equipped with an 11 mpxl olympus sis quemesa camera. fluorescent droplets were incubated in plastic cell counter slides (bio - rad) at room temperature. fluorescence recovery after bleaching was monitored using zen software on a zeiss lsm 510 meta nlo confocal microscope. for intradroplet frap, a circular are of 1m radius was bleached in droplets with a radius between 5m and 10m. raw data was background substracted and normalized using excell, and plotted using prism software. measurements were carried out in a ls55 luminescence spectrometer (perkinelmer), with 200 nm pr - alexa568 n - terminally labeled protein in 50 mm k2hpo4/kh2po4 buffer at ph 7. after the addition of 10m of bulk pr30, fluorescence anisotropy was measured at increasing rna concentration at 578 nm excitation and 603 nm of emission wavelengths. 20m pr30 was titrated with increasing concentration of polyu rna starting at a 0.04g/l concentration in a dynapro nanostar (wyatt technologies) in 50 mm k2hpo4/kh2po4 buffer at ph 7. the size of particles was followed for one hour after every titration step (each titration step represented a 0.5% dilution). to control sedimentation and droplet fusion sample was mixed thoroughly with a pipet randomly between time points. ans (8-anilinonaphthalene-1-sulfonic acid) fluorescence was measured for 40m pr20 or 40m gr20 peptide in the presence and absence of 0.05g/l polyu rna in a biotek mx synergy plate reader in 50 mm k2hpo4/kh2po4 buffer at ph 7. emission spectra were recorded between 400 nm and 600 nm at 380 nm excitation wavelength. pr30 and polyu rna were dissolved in miliq grade water and added together to make a final concentration of 400 m pr and 4.8 mg / ml polyu rna to induce llps. for experiments on the free peptide, pr the samples were then, either immediately or after an incubation time of 1h for the llps samples, diluted with a 20-fold excess of d2o. after the indicated time points the hdx reaction was quenched by bringing the ph of the solution to 1 using formic acid, followed by snap freezing in liquid nitrogen. mass spectrometry analysis of deuterium incorporation was performed on a synapt g2 hdms mass spectrometer (waters, wilmslow, uk) by direct infusion of the undigested sample using nano - electrospray ionization with in - house prepared gold - coated borosilicate needles. hela cells were trypsinized, pelleted and washed three times with 1x pbs (thermo scientific). cells were resuspended in k2hpo4/kh2po4 buffer with edta - free complete protease inhibitor cocktail (roche) and sonicated on ice. the lysate was cleared from the insoluble fraction by centrifugation for 15min at 10,000 rpm at 4c. the supernatans was retrieved and protein concentration was measured using micro bca assay (thermo scientific). 1 mg of protein was added to 0.05 mole of pr30 to a final concentration of 100m, and left incubating for 10 min at rt. paraformaldehyde (sigma - aldrich) was added to a final concentration of 0.5%, and crosslinking was left for 5 min. 500l of 2 m glycine (sigma - aldrich) was added to quench paraformaldehyde, and samples were left for 5 min. the volume of the samples was increased to 1.5ml with k2hpo4/kh2po4 buffer, before gently spinning down the pr droplets at 4,000 rpm for 5 min. pellets were subsequently washed with 1ml pbs with 0.4% triton x-100 (sigma - aldrich) and vortexed before spinning down. samples for sds - page were generated identically, but procedure was downscaled four times. pr llps pellets were resuspended in 1x reducing laemmli containing sds (thermo scientific). 1/8 of pellet or input lysate were loaded onto nupage novex 4%12% bis - tris precast gels (thermo scientific). after running gels were stained using coomassie brilliant blue r-250 according to manufacturer s instructions (bio - rad). uncrosslinked (2 replicates) and crosslinked pellets (3 replicates) were redissolved in 300 mm nacl tris buffer, sonicated and boiled to remove crosslinks. proteins in each sample were reduced with 5 mm dtt and incubation for 30 min at 55c and then alkylated by addition of 10 mm iodoacetamide for 15 min at room temperature in the dark. samples were further diluted to a final urea concentration of 2 m and proteins were digested with trypsin (promega) (1/100, w / w) overnight at 37c. peptides were then purified on omix c18 tips (agilent), dried and re - dissolved in solvent a (25 l 0.1% tfa in water / acetonitrile (98:2, v / v)) of which 10 l was injected for lc - ms / ms analysis on an ultimate 3000 rslcnano system (dionex, thermo fisher scientific) in line connected to a q exactive hf mass spectrometer with a nanospray flex ion source (thermo fisher scientific). trapping was performed at 10 l / min for 4 min in solvent a (on a reverse - phase column produced in - house, 100 m i.d. x 20 mm, 5 m beads c18 reprosil - pur, dr. maisch) followed by loading the sample on a 40 cm column packed in the needle (produced in - house, 75 m i.d. 400 mm, 1.9 m beads c18 reprosil - hd, dr. maisch). peptides were eluted by an increase in solvent b (0.1% formic acid in water / acetonitrile (2:8, v / v)) in linear gradients from 2% to 30% in 100 min, then from 30% to 56% in 40 min and finally from 56% to 99% in 5 min, all at a constant flow rate of 250 nl / min. the mass spectrometer was operated in data - dependent mode, automatically switching between ms and ms / ms acquisition for the 16 most abundant ion peaks per ms spectrum. full - scan ms spectra (375 - 1500 m / z) were acquired at a resolution of 60,000 after accumulation to a target value of 3,000,000 with a maximum fill time of 60 ms. the 16 most intense ions above a threshold value of 22,000 were isolated (window of 1.5 th) for fragmentation at a normalized collision energy of 32% after filling the trap at a target value of 100,000 for maximum 45 ms. the s - lens rf level was set at 55 and we excluded precursor ions with single and unassigned charge states. data analysis was performed with maxquant (version 1.5.3.30) (cox and mann, 2008) using the andromeda search engine with default search settings including a false discovery rate set at 1% on both the peptide and protein level. spectra were searched against the human proteins in the uniprot / swiss - prot database (database release version of april 2016 containing 20,103 human protein sequences, www.uniprot.org). the mass tolerance for precursor and fragment ions were set to 4.5 and 20 ppm, respectively, during the main search. enzyme specificity was set as c - terminal to arginine and lysine, also allowing cleavage at proline bonds with a maximum of three missed cleavages. variable modifications were set to oxidation of methionine residues and acetylation of protein n - termini. only proteins with at least one unique or razor peptide proteins were quantified by the maxlfq algorithm integrated in the maxquant software (cox., 2014). further data analysis was performed with the perseus software (version 1.5.3.0) after loading the protein groups file from maxquant. proteins only identified by site and reverse database hits were removed and replicate samples of uncrosslinked and crosslinked were grouped. proteins with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution around the detection limit. then, a t test was performed (fdr = 0.01 and s0 = 1) to compare samples uncrosslinked and crosslinked and calculate the fold change for each protein between both samples. after removal of potential contaminants, 874 proteins were quantified in total (table s1). cells were transiently transfected using lipofectamine 3000 (invitrogen) according to manufacturer s instructions. cells were fixed 24h after transfection in 4% formaldehyde in pbs and stained according standard protocols (including methanol fixation and permeabilization by pbs - t 0.04%). following antibodies were used : anti - flag (f3165, sigma), rabbit anti - flag (# 2368s, cell signaling), mouse anti - g3bp1 (ab56574, abcam), rabbit anti - ddx6 (ab40684, abcam), rabbit anti - yb1 (ab76149, abcam), rabbit anti - tdp-43 (12892 - 1-ap, proteintech), mouse anti - ataxin-2 (611378, bd biosciences), goat anti - tia1 (sc-1751, santa cruz). alexafluor 555 and alexafluor 488 secondary antibodies (life technologies) were used. control stress granules were induced by incubating the cells for 1h with 0.5 mm naaso2 (sigma). percentage of cells carrying stress granules was assessed by g3bp1 staining in all tested cell lines. stress granule enrichment was quantified as the ratio of fluorescence intensity in the stress granule over the intensity of the surrounding cytoplasm. two stress granules per cell were quantified, of at least 30 cells from 3 independent experiments. for stress granule enrichment of pr100, one stress granule per cell was counted, of 40 cells from 2 independent experiments. pr100 stress granules were visualized for quantification with flag staining, arsenite stress granules were visualized by tia1 staining. for analysis of dose- and length dependency of pr cytoplasmic structures, cells were categorized as having diffuse cytoplasmic pr, small stress granules, large stress granules or coarse / granular / heterogeneous cytoplasmic pr staining. data was subsequently also plotted as the percentage of cells displaying non - diffuse cytoplasmic pr staining after manual binning according to cytoplasmic pr100-flag intensity. percentages were fitted by a one - phase exponential curve, illustrating a strong dose - dependency of cytoplasmic structure formation. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pride : pxd005509., w.r., and p.v.d. provided ideas for the project and participated in writing the paper. s.b.	summaryliquid - liquid phase separation (llps) of rna - binding proteins plays an important role in the formation of multiple membrane - less organelles involved in rna metabolism, including stress granules. defects in stress granule homeostasis constitute a cornerstone of als / ftld pathogenesis. polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of als - linked stress granule proteins. we now identify an active role for arginine - rich domains in these phase separations. moreover, arginine - rich dipeptide repeats (dprs) derived from c9orf72 hexanucleotide repeat expansions similarly undergo llps and induce phase separation of a large set of proteins involved in rna and stress granule metabolism. expression of arginine - rich dprs in cells induced spontaneous stress granule assembly that required both eif2 phosphorylation and g3bp. together with recent reports showing that dprs affect nucleocytoplasmic transport, our results point to an important role for arginine - rich dprs in the pathogenesis of c9orf72 als / ftld.
cutaneous melanoma is a highly aggressive malignant tumor of melanocytic origin with increasing incidence in most countries of the world over the last few decades. in caucasian populations the incidence rates for cutaneous melanoma have risen faster than those for any other malignant entity over the last 30 years. v) or mutations of tumor - suppressor genes (e.g., cdkn2a, encoding for cyclin - dependent kinase inhibitor 2a), which might be reflected by a positive family history of melanoma. when a patient s risk for melanoma needs to be assessed during daily clinical routine, the personal and family history of melanoma, number of common and atypical nevi, skin type, presence of freckles, eye color, hair color, and the presence of non - melanoma skin cancer lesions should be considered. the overall number of nevi induced by ultraviolet radiation in early infancy was shown to be an especially important predisposing risk factor for the development of melanoma. individuals with a high nevus count are endangered notably, whereby the risk of melanoma development increases almost linearly with rising numbers of melanocytic nevi on the whole body. although cutaneous melanoma is less common than other skin cancers, it causes the majority (approximately 90 percent) of deaths related to skin cancer. an advanced tumor thickness (breslow s depth) is strongly associated with an increased mortality. therefore, the early detection of melanoma at early and curable stages is critical for the patients survival. in contrast to many other tumor entities, cutaneous melanoma may already develop metastases at a low tumor volume. once distant metastases have occurred, the median survival is approximately nine months and the five - year survival rate is less than 10 percent. on the contrary, for thin melanomas with a tumor thickness of less than 0.76 mm, these rates markedly decrease to 48 percent for lesions with more than 3 mm tumor thickness. until today, the most common method for detecting melanoma is visual diagnosis. to make a correct diagnosis, the most frequent form of cutaneous melanoma is superficial spreading melanoma (ssm, figure 1). it grows slowly and initially in a horizontal plane and subsequently, in more advanced cases, it will reach a vertical growth phase. with naked eye examination, lesions are often sharply demarcated, polycyclic and multicolored. in contrast, nodular melanoma (nm), a much more aggressive form, exhibits an early vertical growth phase. nm emerges primarily and is then often detected late, at an advanced tumor thickness, or it develops secondarily within a ssm (figure 2). the nodes often grow rapidly, are vulnerable with a tendency to ulceration and bleeding, or might appear non - pigmented. lentigo maligna melanoma (lmm, figure 3) characteristically occurs at older age and is localized in areas of chronically sun - exposed skin, like the face or dorsa of the hands. similar to ssm, it appears polycyclic, sharp - bounded and often shows multiple brown spots. a rare growth variant of cutaneous melanoma, the acral - lentiginous melanoma (alm, figure 4) is localized in palmoplantar skin but may also involve the nail unit. alm represents the most common growth variant in dark - skinned (e.g., asian) populations. due to the architecture of palmoplantar skin with parallel ridges and furrows, the margins are often not clearly defined and the pigmentation is incoherent. moreover, special forms like mucosal and ocular malignant melanoma exist. in summary, the most effective approach to improve the prognosis of cutaneous melanoma the identification and the screening of high - risk patients is an important prerequisite to further enhance efforts to lower the mean tumor - thickness at the time of diagnosis. excisional biopsies of suspicious lesions with subsequent histopathologic examination of specimens will allow for a definite classification. the application of the clinical abcd rule (table 1) devised by friedman in 1985 is a common method for the clinical diagnostics of cutaneous melanoma. the criteria asymmetry, irregular margin, multiple colors and a diameter over 6 mm, cutaneous melanoma has to be considered. however, the abcd rule is characterized by a low specificity as other benign skin lesions such as seborrheic keratosis may also fulfill the aforementioned criteria. moreover, it is not applicable for pigmented lesions on the palms, soles or face due to the particular skin anatomy at these sites. small melanomas with a diameter of 6 mm or less or rare non - pigmented subtypes of melanoma will also not be detected by the abcd rule. certain dynamic changes within melanocytic lesions are also suggestive of cutaneous melanoma, for instance, asymmetrical changes in size, shape or color, or itching and bleeding. in consequence, e for evolving or evolution to the abcde rule. before examination of selected nevi in more detail, it is useful to get an overview of recurrent and predominant patterns of a patient s nevi. nevi in the same individual tend to resemble one another, so - called signature lesions, indicating benign nevi on a regular basis. in contrast, melanomas often deviate from the individual s nevus pattern, so - called ugly duckling sign. recently, a high sensitivity for the early detection of melanoma with the help of the ugly duckling sign could be demonstrated. medical total body photography is often used in dermatology to support the clinical surveillance of high - risk patients. photographically assisted follow - up images help clinicians and patients to detect new and changing pigmented lesions with the unaided eye. in contrast to digital dermoscopy, which allows for monitoring of a few lesions only, full body photography permits monitoring of all lesions of a patient. dermoscopy (also known as epiluminescence microscopy, dermatoscopy, or amplified surface microscopy) allows for a significant improvement in the preoperative diagnostic accuracy of pigmented skin lesions. dermoscopy is a non - invasive technique for the in vivo examination of melanocytic and non - melanocytic skin lesions. the handheld dermoscope traditionally consists of a magnifier, a non - polarized light source and a transparent plate. a liquid medium, for instance, immersion oil or spray for skin disinfection, is needed between the instruments contact plate and the skin to reduce the reflection of light. with the help of dermoscopy further information about the architecture, e.g., the pigmented network, the vascular pattern and the distribution of color of a single lesion is obtained. today, many different dermoscopes are commercially available (e.g., dermatoscope by heine optotechnik ; episcope by welch allyn ; dermogenius by rodenstock przisionsoptik). more recently dermoscopes that emit polarized light to eliminate skin surface reflection entered the market. these instruments do not require skin contact and a liquid medium can be omitted (e.g., dermlite 3 gen). this procedure for classification of pigmented skin lesions was agreed on at an international consensus meeting. in the first step it has to be determined whether a melanocytic or non - melanocytic, the differentiation between benign or malignant / suspicious lesions follows in a second step. for this most algorithms (e.g., the abcd rule of dermoscopy, the 7-point checklist of dermoscopy, or menzies scroring method) use a number of criteria associated with the presence of melanoma as first described for the pattern analysis by pehamberger. especially, three algorithmic methods (qualitative pattern analysis, the abcd rule of dermoscopy, and the 7-point checklist) were shown to be valid and reliable in distinguishing benign and malignant melanocytic tumors. the pattern analysis is based on a detailed, qualitative assessment of numerous dermoscopic criteria, and a high rate of diagnostic accuracy could be obtained by experienced investigators after a significant degree of formal training (19). the abcd - rule of dermoscopy uses a semiquantitative scoring system based on a complex evaluation of asymmetry, border, color, and different dermoscopic structures within the lesion. the 7-point checklist (table 2) was developed as a quantitative scoring system with three major criteria (score of 2 points) and four minor criteria (score of 1 point). the 7-point checklist can be learned and applied more easily and in comparative studies allowed the best sensitivity in the hands of non - experts. two unsuspicious melanocytic nevi are shown in figures 5 and 6, while three superficial spreading melanomas with their typical criteria for malignancy are demonstrated in figures 79. a systematic overview of medline publications between 1983 and 1997 showed that dermoscopy leads to a 1027% increase in sensitivity as compared to the clinical diagnosis with the unaided eye. dermoscopy not only allows for an earlier detection of melanoma but was also shown to avoid unnecessary excisions of benign nevi. depending on the individual experience of the clinician, a sensitivity of up to 92%and a specificity of up to 99% were documented for the detection of cutaneous melanoma by dermoscopy. among dermatologists, dermoscopy has become a routine examination technique in europe and is with gaining acceptance worldwide. when computer hardware became more and more available and affordable, digital dermoscopy devices were developed and rapidly integrated into the clinical setting. digital overview and dermoscopic images offer the advantage of computer storage and retrieval during later examinations of patients. some digital dermoscopy devices even offer a computer - assisted diagnosis [2426 ]. despite a higher impact on financial and personnel resources, the sequential digital dermoscopic examination offers a number of advantages for patients with a high number of atypical nevi and a personal and/or family history of melanoma. due to the multitude of atypical pigmented lesions in these high - risk patients, it is often difficult to detect melanoma at an early stage without excising hundreds of benign nevi (figure 10). by comparison of dermoscopic follow - up images with baseline images a range of studies have proven that digital dermoscopy follow - up of high - risk patients allows for the early detection of melanomas that have not yet acquired melanoma - typical dermoscopic features, thus increasing the sensitivity [2729 ]. with the help of sequential digital dermoscopy, incipient melanomas can be identified by detection of intralesional changes, for instance asymmetrically enlargement or architectural changes (figure 11 a c). this applies especially for melanomas without suspicious dermoscopic features, so - called featureless melanoma. another strategy of sequential dermoscopic follow - up is the short - term follow - up (three - month interval), which targets a restricted number of highly atypical nevi, which should be removed whenever dynamic changes become apparent. numerous systems for sequential digital dermoscopy imaging are commercially available (e.g., molemax, fotofinder, solarscan). the need for a further improved diagnostic accuracy for the assessment of skin tumors has led to the development and investigation of new imaging tools. an innovative technique is the in - vivo confocal laser scanning microscopy (clsm), which represents a relatively novel imaging tool allowing for non - invasive examination of skin morphology in real time and at a near - histopathologic resolution. multiple studies have evaluated the use of reflectance mode clsm for the diagnosis of melanoma in the past decade. in this regard, two different algorithms for melanoma diagnosis have been developed by two independent groups from modena and barcelona, showing similar sensitivity and specificity values for several clsm criteria. the relative simple two - step method developed by segura uses protective, as well as risk factors, whereby the presence of two risk factors results in a sensitivity of 86.1% and a specificity of 95.3% for the diagnosis of melanoma. pellacani identified six criteria, two major and four minor criteria, as independently correlated with a melanoma diagnosis. the two major criteria corresponded to the presence of atypical melanocytic cells within the epidermal basal layer and the papillary dermis or within the basal layer of non - edged papillae. the four minor criteria were represented by the presence of roundish cells in superficial layers spreading upward in a pagetoid fashion, pagetoid cells widespread throughout the lesion, cerebriform clusters within the papillary dermis and bright nucleated cells in the upper dermis. the presence of at least two features, one major and one minor criterion, was essential for melanoma diagnosis. on the other hand, regular dermo - epidermal architecture and absence of pagetoid infiltration and atypical cells were indicative of benign lesions. the evaluation of skin lesions by clsm is based on illumination of the tissue by a point laser light source and reflectance of individual skin structures, of which melanin is providing the strongest contrast [3537 ]. specific morphological pattern can be observed by clsm in nevi and melanoma allowing for differentiation of benign and malignant, but also for differentiating melanocytic from non - melanocytic lesions such as pigmented basal cell carcinoma (figure 12). due to scanning mode of clsm an area of up to 8 8 mm may be evaluated on horizontal sections and on different skin levels (e.g., stratum corneum, stratum spinosum, dermo - epidermal junction, dermis). thereby, the great advantage of clsm lies in the immediate diagnosis that may guide the clinician s decision for further management of a lesion. furthermore, clsm provides the clinician and the researcher with the possibility of monitoring of pigmented lesions over time and may change the way we evaluate melanocytic lesions in the future. to implement the clsm in the clinical setting, the multispectral digital dermoscopy, a new development of conventional dermoscopy, has recently entered the european market (melafind) and offers a fully automated differentiation of melanoma from atypical melanocytic nevi. light of different wavelengths is transmitted and the degree of reflection or absorption by the different components of human skin (e.g., collagen, melanocytes, and blood vessels) is analyzed by a computer algorithm. a diagnosis is made by the assessment of the distribution of melanin, collagen and hemoglobin in a given lesion. a prospective multicenter trial, which led to the approval by the european agencies, showed a high sensitivity of 98.4%, but a low specificity of approximately 10%. however, as reported in the study by monheit, the specificity was superior to that of clinicians formally trained in the use of dermoscopy. another novel non - invasive optical biomedical imaging technique is the optical coherence tomography (oct). the basic principle relies on low - coherence interferometry enabling high resolution, two- or three - dimensional, cross - sectional imaging of microstructural morphology in biological tissue. using conventional oct, the stratum corneum, the epidermis, and upper dermis and blood vessels can be visualized. however, with most of the present devices, it is not possible to investigate skin tissue on the cellular level, whereas the architecture of a pigmented lesion and the distribution of blood vessels can be assessed. cutaneous melanomas often show marked architectural disarray, the dermo - epidermal border is blurred and exhibits finger - shaped elongated rete ridges. more precise data concerning sensitivity and specificity of oct for the diagnosis of cutaneous melanoma are missing. of note, for the diagnosis of intraocular tumors, optical coherence tomography was shown to provide valuable information. early detection of cutaneous melanoma is one of the most effective ways of reducing mortality rates from this disease. in addition to the clinical assessment with the unaided eye, dermoscopy has become a standard examination technique throughout most european countries, as it was shown to significantly improve the diagnostic accuracy of the clinical examination. total body photography notably facilitates the detection of newly developed pigmented lesions in patients with a high nevi count. with the help of sequential digital dermoscopy imaging, even subtle dynamic changes are detectable, which is shown to improve the differentiation of atypical nevi and early melanomas in high - risk patients. a novel imaging tool is the confocal laser scanning microscopy, allowing examination of skin tumor morphology on a cellular level in real time. in the near future the recently approved melafind device will probably be applied for difficult - to - diagnose melanocytic lesions in selected centers. above all, it has to be emphasized that none of the aforementioned examination techniques are currently able to provide a definite and final diagnosis or to replace excisional biopsy of suspicious lesions followed by histological analysis.	cutaneous melanoma is a highly aggressive malignant tumor of skin melanocytes with an increasing incidence in most countries of the world, especially in the fair - skinned populations. despite all preventive and therapeutic efforts, malignant melanoma is still the most lethal skin cancer. a delayed diagnosis results in an advanced stage and worsened prognosis. once distant metastases are present, the five - year survival rate is less than 10 percent. at the same time, patients may be cured by an early diagnosis of cutaneous melanoma followed by a wide excision. therefore, the early detection of melanoma at curable stages is crucial for the patients survival. besides the investigation of pigmented lesions with the unaided eye, a wide range of examination techniques for improved diagnostic accuracy have been developed and validated in clinical trials. however, none of these techniques are able to provide a definite and final diagnosis or to replace an excisional biopsy of suspicious lesions followed by histological analysis. this review provides a concise overview of general principles as well as current and future strategies for an improved early diagnosis of cutaneous melanoma
dendritic spines were first discovered by cajal in 1888 when studying the dendrites of purkinje cells in hens (figures 1(a) and 1(b)) :... la superficie... aparece erizada de puntas espinas cortas... (cajal, 1888).... la superficie... aparece erizada de puntas espinas cortas... (cajal, 1888). cajal then extended the study of dendritic spines to many different types of cells and species (figures 1(c) and 1(d), see) suggesting a functional role for the dendritic spines. his concept of these structures evolved from their role in canalizing nerve fibers towards dendrite profiles (see, figure 2(a) green lines) to a connective function (see, figures 2(a), blue lines and 2(b)), incrementing the surface area of neurons and/or extending the dendrite to make contact with distant nerve fibers [57 ]. the role of dendritic spines in connecting nerve fibers was later demonstrated by means of electron microscopy (see, figure 2(c)), which led to the general agreement that the majority of excitatory connections in the cerebral cortex are established at dendritic spines [9, 10 ], even though excitatory connections can also end on dendritic shafts [11, 12 ]. many authors have contributed to the knowledge of the dendritic spines using light microscopy, electron microscopy and three - dimensional (3d) reconstructions on serial ultrathin sections ; studying plastic changes, distribution, pathological changes, and so forth, (see). understanding how dendritic spines emerge and develop, and the relationship between synaptogenesis and spinogenesis, would provide insight into : (i) the correct wiring of brain circuits during development, including the selective formation and retention of synaptic contacts between neurons ; (ii) the timing of the appearance of dendritic spines in relation to the initial establishment of axodendritic contacts ; (iii) the mechanisms to arrange new connections in response to experience in the adult animal ; and (iv) the underlying mechanisms of severely altered synaptogenesis and spinogenesis that occur in various disease states (fragile x syndrome, down 's syndrome, etc.). different models of both spinogenesis and synaptogenesis have been proposed, and we will principally examine the former, discussing the different factors thought to be implicated in spinogenesis (genetic factors, dendrite - axon interactions, nervous activity, etc.). then, we will review studies on the development of dendritic spines in the cerebellum (purkinje cells, granule cells, basket cells, and golgi cells), cerebral cortex (pyramidal cells, double bouquet cells, basket cells) and olfactory bulb (granule cells), finally proposing a unifying model of spinogenesis. these slides, stored at the cajal institute, contain sections of the developing cerebellum, cerebral cortex and olfactory bulb, which allowed us to follow the evolution of dendritic spines at different points in the development of different animal species, including birds, the cats, dogs, humans, and rabbits. the application of the so - called ontogenic method by cajal constitutes, from our point of view, one of the most important reasons for his success in neuroscience. instead of studying the complexity of the adult brain, he focused first on the nervous system of lower animals, embryos and young higher animals. thanks to this approach, many of his histological slides are from different species, which allowed us to readily follow the evolution of filopodia and dendritic spines. we have obtained very high - quality extended focus images, 3d - reconstructions and rendered images that we have correlated with recent live imaging studies. ultrastructural data have provided insight into the fine morphology of synapses, whereas live - imaging techniques shed light on the dynamics of both the presynaptic terminal and the postsynaptic dendrite, as well as on spinogenesis and its relation to synaptogenesis. these data suggest that different mechanisms underlie spinogenesis and synaptogenesis depending on the cell type and area of study. furthermore, different models of spinogenesis could even coexist in the same cell, reflecting the high degree of plasticity in dendritic spine formation. three main models of spinogenesis have been proposed : (i) the sotelo model (ii) the miller / peters model and (iii) the filopodial model (see, figure 3). in 1978, sotelo proposed that dendritic spines could grow from dendritic trees, independently of the fiber terminals, according to an autonomous cell program of spinogenesis (figure 3(a)). this model is particularly characteristic for purkinje cell spinogenesis and it was generated on the basis of morphological data gained from experiments on weaver mutant mice that lack granule cells [2830 ], reeler mutant mice in which granule cell migration is perturbed, and neonatal rats irradiated to eliminate the granule cells. the purkinje cells in these animals bear dendritic spines that have differentiated postsynaptic densities, even though they do not have presynaptic terminals [2830, 33 ]. other experiments have provided data to support this model, whereby dendritic spines preferentially form regular linear arrays that trace helical pathways with a short pitch, as suggested earlier. these helical paths have a similar periodicity in fish and mammalian purkinje cells (both weaver and wild - type mice), suggesting that the ordering of the spines around the dendrite is an inherent property of the dendrite to maximize the capacity of the dendritic spines to interact with axons. nevertheless, in normal animals, purkinje cell branchlets give rise to long spine - like processes that form synaptic contacts with parallel fibers. following the onset of these synaptic contacts, the long spine - like processes develops a terminal head, and the parallel fibers form axonal swellings that contain synaptic vesicles. the mature dendritic spine has a big terminal head and a short neck (figure 4(a)). cajal described similar long spine - like processes in the purkinje cell branchlets impregnated by the golgi method of the 15-day - old cat (figure 10(b)). larramendi also found that parallel fibers often form long synaptic adhesions with developing spines (figure 4(b)). these asymmetric synapses become shorter as the dendritic spine reaches maturity through a phenomenon referred to as, it was found that isolated purkinje cells had a low rate of survival and they developed a poor dendritic tree covered only with filopodia. however, when purkinje cells were cultured together with granule cells, they developed a characteristically complex and spiny dendritic tree. a degree of ramification in the dendritic tree might also be necessary to form dendritic spines, and it is noteworthy that when the branchlets of purkinje cell are not well - developed, dendritic spines were also immature. moreover, the size of the dendritic spines and their postsynaptic densities appears to be directly related to the activity of the parallel fibers. indeed, dendritic spines on purkinje cells undergo changes in morphology and density in association with motor learning. while these alterations mainly affect the dendritic spines of the purkinje cells connecting with parallel fibers, the population of spines in proximal purkinje dendritic segments that receive inputs from the climbing fibers (figure 7(c)) also show different morphological parameters. although there is a strong genetic regulation of distal spine formation, input activity has an enormous influence on their development and such dendritic spines that contact parallel fibers are smaller and thicker (see [21, 38 ] ; figure 7(b)) and they are less numerous. in the adult rat, the proximal segment of the purkinje cell tree has few spines, about 0.01 spines/m, whereas the spiny branchlets have a density of 0.46 spines/m. purkinje cells spines that contact climbing fibers appear in clusters of five or six that connect with the varicosity of a climbing fiber (see [22, 40 ] ; figure 7(d)). these postsynaptic structures are very numerous during development, (as represented in the scientific drawings by cajal (see [6, 7 ] ; figure 7(a)) and they are readily observed in his histological slides (figures 9 (b)9(d)). however, during development the density of these dendritic spines decreases. following the chemical destruction of the inferior olive by intraperitoneal injection of 3-acetyl - pyridine, the sectioning of olivary fibers [24, 42 ], or the blockage of nerve activity with tetrodotoxin [36, 3840, 43 ] there was an increase in the number of dendritic spines on the proximal segments and soma of the purkinje cells. thus, climbing fibers have an inhibitory effect on spinogenesis in the proximal branches and soma of the purkinje cells, regulating the density of the dendritic spines through activity - dependent mechanisms [38, 39 ]. this model proposes that the dendritic spines of pyramidal neurons directly evolve from synapses on dendritic branches by successive outgrowth of a sessile to a pedunculated appendage (figure 3(b)). first, synapses are established on the dendritic shafts while in a second step, the presynaptic region of the axon develops a swelling where synaptic vesicles accumulate. most of these immature dendritic spines are stubby spines recognized by their flocculent material. in the third stage, many thin or mushroom - shaped dendritic spines appear and axon terminals show well - developed varicosities. the same sequence of spine formation was observed through electron microscopy in 3, 6, 9, 12, 15, and 21-day - old rat pups and on spiny stellate neurons of the primary visual cortex of one - week - old monkeys (macaca nemestrina ;). this model appears to be supported by the large quantity of synapses on dendrites during the initial stages of development [4550 ] and the higher number of sessile spines in the initial stages of development when compared to the adult [20, 51 ]. however, in 19-day - old mice, spinogenesis and synaptogenesis follow a different pattern to that described by miller and peters or mates and lund. in a quantitative electron microscopy study using serial thin sections, freire distinguished three phases in the development of dendritic spines in the visual cortex of 19-day - old mice and all 76 of the dendritic spines studied were contacted by at least one axon (figure 5). in the 1st phase, the volume of the dendritic spines was less than 5 10 m (figures 5, 1(a), 1(b), 2(a), and 2(b)), there were no cisterns in the spine apparatus, and there were no synaptic specializations when the volume was less than 3 10 m (figures 5, 1(a), and 1(b)). in the 2nd phase, the dendritic spines had a volume between 5 10 m and 19 10 m, the spine apparatus had one or two cisterns, and the surface area of their synaptic zones varied between 5 10 m and 23 10 m. in the 3rd phase, the dendritic spines had a volume greater than 19 10 m, at least three cisterns in the spine apparatus, and a larger surface area of the synaptic zone (greater than 23 10 m ; figures 5, 3(a), and 3(b)). the linear correlations between the volume of dendritic spines and the surface area of their synaptic zones, and between the volume of the spines and the number of sacs in the spine apparatus, were highly significant (p <.01). therefore, the large variation in size of the dendritic spines in 19-day - old mice might be due to the coexistence of spines at different stages of development. thus, it was suggested that the dendritic spines forms directly from the dendritic shaft when it is contacted by an axon that induces the clustering of rac1, a small rhogtpase that pushes the axon and the clustered rac1 molecules away from the dendrites, forming the spines and recruiting ampa receptors to synapses. protracted synaptogenesis was demonstrated after local electrical theta - burst stimulation, by correlating time - lapse two - photon microscopy of newly formed spines on ca1 pyramidal neurons in mice organotypic hippocampal slices with electron microscopy. among the young spines, only a few hours old (3.2 0.7 h, n = 13), 9 were in contact with axons but did not form synapses, while 4 of those analyzed were not contacted by axons. indeed, only old spines (16.3 0.4 h, n = 9) form synapses resembling those of the preexisting spines. these old spines had a significantly larger volume than the younger ones and they were indistinguishable from the controls (old, 0.1646 0.0262, n = 9 ; young, 0.0534 0.0124, n = 13, p <.001 ; control spines, 0.1421 0.0240 m, n = 14, p =.55). these data were considered to be evidence that a shaft synapse is not normally converted to a spine synapse by budding from a dendrite. furthermore, time - lapse imaging in live developing hippocampal slices, identified the migration of the psd (postsynaptic density) along the shaft of the dendrites through a dendritic spine or a protospine using gfp - tagged psd-95 (postsynaptic density 95) protein (see ; figure 6(b)). however, there was no evidence that psds on the dendritic shaft represent a synaptic specialization. another possible option is that these psds could be synthesized at the dendritic shaft and then moved to those dendritic spines or filopodia where synapses are formed. further support for this model came during dendritic spine loss, following exposure of cortical neuron cultures to nmda, ampa, or oxygen and glucose deprivation (similar characteristics of ischemia), the axon terminals remain joined to the swelling formed at the site of the dendritic spine. two hours after agonist exposure, dendritic spines reemerged in the same location where they disappeared, suggesting that psds may contain information for the production of functional dendritic spines. nevertheless, it should be borne in mind that these pathological conditions might be different from those that might occur during normal development. firstly, there are many fewer synapses on dendritic shafts during development than the total number of synapses that exist in the adult neuron, and, hence, sampling during early stages of development might not be representative of the total adult population. it is also possible that prior to the appearance of filopodia and dendritic spines, synapses could only have been established on dendritic shafts. in hippocampal slices, time - lapse imaging studies indicated that dendritic spines may emerge spontaneously from dendrites [19, 27, 54, 56, 57 ] and also in anesthetized animals [5860 ]. two of these studies focused particularly on the role of the presynaptic terminals. by correlating in vivo imaging studies and electron microscopy, adult ca1 pyramidal neurons in organotypic cultures were seen to give rise to small spines or filopodia after local electrical theta - burst stimulation (tbs) of afferent fibres [54, 60 ]. these spines initially grow without synapses, and they later formed these functional structures preferentially on preexisting boutons (multisynaptic boutons). moreover, it was confirmed that in the adult neocortex, the density and the packing of neurites is so high that it might be impossible for axons to first establish synapses on shafts and for them then to move the dendritic spines away, crossing over many other neurites. if this mechanism is active during development, it must be tightly and sequentially regulated so that axons do not block one another. although synaptic contacts appeared after the emergence of the dendritic spine [54, 60 ], these studies did not rule out the role of the presynaptic axon in the induction of spinogenesis. since these experiments were performed under tbs condition, nervous activity of the presynaptic terminal could be essential for the dendritic spine formation. many years ago, several experiments also showed the importance of sensory activity in the development and maintenance of dendritic spines [52, 6167 ]. in many neurons, dendritic trees are initially smooth prior to the appearance of dendritic spines and they are only later covered with other types of dendritic appendages called dendritic filopodia (figure 3(c)). dendritic filopodia can be distinguished from dendritic spines as they are fine structures (diameter less than 0.3 m) of 340 m length that do not usually have a bulbous head. dendritic filopodia are much more dynamic than dendritic spines, and in hippocampal pyramidal neurons, most of the dendritic filopodia continuously extend from and retract back to the shaft of the dendrite with a half - life of about ten minutes [18, 27 ]. the existence of dendritic filopodia during development has often been described [18, 19, 24, 26, 27, 6874 ], and their morphological similarity coupled with the sequential appearance of dendritic filopodia and spines suggest that filopodia might be precursors of dendritic spines. accordingly, the filopodial model proposes that filopodia differentiate into dendritic spines following their interaction with axons (figure 3(c)). by contrast, electron microscopy studies have shown that there are many synapses on dendritic shafts and at the base of filopodia, suggesting that filopodia may be particularly implicated in synaptogenesis by recruiting synapses to the shafts. thus, dendritic filopodia would retract and incorporate the synapses into the dendritic tree, and, only later, the dendritic spines grow at the sites at which these synapses are found (in a manner similar to that of miller / peters model). studies on postembryonic dendritic remodelling of motoneuron 5 during the metamorphosis of the hawkmoth manduca sexta, indicated that presynaptic terminals readily accumulate along the filopodia towards the dendrites. in recent years, research has focused on dendritic filopodia to shed light on the function of these structures. ultrastructural analyses have demonstrated their implication in synaptogenesis whereas in vivo time - lapse imaging has defined the dynamics of these structures. however, long ago scientists had already proposed their existence, motility and probable implication in synaptogenesis. it is noteworthy that cajal referred to similar developmental structures many years ago, and although they appeared in his drawings (figure 6(a)), he did not specifically name them. cajal proposed that they were transient structures, as later confirmed, and in reference to figure 6(a), cajal wrote : mostramos en la figura 50 algunos dibujos tomados de tallos de pirmides cerebrales adultas o jvenes. en a presentamos el tallo de una pirmide de la regin visual del conejo casi adulto. ntense cun cortas son las espinas y cmo empiezan delgadas y acaban por un bulbo final. en b hemos dibujado otro tallo del nio de dos meses (pirmide de la regin visual). llama la atencin, no slo la mayor longitud de los apndices, sino su frecuencia con que se dividen y los cambios de direccin de sus ramillas secundarias. confrmase la disposicin mostrada en a ; las espinas aparecen un poco ms largas y con frecuencia incurvadas. como trmino de comparacin hemos dibujado en d una dendrita de una clula motriz de la mdula (gato de un mes). advirtase que la superficie est erizada de proyecciones irregulares y rara vez acabadas mediante bulbos. es casi seguro que esta disposicin es transitoria. en fin, dibujamos tambin algunas colaterales nerviosas cruciales u oblicuas (cerebro), sin que sea dable apreciar su fusin con las espinas (cajal, 1933). mostramos en la figura 50 algunos dibujos tomados de tallos de pirmides cerebrales adultas o jvenes. en a presentamos el tallo de una pirmide de la regin visual del conejo casi adulto. ntense cun cortas son las espinas y cmo empiezan delgadas y acaban por un bulbo final. en b hemos dibujado otro tallo del nio de dos meses (pirmide de la regin visual). llama la atencin, no slo la mayor longitud de los apndices, sino su frecuencia con que se dividen y los cambios de direccin de sus ramillas secundarias. en c confrmase la disposicin mostrada en a ; las espinas aparecen un poco ms largas y con frecuencia incurvadas. como trmino de comparacin hemos dibujado en d una dendrita de una clula motriz de la mdula (gato de un mes). advirtase que la superficie est erizada de proyecciones irregulares y rara vez acabadas mediante bulbos. dibujamos tambin algunas colaterales nerviosas cruciales u oblicuas (cerebro), sin que sea dable apreciar su fusin con las espinas previously, cajal had also described filopodia in growth cones : en la porcin terminal base, no es raro ver una prolongacin membranosa ms larga, especie de avanzada protoplsmica insinuada en los intersticios intercelular interepitelial (cajal, 1899) [6, page 514 ]. en la porcin terminal base, no es raro ver una prolongacin membranosa ms larga, especie de avanzada protoplsmica insinuada en los intersticios intercelular interepitelial the filopodial motility was suspected by several scientists, including morest, who even proposed a role for these structures in synaptogenesis :... they help to pull the afferent axonal end - branches to their definitive synaptic location. (morest, 1969a, b) [68, 69 ].... they help to pull the afferent axonal end - branches to their definitive synaptic location. (morest, 1969a, b) [68, 69 ]. filopodia motility was also demonstrated in growth cones, although the motility of dendritic filopodia was demonstrated by dailey and smith in 1996, who found that :... most of the filopodial protrusions (up to 10 m long) extended (maximal rate : 2.5 m / min) from dendrite shafts, then retracted back to the shaft within 30 minutes or less (median lifetime, 10 min). in addition to the great abundance of dendritic filopodia during active synaptogenesis [24, 26, 68, 69, 73, 77 ], their elongated morphology suggests that these structures could play a role as bridging structures to facilitate axodendritic synaptic contacts [18, 26, 27, 68, 69, 74, 7883 ]. moreover, filopodia could act either passively to form a virtual dendrite or actively to define the dendritic capture volume [18, 27 ]. the implication of filopodia in synaptogenesis has been elucidated by electron microscopy studies that identified synapses in both dendritic growth cone filopodia [78, 81, 83, 84 ] and collateral dendritic filopodia in different areas of the nervous system (see [20, 74, 85 ], figure 6(e)). interestingly, high - frequency focal synaptic stimulation induced enhanced growth of small filopodia - like protrusions in ca1 hippocampal dendrites of organotypic slices prepared from rat pups at postnatal day 7 and cultured for 7 to 9 days. this process is stimulated by synaptic transmission mediated by activation of nmda receptors, since the presence of apv (dl-2-amino-5-phosphonovaleric acid), a specific antagonist for nmda receptors, abolished the growth of filopodia. the synapses in these filopodia are more likely to connect with active presynaptic axons during the synaptic stimulus, providing a mechanism to explain hebbian rules of synaptic plasticity. overexpression of glur2 receptors (the glutamate receptor 2 subunit of ampa receptors) increases the length of spines, the width of spine heads, and the density of spines in mature cultured hippocampal neurons (22 days in vitro) that normally exhibit mushroom - like spines. however, in younger neurons (11 days in vitro), glur2 overexpression only induces an increase in the density of filopodia - like protrusions that were longer and wider than those found on control neurons (see). the first n - terminal 92 amino acids of the extracellular domain of glur2 are necessary and sufficient to promote the appearance of spines through the formation of a synaptic complex with n - cadherin. in addition, during the development of the rat barrel cortex in vivo, sensory deprivation by whisker trimming markedly reduces the protrusive motility of filopodia and dendritic spines in layer 2/3 pyramidal neurons of deprived regions during a critical period around postnatal days (p) 11/13, but no effect was observed in either younger or older animals. thus, it was concluded that sensory deprivation does not modulate synapse number itself, but, rather, it modifies the experience - dependent rearrangements of synaptic connections required to form precise sensory maps, as shown by the electrophysiological study of layer 2/3 receptive fields. in addition to glur2 receptors, overexpression or clustering of tm - agrin (a transmembrane form of the extracellular matrix heparan sulfate proteoglycan agrin) induces the formation of highly dynamic filopodia by the activation of intracellular signalling cascades (involving lipid rafts, src family kinase fyn, mapk), supporting the hypothesis that tm - agrin is a receptor or coreceptor in neurons. indeed, activity - dependent presynaptic exocytosis of neurotrypsin and the resulting proteolytic cleavage of agrin are a mechanism to promote the activity - dependent formation of dendritic filopodia. the cleavage of agrin requires the additional activation of the postsynaptic cell, and, thus, neurotrypsin - dependent cleavage of agrin could represent a molecular detector for concomitant presynaptic and postsynaptic activation. the activation of sk3 channels (small conductance ca activated k) also leads to immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions in neural progenitor cells. diminished calcium transients in dendrites facilitate filopodial outgrowth during development of organotypic slices of the neonatal rat hippocampus (p 02 + div 13). filopodia can generate calcium transients that probably lead to immature synapse formation when transmitted into the dendrite, preventing the growth of additional filopodia in a range of 10 m, thereby impeding the overload of synaptic inputs on some dendrites as well as underrepresentation of inputs on others. however, since local calcium transients are not completely abolished by blocking gaba receptors, other signalling molecules must also be involved in inducing local calcium transients, such as receptors that recognize adhesion molecules and extracellular matrix components like 1 integrin or neurotrophins. in addition to the similar morphology and sequential appearance of dendritic spines and filopodia [26, 51, 96, 97 ], other studies also suggest that dendritic filopodia evolve to form the definitive dendritic spines [18, 27 ]. on the one hand, there was a progressive decrease of dendritic filopodia and an increase in dendritic spines during development, suggesting that filopodia could transform into dendritic spines. in addition, an intermediate transient stage was detected, the protospine, with the dynamic characteristics of both dendritic spines and filopodia. protospines were stable for up to 22 hrs (the half - life of filopodia is only 10 minutes), but they had the motility of filopodia. dendritic filopodia were also seen to interact with synaptic boutons 510 m away from the dendrite, and the dendritic filopodia that establish synaptic contacts were more stable and less motile with a differentiated head (figure 6(b) ;). some dendritic filopodia have a swelling on their synaptic contacts [20, 98 ], and, thus, a bulbous head appears to be a characteristic of dendritic spines and protospines that promote the stabilization of the filopodia. dendritic spines with several heads, multispines, were described in golgi - impregnated or lucifer yellow - filled neurons of the medial hyperstriatum ventrale of 3-day - old chicks, a brain area implicated in learning and memory processes in the chick. the multispine pedicle or neck can be branched or unbranched, and the number of heads per multispine ranged from 2 to 8 (mean = 3) for branched multispines, and from 2 to 4 multispines represented 16.4% of the total number of spines, but if the number of spine heads is considered, this percentage increases to 34.69%. multispines are also found in all neuronal nuclei studied in 3-day - old chicks, for example, the ectostriatum, field l, hyperstriatum ventrale, lobus parolfactorius, and neostriatum. these multispines are similar structures to the protospines, and they were previously considered as a transitory phase in the development of dendritic spines. for instance, cajal drew dendritic appendages with intermediate characteristics between filopodia and spines in the visual cortex of a 2-month - old child (figures 6(a) and 6(b)). in a trisomic infant, long (hair - like) dendritic spines with several dilatations in their pedicles as retaining their primitive developing appearance, suggesting that inadequate en passant contacts formed because the spines failed to develop mature spines (shorter and one - headed spines). multispines or protospines can also be seen in his pictures and drawings of neurons in normal developing cerebral cortex (see figures 2(c) and 2(e) in). we have found long appendages similar to the protospines, with one or more heads and morphological characteristics between dendritic spines and filopodia, in cajal 's histological slides (figures 10(c), 13(d), 14, 15, 16, 17, 18, and 19). interestingly, de novo synthesis of gfp - tagged psd-95 may occur in filopodia (figure 6(d)) that sometimes develop shapes similar to those we have seen in cajal 's histological slides, exhibiting characteristics of both dendritic spines and filopodia. however, there is no clear evidence that dendritic filopodia are the precursors of all dendritic spines. although dailey and smith occasionally observed the apparent conversion of an actively protrusive dendritic filopodium to a more stable spine - like structure, they could not correlate this conversion with functional synapse formation in their slice preparation. using two - photon time - lapse imaging of developing hippocampal pyramidal neurons, it was confirmed that most filopodia turn into sessile and pedunculated spines, although filopodia could also extend out of existing spines. established that filopodia only occasionally led to the formation of durable dendritic spines in a more general process that stabilized new protrusions, as determined by the enlargement of the spine head and the expression of psd-95 within a critical 24 h period. psd-95 controls synapse formation by regulating nnos (neuronal nitric oxide synthase) expression at the synapse, releasing no (nitric oxide), and activating cyclic guanosine monophosphate (cgmp) signalling in presynaptic axons. multi - innervation (up to seven presynaptic terminals) of large dendritic spines occurs when psd-95 is overexpressed in rat hippocampal slice cultures maintained for 1115 days in vitro. there is also other data suggesting that filopodia are probably not the precursors of all dendritic spines. some studies indicated that dendritic filopodia disappear completely before the appearance of the dendritic spines [20, 68, 69 ]. moreover, many neurons that do not have dendritic spines in adulthood bear dendritic filopodia during the synaptogenic period [24, 70, 73, 77, 105107 ]. however, although we have observed dendritic filopodia in double bouquet cells and basket cells of the cerebral cortex and on basket cells and golgi cells of the cerebellar cortex, the number of filopodia is very low when compared with the number of filopodia of pyramidal cells in the cerebral cortex. in contrast to the filopodial model, some experiments show that dendritic spines could develop de novo from dendritic branches [27, 57 ]. however, these experiments do not rule out the possibility that spines are born de novo without synapses, as demonstrated in adult animals in which spines emerge as thin dendritic spines or as short filopodia that later appear to form a differentiated head. similarly, nonsynaptic dendritic spines in the neocortex that resemble dendritic filopodia have been seen, although smaller than those observed during development, suggesting that they could be new spines formed in adult animals. it is possible that adult neurons show smaller dendritic protrusions than the dendritic filopodia observed during development, possibly because during development there is more free space in the neuropil and the axons are further apart (see supplementary video 1 available online at doi : 10.1155/2011/769207). besides participating in synaptogenesis and spinogenesis, dendritic filopodia are also implicated in the selection between presynaptic candidates when forming the correct nervous connections. thus, dendritic filopodia can select synaptic partners between axons (excitatory versus inhibitory :). indeed, when filopodial contacts are stabilized with excitatory terminals, high - frequency local calcium transients independent of glutamate are evident whereas short - lived contacts, especially with inhibitory terminals, are associated with low - frequency local calcium transients. filopodia can be formed on the terminal dendritic growth cones, and according to the synaptotropic hypothesis, filopodia could receive synaptic contacts forming a dendritic branch holding the synaptic contacts. by successive iteration two kinds of filopodia might exist, each with different properties and functions : collateral filopodia and dendritic growth cones filopodia. filopodia of the dendritic growth cones are involved in dendrite growth and branching in an activity independent manner whereas shaft filopodia are responsible for activity - dependent synaptogenesis, and, in some cases, they may become dendritic spines. the cajal museum holds 4529 histological slides personally prepared by santiago ramn y cajal. of these, 809 are stained by the golgi method, which enables us to visualize splendidly preserved dendritic spines and filopodia. although there are 109 histological slides stained with the methylene blue method, which also enables filopodia and dendritic spines to be observed, these preparations were not used in this study due to their poor preservation. here, we have employed the following methods. the age of the animalsthe age of the animals had to be determined by the labeling of cajal 's histological slide. sometimes, cajal did not write the exact prenatal and postnatal day of development of animal, he used the month or phrases indicating the stage of development like almost newborn, we have found that this was sufficient for the type of analysis carried out in this work. the age of the animals had to be determined by the labeling of cajal 's histological slide. sometimes, cajal did not write the exact prenatal and postnatal day of development of animal, he used the month or phrases indicating the stage of development like almost newborn, we have found that this was sufficient for the type of analysis carried out in this work. focus extended imagingwe have obtained stacks of digital images of the histological slides and the areas out of focus in each image were eliminated using image j software. we have obtained stacks of digital images of the histological slides and the areas out of focus in each image were eliminated using image j software. three - dimensional reconstructions and renderingwe used the neuronal coding, neuronal quantification, and neuronal transformation programs [16, 112, 113 ] for the 3d - reconstruction and quantification of neuronal structures (dendrites, axon, dendritic spines, dendritic filopodia, and varicosities). we also developed a computer program to convert the codes used in the neuronal programs to the pov - ray (persistence of vision raytracer : http://www.povray.org/), a freely available program that we used for rendering. we used the neuronal coding, neuronal quantification, and neuronal transformation programs [16, 112, 113 ] for the 3d - reconstruction and quantification of neuronal structures (dendrites, axon, dendritic spines, dendritic filopodia, and varicosities). we also developed a computer program to convert the codes used in the neuronal programs to the pov - ray (persistence of vision raytracer : http://www.povray.org/), a freely available program that we used for rendering. we have studied developmental stages iii to vi since we were unable to find cells in the two first stages in the golgi - impregnated histological slides. cajal described the first or fusiform phase using the reduced silver nitrate method, but as it does not stain dendritic appendages, we did not study any of the histological preparations stained in this way. in addition, an intermediate phase (phase of regressive atrophic dendrites) between the fusiform phase (i) and the stellate cell phase with disoriented dendrites (iii) has also been described [115117 ]. thus, the successive phases of the development of the purkinje cell are listed below. (i) fusiform stageduring this phase, the dendrites extending from both sides of the soma are smooth, and they do not have any appendages. this stage of development extends from 12 to 14 days of incubation in the chicken according to cajal. during this phase, the dendrites extending from both sides of the soma are smooth, and they do not have any appendages. this stage of development extends from 12 to 14 days of incubation in the chicken according to cajal. (ii) regressive - atrophic dendrite phase.there is an intense regressive process with the reabsorption or retraction of the long dendrites. the purkinje cell becomes reduced to the soma with no branches or with short processes. there is an intense regressive process with the reabsorption or retraction of the long dendrites. the purkinje cell becomes reduced to the soma with no branches or with short processes. (iii) dendritic disorientation stagethis stage is characterized by the explosive outgrowth of short perisomatic protrusions emerging in all directions. we observed some cells with this morphology in histological preparations from a newborn dog, and the dendrites evidently do not develop spines (figures 8(a)8(g)), although they have both lateral (figure 8(b)) and terminal dendritic filopodia, the latter being especially notable (figures 8(c) and 8(d)). this stage is characterized by the explosive outgrowth of short perisomatic protrusions emerging in all directions. we observed some cells with this morphology in histological preparations from a newborn dog, and the dendrites evidently do not develop spines (figures 8(a)8(g)), although they have both lateral (figure 8(b)) and terminal dendritic filopodia, the latter being especially notable (figures 8(c) and 8(d)). (iv) dendritic regularization stagethe size of the soma increases to 1520 m, and the purkinje cells have a differentiated apical trunk with secondary and tertiary branches (figure 9(a)). basal dendritic branches also emerge from the soma, but they are usually shorter than the apical ones. both the basal and apical branches bear dendritic spines (figures 9(b)9(d)), and dendritic filopodia are frequently seen along the dendritic branches and at their tips (figures 9(g) and 9(h)). the branches with filopodia are typically observed at the base of the soma, near the emerging axon (figures 9(e)9(g)). cajal did not identify dendritic spines or filopodia at this developmental stage, although both spines and filopodia are carefully represented in his scientific drawings (figures 9(b)9(g)). it is interesting to note that the perisomatic branches also develop dendritic spines, although these will later be reabsorbed. the size of the soma increases to 1520 m, and the purkinje cells have a differentiated apical trunk with secondary and tertiary branches (figure 9(a)). basal dendritic branches also emerge from the soma, but they are usually shorter than the apical ones. both the basal and apical branches bear dendritic spines (figures 9(b)9(d)), and dendritic filopodia are frequently seen along the dendritic branches and at their tips (figures 9(g) and 9(h)). the branches with filopodia are typically observed at the base of the soma, near the emerging axon (figures 9(e)9(g)). cajal did not identify dendritic spines or filopodia at this developmental stage, although both spines and filopodia are carefully represented in his scientific drawings (figures 9(b)9(g)). it is interesting to note that the perisomatic branches also develop dendritic spines, although these will later be reabsorbed. (v) disappearance of perisomatic branches, emergence of secondary and tertiary branches on the apical trunk, and appearance of dendritic spinesas development progresses, the dendritic tree increases in size (figures 1012) engendering a single apical trunk with higher - order branches. the branchlets (small terminal branches) and the dendritic spines contact the parallel fibers. we have found different neuronal types in this phase of development, the more immature of which were observed by cajal in newborn cats (figure 10(b)). these neurons have dendritic spines (figure 10(a)2 - 3), especially on the proximal primary branches whereas the high - order branches bear several thin dendritic spines (figure 10(a)2). there are numerous filopodia located both laterally and on the terminal portions of the dendritic branches (figures 10(a)1, 10(b), and 10(c)). in 17-day - old dogs (figures 11(a)11(d)) and in young almost adult cats (figures 11(e)11(h)), the ramifications and density of the dendritic appendages in the dendritic tree increase, and the filopodia are predominantly located at the terminal portion of the branchlets (figures 11(b)11(f)). some collateral filopodia can be found in the purkinje cells of 17-day - old dogs (figures 11(b) and 11(d)2) and their branchlets may also bear thin dendritic spines (figures 11(c)2 and 11(d)3). there were many sessile dendritic spines or spines with no clear differentiation between the head and neck that decrease in purkinje cells of the young almost adult cats (figures 11(f), 11(g)2 - 3, and 11(h)3 - 4). furthermore, there were some thorny structures (figure 11(c)4) that were defined as.in contrast to the cat and dog, the 12-day - old mouse has more types of dendritic spines (i.e., sessile, thin, mushroom, and ramified) that are more mature than in a cat or a dog of a similar age (data not shown). as development progresses, the dendritic tree increases in size (figures 1012) engendering a single apical trunk with higher - order branches. the branchlets (small terminal branches) and the dendritic spines contact the parallel fibers. we have found different neuronal types in this phase of development, the more immature of which were observed by cajal in newborn cats (figure 10(b)). these neurons have dendritic spines (figure 10(a)2 - 3), especially on the proximal primary branches whereas the high - order branches bear several thin dendritic spines (figure 10(a)2). there are numerous filopodia located both laterally and on the terminal portions of the dendritic branches (figures 10(a)1, 10(b), and 10(c)). in 17-day - old dogs (figures 11(a)11(d)) and in young almost adult cats (figures 11(e)11(h)), the ramifications and density of the dendritic appendages in the dendritic tree increase, and the filopodia are predominantly located at the terminal portion of the branchlets (figures 11(b)11(f)). some collateral filopodia can be found in the purkinje cells of 17-day - old dogs (figures 11(b) and 11(d)2) and their branchlets may also bear thin dendritic spines (figures 11(c)2 and 11(d)3). there were many sessile dendritic spines or spines with no clear differentiation between the head and neck that decrease in purkinje cells of the young almost adult cats (figures 11(f), 11(g)2 - 3, and 11(h)3 - 4). furthermore, there were some thorny structures (figure 11(c)4) that were defined as in contrast to the cat and dog, the 12-day - old mouse has more types of dendritic spines (i.e., sessile, thin, mushroom, and ramified) that are more mature than in a cat or a dog of a similar age (data not shown). (vi) adult stagewe found three histological slides prepared by cajal of the translobular cerebellum from adult animals (cat, bird, and man) impregnated by the golgi method. the dendritic tree of the purkinje cell is completely developed, although the degree of ramification varies with the species (figures 12(a) and 12(b)), and in birds, purkinje cells are less ramified than those of the cat or human. in the adult phase, the dendritic trees are densely covered with different types of dendritic spines (thin, ramified, mushroom, sessile : figures 12(c)12(e), 12(i)12(n)), but there is also a significant difference between the proportion of each type in each species (figures 12(f)12(h)). thus, the purkinje cell tree in birds has more sessile dendritic spines than in mammals indicating a progressive differentiation of the neck and the head of the dendritic spine along the phylogenetic scale. we found three histological slides prepared by cajal of the translobular cerebellum from adult animals (cat, bird, and man) impregnated by the golgi method. the dendritic tree of the purkinje cell is completely developed, although the degree of ramification varies with the species (figures 12(a) and 12(b)), and in birds, purkinje cells are less ramified than those of the cat or human. in the adult phase, the dendritic trees are densely covered with different types of dendritic spines (thin, ramified, mushroom, sessile : figures 12(c)12(e), 12(i)12(n)), but there is also a significant difference between the proportion of each type in each species (figures 12(f)12(h)). thus, the purkinje cell tree in birds has more sessile dendritic spines than in mammals indicating a progressive differentiation of the neck and the head of the dendritic spine along the phylogenetic scale. we will present our conclusions on the development of purkinje cells dendritic spines under three headings in relation to the three known types of dendritic appendages : dendritic filopodia, dendritic spines contacting climbing fibers, and dendritic spines contacting parallel fibers. (1) dendritic filopodiawe observed dendritic filopodia associated with purkinje cells at developmental stages (iii), (iv), and (v) (figures 810), and the density of filopodia along the dendritic tree of the purkinje cell changed according to these developmental stages. filopodia found at the tip and the terminal portion of the dendrites (figures 8(c), 8(d), and 8(f)), and at the lateral branches of the definitive apical trunk (figure 9(h)) are probably implicated in the orientation and growing of the dendrites. while filopodia on branches that emerge laterally from the soma (figures 9(e) and 9(f)) are probably implicated in the interaction with the climbing fibers at this developmental stage. later on, in the purkinje cells, we found dendritic appendages with morphological characteristics of both dendritic filopodia and spines (figures 11(b), 11(c)1 and 11(d)1), like the protospines in hippocampal tissue slices. protospines have a gelatinous appearance, they are stained light brown by the golgi method, and they contain one or more bulbous heads. previously, the collateral filopodia and protospines were also observed in the cerebellum of 5, 10, and 15-day - old rats whereas they are rare in 30-day - old rats (figure 10(d) :). the presence of these dendritic appendages suggests that synaptogenesis and spinogenesis can be also mediated by filopodia in the purkinje cell. interestingly, cajal observed similar collateral filopodia in his drawings (figure 10(b)), and describing the purkinje cell dendrites in a 15-day - old cat, he wrote : casi todas sus ramas (figure 2(b), figure 163 de este trabajo) poseen ligeras espinas perpendi - cularmente insertas en su contorno. estas espinas aparecen teidas en caf claro y son ms grandes que las de las ramitas terminales de los corpsculos adultos (cajal, 1889). we observed dendritic filopodia associated with purkinje cells at developmental stages (iii), (iv), and (v) (figures 810), and the density of filopodia along the dendritic tree of the purkinje cell changed according to these developmental stages. filopodia found at the tip and the terminal portion of the dendrites (figures 8(c), 8(d), and 8(f)), and at the lateral branches of the definitive apical trunk (figure 9(h)) are probably implicated in the orientation and growing of the dendrites. while filopodia on branches that emerge laterally from the soma (figures 9(e) and 9(f)) are probably implicated in the interaction with the climbing fibers at this developmental stage. later on, in the purkinje cells, we found dendritic appendages with morphological characteristics of both dendritic filopodia and spines (figures 11(b), 11(c)1 and 11(d)1), like the protospines in hippocampal tissue slices. protospines have a gelatinous appearance, they are stained light brown by the golgi method, and they contain one or more bulbous heads. previously, the collateral filopodia and protospines were also observed in the cerebellum of 5, 10, and 15-day - old rats whereas they are rare in 30-day - old rats (figure 10(d) :). the presence of these dendritic appendages suggests that synaptogenesis and spinogenesis can be also mediated by filopodia in the purkinje cell. interestingly, cajal observed similar collateral filopodia in his drawings (figure 10(b)), and describing the purkinje cell dendrites in a 15-day - old cat, he wrote : casi todas sus ramas (figure 2(b), figure 163 de este trabajo) poseen ligeras espinas perpendi - cularmente insertas en su contorno. estas espinas aparecen teidas en caf claro y son ms grandes que las de las ramitas terminales de los corpsculos adultos (cajal, 1889). casi todas sus ramas (figure 2(b), figure 163 de este trabajo) poseen ligeras espinas perpendi - cularmente insertas en su contorno. estas espinas aparecen teidas en caf claro y son ms grandes que las de las ramitas terminales de los corpsculos adultos (cajal, 1889). (2) dendritic spines contacting climbing fiberswe observed dendritic spines on the lateral dendrites of the soma (figure 9(d)). according to larramendi and victor and laxson and king, these dendritic spines receive synaptic contacts (gray type i) from the climbing fibers. later on in development, the perisomatic primary dendrites will be reabsorbed, and only perisomatic dendritic spines will remain, receiving connections from climbing fibers (see [21, 120 ] ; figures 7(b)7(d)). in fact, we did not observe these dendritic spines in adult animals, although a small population should still be present. we observed dendritic spines on the lateral dendrites of the soma (figure 9(d)). according to larramendi and victor and laxson and king, these dendritic spines receive synaptic contacts (gray type i) from the climbing fibers. later on in development, the perisomatic primary dendrites will be reabsorbed, and only perisomatic dendritic spines will remain, receiving connections from climbing fibers (see [21, 120 ] ; figures 7(b)7(d)). in fact, we did not observe these dendritic spines in adult animals, although a small population should still be present. (3) dendritic spines contacting parallel fibersthe greater density of sessile dendritic spines on purkinje cell branchlets of cat almost adult (figures 11(f), 11(g)2, 3, and 11(h)3, 4) suggests that the majority of dendritic spines could emerge from dendrites through progressive growth, and only later will many dendritic spines differentiate their head and neck, becoming pedunculated spines. however, there are many collateral filopodia and protospines in newborn cats (figure 10(c)) that could evolve directly into dendritic spines. the greater density of sessile dendritic spines on purkinje cell branchlets of cat almost adult (figures 11(f), 11(g)2, 3, and 11(h)3, 4) suggests that the majority of dendritic spines could emerge from dendrites through progressive growth, and only later will many dendritic spines differentiate their head and neck, becoming pedunculated spines. however, there are many collateral filopodia and protospines in newborn cats (figure 10(c)) that could evolve directly into dendritic spines. pyramidal cells follow a stereotypic developmental program, and the dendritic appendages initially described by cajal [6, 7 ] serve to characterize the different stages of development. however, there are two phases that cajal described with the reduced silver nitrate method that we have been unable to study, as this procedure does not impregnate neuronal appendages : the primitive bipolar stage and the neuroblast stage. (i) primitive bipolar phasecajal studied this stage through the reduced silver nitrate method in the cerebral rostral vesicle of 34-day - old chicken embryos. two opposite processes emerge from the soma, one directed towards the cerebral vesicle surface whereas the other is directed towards the inner side. cajal studied this stage through the reduced silver nitrate method in the cerebral rostral vesicle of 34-day - old chicken embryos. two opposite processes emerge from the soma, one directed towards the cerebral vesicle surface whereas the other is directed towards the inner side. (ii) neuroblast phase.the peripheral process disappears whereas the inner one remains and develops into an axon with a growth cone at its tip. this axon can only be observed during the initial stages of development, as it is very difficult to detect in the fetus and newborn animals. the peripheral process disappears whereas the inner one remains and develops into an axon with a growth cone at its tip. this axon can only be observed during the initial stages of development, as it is very difficult to detect in the fetus and newborn animals. (iii) bipolar phase (figures 13(a) and 13(b)).the first outgrowth from the soma forms the axon, which is followed by the emission of an apical dendrite that reaches layer i, giving the neuron its bipolar shape. during this stage, however, in some cells, small appendages emerge from the soma and the apical trunk (figure 13(b)). some bipolar cells also contain a basilar branch that usually ends in a ball. the first outgrowth from the soma forms the axon, which is followed by the emission of an apical dendrite that reaches layer i, giving the neuron its bipolar shape. during this stage, the neurons appear to have a smooth surface with no dendritic appendages. however, in some cells, small appendages emerge from the soma and the apical trunk (figure 13(b)). some bipolar cells also contain a basilar branch that usually ends in a ball. (iv) phase of basilar dendrite appearance and of collateral oblique dendrites of the apical trunkthe pyramidal cells emit basal dendrites, oblique branches of the apical trunk (initially those more proximal to the soma), and the apical tuft, as observed in cajal 's histological preparations of the newborn rabbit (figure 13(c)) and human fetus (figure 13(e)). during this stage the dendritic branches are smooth or with some appendages (filopodia, protospines, and thin and mushroom spines ; figure 13(d)18). the pyramidal cells emit basal dendrites, oblique branches of the apical trunk (initially those more proximal to the soma), and the apical tuft, as observed in cajal 's histological preparations of the newborn rabbit (figure 13(c)) and human fetus (figure 13(e)). during this stage the dendritic branches are smooth or with some appendages (filopodia, protospines, and thin and mushroom spines ; figure 13(d)18). (v) phase in which nervous [axonal ] collaterals appearthese collaterals first appear on big pyramidal cells and during the following days, the small pyramidal cells also develop axon collaterals. during this phase, the dendrites (apical tuft, figures 14(a) and 14(b) ; apical trunk, figures 14(c)14(e) ; basilar dendrites, figures 14(f) and 14(g) ; oblique apical dendrites, figure 14(h) ; see also figures 15(a)15(d) and 16(a)16(c), supplementary video 2) develop many dendritic filopodia, protospines, and some dendritic spines. later, the ratio is reversed with many more dendritic spines than dendritic filopodia and protospines (figures 15, 16(d)16(f), and 17(e), supplementary video 3). in cajal 's histological slides, we were able to see different forms simultaneously (dendritic spines, protospines, and filopodia), although there were successive periods when dendritic filopodia and protospines or dendritic spines were very abundant, and the evolution was gradual. moreover, spinogenesis did not occur simultaneously in each neuron, and some dendritic branches developed spines earlier (figure 17(e)) than others (figure 17(d)). we also found that dendritic spines and filopodia coexist, especially in neurons of newborn animals and in the first few day of life. in addition, there are many protospines more than 5 m long, sometimes ramified with terminal bulbous heads (figures 14, 15, 16(b), and 17) and/or with heads along the protospine stalk (figures 14, 15(b), and 16(c)). these kinds of protospines were also visible in later stages of development.the density of dendritic appendages increased during development, although the proportion of dendritic filopodia fell. we found more dendritic spines in more mature stages, such as cajal 's histological slides shown in figures 17(d) and 17(e). interestingly, we also found many dendritic spines emerging from the dendritic tree that did not have a clear distinction between head and neck. for instance, the distinct proportions of the dendritic appendages were 64.7% thin spine, 16.8% sessile spine, 14.7% mushroom spine, 2.41% branched spine, and 1.39% dendritic filopodia, in a young mouse pyramidal cell visualized in a histological preparation of cajal. these sessile spines are probably dendritic spines emerging de novo from the dendritic branches. these collaterals first appear on big pyramidal cells and during the following days, the small pyramidal cells also develop axon collaterals. during this phase, the dendrites (apical tuft, figures 14(a) and 14(b) ; apical trunk, figures 14(c)14(e) ; basilar dendrites, figures 14(f) and 14(g) ; oblique apical dendrites, figure 14(h) ; see also figures 15(a)15(d) and 16(a)16(c), supplementary video 2) develop many dendritic filopodia, protospines, and some dendritic spines. later, the ratio is reversed with many more dendritic spines than dendritic filopodia and protospines (figures 15, 16(d)16(f), and 17(e), supplementary video 3). in cajal 's histological slides, we were able to see different forms simultaneously (dendritic spines, protospines, and filopodia), although there were successive periods when dendritic filopodia and protospines or dendritic spines were very abundant, and the evolution was gradual. moreover, spinogenesis did not occur simultaneously in each neuron, and some dendritic branches developed spines earlier (figure 17(e)) than others (figure 17(d)). we also found that dendritic spines and filopodia coexist, especially in neurons of newborn animals and in the first few day of life. in addition, there are many protospines more than 5 m long, sometimes ramified with terminal bulbous heads (figures 14, 15, 16(b), and 17) and/or with heads along the protospine stalk (figures 14, 15(b), and 16(c)). these kinds of protospines were also visible in later stages of development. the density of dendritic appendages increased during development, although the proportion of dendritic filopodia fell. we found more dendritic spines in more mature stages, such as cajal 's histological slides shown in figures 17(d) and 17(e). interestingly, we also found many dendritic spines emerging from the dendritic tree that did not have a clear distinction between head and neck. for instance, the distinct proportions of the dendritic appendages were 64.7% thin spine, 16.8% sessile spine, 14.7% mushroom spine, 2.41% branched spine, and 1.39% dendritic filopodia, in a young mouse pyramidal cell visualized in a histological preparation of cajal. these sessile spines are probably dendritic spines emerging de novo from the dendritic branches. such appendages could be filopodia sensing the local environment in order to initiate the formation of basilar branches in the correct position and orientation. we found few dendritic spines during fetal periods and in newborn animals (figure 13(d)) despite the presence of some dendritic filopodia. in successive developmental stages, dendritic appendages increase in number, and our observations indicate an initial proliferation of dendritic filopodia. although, some studies suggest that the dendritic filopodia completely disappear before the appearance of dendritic spines [20, 68, 69 ], we observed filopodia and dendritic spines coexisting in many different neurons. the progressive decrease in filopodia and the progressive increase in dendritic spines could suggest that filopodia give rise to dendritic spines. it is noteworthy that we found many appendages with a mixed morphology between dendritic spines and filopodia (protospines). protospines could either evolve into dendritic spines (filopodial model) or retract to the dendritic shaft, and thus, the formation of dendritic spines could follow the miller / peters model. however, dendritic spines also appear to emerge directly from the dendritic shafts, especially in advanced stages of development (figure 15(c)). during adulthood, owing to the reduction of the extracellular space, dendritic spines could emerge from the dendritic shafts as short filopodia or as spines with no synapses to form the definitive dendritic spines. thus, small dendritic protrusions with no clear difference between the head and neck were described in, the adult as resembling the structures we found during development. also in the adult, the emergence of de novo dendritic spines from dendritic trunks was also described to only later develop a synapse. we have studied eleven golgi - impregnated histological slides of the olfactory bulb of newborn and one - month - old dog, and mice. we only found granule cells in the developmental stage when the dendritic tree is completely formed, and these granule cells have a peripheral dendrite that ends in a dendritic tuft and a number of short inner dendritic branches (figure 18(a) ; supplementary video 4). the basilar and apical branches of these granular cells have many long filopodia and protospines (mean size = 5.77 m) that are frequently ramified (figure 18(b)). these branches also bear different types of dendritic spines, especially thin and mushroom spines. like the pyramidal cells, there are many filopodia at the primary stages of development, while later, the granule cells become covered by protospines and dendritic spines (figure 18(b)). sometimes, these protospines have more than one varicosity (figures 18(b)-16 and 18(b)-18). interestingly, there are also some dark - brown dendritic appendages that differ from the light - brown golgi - impregnated dendritic filopodia found on the apical tuft. these also have morphological characteristics between those of filopodia and dendritic spines, with very long necks and big heads. however, based on the dark color of the golgi staining, we think that they are more stable structures than the protospines and that they could be the gemmules described by rall and shepherd in the adult dendritic tree of the granule cell (figures 18(b).18, arrow). a 3-d reconstruction of a granule cell from a one - month - old dog can be seen in the supplementary video 4. granule cells of the olfactory bulb are characterized by their small size and the absence of axon. cajal confirmed this discovery of golgi and described the connection of these granule cells with mitral cells and the existence of dendritic spines on the granule cell branches :... la expansin perifrica de los granos posee una orientacin y conexin invariables, toda vez que se dirige constantemente la zona plexiforme, donde se termina favor de un penacho de ramas fuertemente espinosas, en contacto con las dendritas secundarias nacidas en las clulas mitrales (cajal, 1904) [7, page 927 ].... la expansin perifrica de los granos posee una orientacin y conexin invariables, toda vez que se dirige constantemente la zona plexiforme, donde se termina favor de un penacho de ramas fuertemente espinosas, en contacto con las dendritas secundarias nacidas en las clulas mitrales (cajal, 1904) [7, page 927 ]. filopodia are very common during the initial steps of development, protospines are especially common during the next phases whereas dendritic spines are commonly found in the histological preparations of more mature animals. these protospines could interact with different presynaptic partners in a kind of synaptogenic competition, as supported by the confirmation of filopodia as multisynaptic structures [20, 123 ]. besides the dendritic filopodia and protospines, long dendritic spines with a big head (gemmules) these dendritic spines were 5 to 6 m in length, with a head of 12 microns. electron microscopy [125127 ] confirmed that gemmules are both inhibitory presynaptic and excitatory postsynaptic elements, and reciprocal synapses between granule cell gemmules and mitral cell dendrites are implicated in mediating feedback and lateral inhibition of the mitral and tufted cells [121, 128 ]. in conclusion, based on the strong presence of filopodia and protospines and the weak presence of sessile dendritic appendages on these cells, spinogenesis in olfactory bulb granule cell seems to mainly follow the filopodial model. besides the projecting neurons of the cerebral and cerebellar cortices, interneurons must also be considered. indeed, their dendrites also develop filopodia and spines but at a lower density than purkinje or pyramidal cells. besides the purkinje cells, we have identified many other cerebellar neuronal types in cajal 's slides : granule cells (figures 19(c) and 19(d)), basket cells (figures 19(a)), star cells (figures 19(b)), golgi cells, lugaro cells, and so forth. these cells also develop filopodia (figures 19(a) and 19(b)) and dendritic spines, albeit at a lower density than purkinje cells. some dendritic spines remain in adult cells, and the spine density is dependent on the cell type. basket and star cells have the greatest spine density followed by golgi cells whereas lugaro cells only occasionally have dendritic spines. it has been pointed out that these cells are smooth in the mature animal while they have filopodia during development, arguing against the role of filopodia in spinogenesis. the fact that dendrites of mature cells have few spines and that these cells develop filopodia during development, makes it possible that some of these filopodia could originate dendritic spines. it should be noted that adult granule cells do not bear dendritic spines, although their dendrites contain pedunculated appendages with head and neck. the formation of these appendages is protracted and depends on an interaction with mossy fibers. at the beginning of its development, the tip of the granule cell dendrites contains a bulb, and it may contain some dendritic filopodia. later, some pedunculated appendages similar to dendritic spines appear whereas the dendritic terminals become more complex, forming the adult claw endings (figures 19(c) and 19(d) ; supplementary videos 5 and 6). we have also studied some interneurons in the cerebral cortex, such as the double bouquet cells (figure 19(e) ; supplementary video 7), basket cells (figure 19(f) ; supplementary video 8), and martinotti cells. these cells also have spines like pyramidal cells, although the proportion of filopodia in the initial stages of development and of dendritic spines at advanced development stages is very much lower. interestingly, protospines are also present, suggesting that the filopodial model also operates in these cells, although some sessile spines seem to emerge directly from the dendritic shaft. according to kawaguchi., spine density also varies in the different interneurons of the cerebral cortex (martinotti cell, double bouquet cell, and basket cell), and many of these dendritic spines establish functional synaptic connections (70% in the martinotti cell). in hippocampal cultures, parvalbumin - positive interneurons transfected with the glur2 subunit of the ampa receptor express many dendritic spines, suggesting that these interneurons have the potential to form dense spines on their dendrites like pyramidal cells, and that their spinogenesis is also highly regulated at the genetic level. the three models of spinogenesis proposed are based on partial visions of a unique but more general model common to all regions of the nervous system. the model of sotelo proposes that cerebellar dendritic spines emerge from dendritic trees through an autonomous cell program, independent of axon terminals (figure 3(a)). however, in normal animals, purkinje cell branchlets grow long spine - like processes that only form synaptic contacts and a terminal head after contacting parallel fibers (figure 4(a)). in the present scientific nomenclature, thus, the synapse between the parallel fiber and dendritic spine provides a simple example of spinogenesis via filopodia and protospine (figure 4(a)). cajal described similar small filopodia in a purkinje cell from a 15 day - old cat (figure 10(b)), and we found filopodia and protospines in the cerebellum in cajal 's preparations (this chapter, figures 10, 11, and 12). filopodia and protospines were observed by berry and bradley in the rat cerebellum (figure 10(d)), and larramendi found long synaptic adhesions between parallel fibers and small dendritic appendages that become mature dendritic spines through a process of synaptic adhesion waning (figure 4(b)). the miller / peters model is based on the maturation of the rat visual cortex (figure 3(b)). it proposes that the formation of dendritic spines begins with a symmetric synapse between a dendrite and an axon, which gives rise to a dendritic elevation that gradually elongates and converts into a spine, at the same time the synapse becomes asymmetric. in the rat visual cortex, dendrites form synapses with symmetric densities as early as day 3 and asymmetric ones by day 9. described similar phases of dendritic spine formation in the dentate gyrus of the rat, as did west and del cerro in the molecular layer of the foetal rat cerebellum, and j. w. hinds and p. l. hinds in the mouse olfactory bulb. the latter authors described isolated postsynaptic densities that can be converted to synapses when a presynaptic specialization develops opposite to them at the beginning of synaptogenesis (e15). indeed, they calculated that symmetric synapses take 9 - 10 hours to transform into asymmetric ones. however, in the visual cortex of 19-day - old mice, asymmetric synapses between a dendritic spine and axon form as the spine matures.. found that dendritic spines grow directly from the dendritic shaft, previously contacted by an axon that induced the clustering of rac1, and subsequent morphological changes led to spinogenesis. also found that synapses appear after the emergence of the dendritic spine in newly formed spines in the barrel cortex of adult mice in vivo and in ca1 pyramidal neurons in organotypic hippocampal slices, respectively the intermediate phase in the development of a pedunculated spine is similar to that of a stubby spine (figures 5 and 2(a)). miller and peters suggested if the stubby spines are developing ones, spine formation and remodelling may be occurring even in the adult animal. parnass. found considerable morphological conversion between each category of dendritic appendages (filopodia, stubby spine, and pedunculated spine) using two - photon time - lapse imaging of developing hippocampal pyramidal neurons transfected with e - gfp in cultured slices, although the period studied was only between 24 h. in the rat visual cortex, miller and peters found filopodia between 3 to 12 days, extending from all dendrites, but rarely at days 15 and 21. although filopodia can form synaptic contacts, they were considered transient structures not related to dendritic spine formation. we found filopodia, protospines, and dendritic spines (thin and mushroom) in pyramidal cells of the newborn rabbit cerebral cortex (figure 13(d)18) during developmental phase (iv). there are many dendritic filopodia, protospines and some dendritic spines (figure 14) in the following developmental phase (v), but the ratio then reverses, many more dendritic spines appearing than dendritic filopodia and protospines (figures 1517). in addition, fiala. found dendritic filopodia forming asymmetric synaptic contacts with axons, especially during the first postnatal week in the rat hippocampal area ca1. the filopodial model is based on the data gathered from developing hippocampal tissue slices (figure 3(c)). lateral filopodia were replaced by spine - like structures (protospines) with a slow turnover. the ultimate fate of filopodia and protospines is still unclear ; although it was proposed that filopodia recruit shaft synapses that later give rise to spines through a process of outgrowth. larramendi interpreted his data as if filopodia could give rise to dendritic spines (figure 4), and parnass. confirmed that most filopodia produce sessile and pedunculated spines, although filopodia could also extend out of existing spines.. found that filopodia only occasionally lead to the formation of stable dendritic spines by enlargement of the spine head and the expression of psd-95 within a critical period of 24 h. the unifying model of spinogenesis that we propose takes into account the development of the neuron and the asynchrony and plasticity in the formation of dendritic spines. mode 1at the very beginning of synaptogenesis (e15, mouse), isolated postsynaptic densities can convert to synapses when a presynaptic specialization develops opposite them. the initial symmetric contact is transformed into an asymmetric one by 9 - 10 hours. the outgrowth of these contacts gives rise to the first dendritic spines. during the maturation of the dendritic spine, the length of the synaptic contact diminishes, a process called, the first axons arriving could easily contact with the dendrites because there are abundant spaces in the neuropil. at the very beginning of synaptogenesis (e15, mouse), isolated postsynaptic densities can convert to synapses when a presynaptic specialization develops opposite them. the initial symmetric contact is transformed into an asymmetric one by 9 - 10 hours. the outgrowth of these contacts gives rise to the first dendritic spines. during the maturation of the dendritic spine, the length of the synaptic contact diminishes, a process called, the first axons arriving could easily contact with the dendrites because there are abundant spaces in the neuropil. mode 2 in newborn animals and during the first postnatal week, lateral dendritic filopodia can develop varicosities and heads giving rise to multispines or protospines after contacting with axons. in this developmental period, the intercellular spaces in the neuropil diminish, and the emission of filopodia could be a neuronal strategy to capture and recruit axonal endings to new dendritic spines (see [15, 20, 68, 69 ]) and at the same time increasing the dendritic capture volume. filopodia contacting several axons can choose the most active ones and, perhaps, discriminate between distinct types of axonal endings. in newborn animals and during the first postnatal week, lateral dendritic filopodia can develop varicosities and heads giving rise to multispines or protospines after contacting with axons. in this developmental period, the intercellular spaces in the neuropil diminish, and the emission of filopodia could be a neuronal strategy to capture and recruit axonal endings to new dendritic spines (see [15, 20, 68, 69 ]) and at the same time increasing the dendritic capture volume. filopodia contacting several axons can choose the most active ones and, perhaps, discriminate between distinct types of axonal endings. mode 3 in young [52, 19-day - old mouse ] and adult animals and also in organotypic hippocampal slices [19, 27, 54, 56, 57 ], young dendritic spines contacting with axons do mature at the same time as that of the synaptic contacts. these three modes or strategies to form dendritic spines have a specific temporal window, but with a considerable overlap and a characteristic intensity peak. in addition, the development of dendritic structures (spines, filopodia and protospines) is asynchronous. thus, we can distinguish distinct phases of maturation of these structures, which also makes it a little more difficult to interpret the experimental results. even in the adult mouse neocortex, 3.6% of the spines clearly lacked synapses, and they resembled small dendritic filopodia. in young [52, 19-day - old mouse ] and adult animals and also in organotypic hippocampal slices [19, 27, 54, 56, 57 ], young dendritic spines contacting with axons these three modes or strategies to form dendritic spines have a specific temporal window, but with a considerable overlap and a characteristic intensity peak. in addition, the development of dendritic structures (spines, filopodia and protospines) is asynchronous. thus, we can distinguish distinct phases of maturation of these structures, which also makes it a little more difficult to interpret the experimental results. even in the adult mouse neocortex, 3.6% of the spines clearly lacked synapses, and they resembled small dendritic filopodia. dendritic spines can develop without the assistance of filopodia. in this regard, what is the role of dendritic filopodia ? some authors have speculated about the role of filopodia as a bridging structure to facilitate axodendritic synaptic contacts, increasing the dendritic capture volume. current data corroborated the implication of filopodia in synaptogenesis and their eventual transformation into dendritic spines. are the filopodial synapses incorporated into the dendrite shaft prior to the spine growing ? do contacting axons follow the incorporation of filopodial synapses to the dendritic shaft ? the meaning of the intermediate phase between filopodia and dendritic spines (multispine or protospine) is still uncertain. the question arises as to whether this phase is necessary to discriminate active axonal endings when there is a period of large - scale arrival of specific axons. alternatively, could multispines also be implicated in sensing and/or integrating different active axons prior to moving them to the dendritic shaft ? new improvements in microscopy, such as stimulated emission depletion (sted) microscopy, with greater resolution than confocal or two - photon microscopy, will allow us to visualise activity - driven structural changes of spines with unprecedented clarity. this technique should reveal fine details such as the shape of the spine head or the width of the spine neck, which together with the development of visualization methods showing complete axons will help advance studies of the interaction of axons with filopodia, protospines, and spines. further studies are needed to determine the mechanisms implicated in the initial formation of dendritic spines at the very beginning of synaptogenesis during embryogenesis (mode 1 of the unifying model), as well as the possible influence and/or the shared mechanisms in the development of lateral filopodia (mode 2) and dendritic spines in young and adult animals (mode 3). high - frequency focal synaptic stimulation by activation of nmda receptors, overexpression of glur2 and tm - agrin, proteolytic cleavage of agrin by neurotrypsin, or activation of sk3 channel, all induce filopodia growth. however, more studies are needed to better understand the mechanisms of filopodia formation and retraction. clustering of rac1 has been implicated in the formation of spines directly from the dendritic shaft previously contacted by an axon, recruiting ampa receptors to synapses and providing a link between presynaptic and postsynaptic developmental changes. a basic mechanism to form stable dendritic spines is the expression of psd-95 within a critical period of 24 h. this protein may help control synapse formation by regulating nnos expression at the synapse, thereby promoting no release and/or activation of cgmp signalling in presynaptic axons. however, the details of activity - dependent growth and evolution of dendritic spines related to learning and memory are far from clear.	dendritic spines receive the majority of excitatory connections in the central nervous system, and, thus, they are key structures in the regulation of neural activity. hence, the cellular and molecular mechanisms underlying their generation and plasticity, both during development and in adulthood, are a matter of fundamental and practical interest. indeed, a better understanding of these mechanisms should provide clues to the development of novel clinical therapies. here, we present original results obtained from high - quality images of cajal 's histological preparations, stored at the cajal museum (instituto cajal, csic), obtained using extended focus imaging, three - dimensional reconstruction, and rendering. based on the data available in the literature regarding the formation of dendritic spines during development and our results, we propose a unifying model for dendritic spine development.
acute cholecystitis as a major complication of gallstones is diagnosed in 10% to 35% of patients admitted for cholecystectomy. the anatomy at calot 's triangle in acute cholecystitis is distorted due to adhesions. as such, cholecystectomy for this condition is technically demanding, time consuming, and results in high morbidity. in the prelaparoscopy era, the traditional management of patients with acute cholecystitis included initial conservative treatment to cool down the inflamed gallbladder followed by delayed open cholecystectomy (oc) several weeks later. this approach, however, was challenged by early oc, first advocated by essenhigh in 1966. since then, several randomized studies have shown that early oc for acute cholecystitis is as safe as delayed oc with reduced morbidity and hospital stay, lower costs, and rapid recovery. it appears that laparoscopic treatment of acute cholecystitis is following the same trend as oc. initially, laparoscopic cholecystectomy (lc) was contraindicated in patients with acute cholecystitis because of the fear of increased morbidity and high rates (60%) of conversion to oc that negate the advantages of laparoscopic surgery. indeed, the bile duct injury of 5.5% during lc for acute cholecystitis was a major concern. on the other hand, initial medical treatment for acute cholecystitis followed by delayed lc is associated with several shortcomings. first, 20% to 26% of patients fail to respond to conservative treatment or develop early complications during the first admission and require an urgent and technically demanding cholecystectomy. secondly, another 15% to 30% of patients are readmitted with recurrent symptoms and undergo an unplanned emergency cholecystectomy while waiting for their scheduled elective procedure. thirdly, a small proportion (2.2%) of patients is lost during the interval period. finally, at times the shrunken, scarred gallbladder and dense fibrotic adhesions at calot 's triangle make interval lc extremely difficult and unsafe. as a result of these events and because of increasing experience and confidence in lc, the indications were extended to include patients with acute cholecystitis. several randomized and nonrandomized studies have documented the feasibility and safety of early lc for acute cholecystitis in experienced hands. in our setting, we have offered lc for all patients with symptomatic gallstones, and our initial experience with early lc in 45 cases of acute cholecystitis showed a low conversion rate and no major bile duct injury or mortality. the aim of this study was to determine the optimal timing of early lc for acute cholecystitis. this study included all consecutive patients who underwent early lc for acute cholecystitis at king fahd hospital of the university, al - khobar, saudi arabia, between january 2001 and december 2006. the diagnosis of ac was based on the presence of at least 2 of the following criteria : (1) acute upper abdominal pain and murphy 's sign, (2) fever > 37.5c and white blood cell count > 10x10/l, and (3) ultrasound findings of thick - walled gallbladder, ultrasound murphy 's sign, and pericholecystic fluid, in the presence of gallstones. exclusion criteria included (1) patients who had no gallstones, (2) those who were not operated on, (3) those who had incomplete data, (4) those who had an oc to start with, (5) those who had delay due to obstructive jaundice, ascending cholangitis, biliary pancreatitis, or comorbid diseases. because previous reports, including our own experience, have shown that lc for acute cholecystitis is best performed within 72 hours of admission, the patients were divided into 2 groups, depending on the timing of lc after admission : group 1 within 72 hours and group 2 after 72 hours. on admission, all patients received intravenous second - generation cephalosporin plus metronidazole, which were continued for 24 hours after surgery. laparoscopic cholecystectomy was performed in both groups by 5 surgeons with similar distribution of early and delayed lcs and the same technique described previously. patients were followed up for 6 weeks and were discharged home unless they had postoperative complications. patient demographics, white blood cell count, ultrasound findings, time from admission to surgery, operation time, conversion to oc, complications, postoperative stay, and total hospital stay were analyzed. statistical analysis was performed using the t test, chi - square test, and fisher 's exact test, with significance set at p<0.05. there were 196 patients with acute cholecystitis ; 56% were females, and the mean age was 41.3 years (range, 13 to 81). laparoscopic cholecystectomy was performed within 72 hours of admission in 82 patients (group 1) and after 72 hours in 114 patients (group 2). both groups were matched in terms of age, sex, body mass index, systemic manifestations, and ultrasound findings (table 1). the mean operation time, including conversions, was 117.254.6 minutes (range, 30 to 300). the mean postoperative stay was 3.72.5 days (range, 1 to 17), and the mean total hospital stay was 9.25.6 days (range, 2 to 28) for all patients. early laparoscopic cholecystectomy (lc) for acute cholecystitis : patient demographics data are presented as mean standard deviation unless otherwise indicated. table 2 shows that there were 2 conversions (2.4%) in group 1 and 8 in group 2 (7%) (p=0.3). obscure anatomy at calot 's triangle was the sole reason for conversion in group 1. the reasons for conversion in group 2 were obscure anatomy at calot 's triangle in 4 patients and difficulty to expose the gallbladder due to severe omental adhesions, inability to grasp the friable gallbladder, liver bleeding, and common bile duct injury in one case each. results of early laparoscopic cholecystectomy (lc) for acute cholecystitis data are presented as mean standard deviation unless otherwise indicated. however, 7 (6%) complications occurred in group 2 (p=0.02) and included 2 cases of respiratory infection, and a case each of cbd injury, retained cbd stone, subhepatic collection due to cystic duct leak, wound infection, and liver bleeding. the cbd injury was managed with immediate conversion and hepatico - jejunostomy. the liver bleeding required conversion and blood transfusion. retained cbd stone was managed with endoscopic retrograde cholangiopancreatography (ercp) and stone extraction. the case of cystic duct leak required ercp and percutaneous drainage der ultrasound / ct guidance. the mean operation time was 10549.5 minutes in group 1and 12656.9 minutes in group 2 (p=0.008). the mean postoperative stay was 3.42.2 days in group 1 and 42.7 days in group 2 (p=0.1). the mean total hospital stay in group 1 was 5.12.3 days compared with 12.25.3 days in group 2 (p=0.001). there were 196 patients with acute cholecystitis ; 56% were females, and the mean age was 41.3 years (range, 13 to 81). laparoscopic cholecystectomy was performed within 72 hours of admission in 82 patients (group 1) and after 72 hours in 114 patients (group 2). both groups were matched in terms of age, sex, body mass index, systemic manifestations, and ultrasound findings (table 1). the mean operation time, including conversions, was 117.254.6 minutes (range, 30 to 300). the mean postoperative stay was 3.72.5 days (range, 1 to 17), and the mean total hospital stay was 9.25.6 days (range, 2 to 28) for all patients. early laparoscopic cholecystectomy (lc) for acute cholecystitis : patient demographics data are presented as mean standard deviation unless otherwise indicated. table 2 shows that there were 2 conversions (2.4%) in group 1 and 8 in group 2 (7%) (p=0.3). obscure anatomy at calot 's triangle was the sole reason for conversion in group 1. the reasons for conversion in group 2 were obscure anatomy at calot 's triangle in 4 patients and difficulty to expose the gallbladder due to severe omental adhesions, inability to grasp the friable gallbladder, liver bleeding, and common bile duct injury in one case each. results of early laparoscopic cholecystectomy (lc) for acute cholecystitis data are presented as mean standard deviation unless otherwise indicated. no complications occurred in group 1. however, 7 (6%) complications occurred in group 2 (p=0.02) and included 2 cases of respiratory infection, and a case each of cbd injury, retained cbd stone, subhepatic collection due to cystic duct leak, wound infection, and liver bleeding. the cbd injury was managed with immediate conversion and hepatico - jejunostomy. the liver bleeding required conversion and blood transfusion. retained cbd stone was managed with endoscopic retrograde cholangiopancreatography (ercp) and stone extraction. the case of cystic duct leak required ercp and percutaneous drainage der ultrasound / ct guidance. the mean operation time was 10549.5 minutes in group 1and 12656.9 minutes in group 2 (p=0.008). the mean postoperative stay was 3.42.2 days in group 1 and 42.7 days in group 2 (p=0.1). the mean total hospital stay in group 1 was 5.12.3 days compared with 12.25.3 days in group 2 (p=0.001) a review of the literature over the past decade shows that early and delayed lc for acute cholecystitis are safe with similar conversion rates, operation time, and overall complications. however, early lc results in significantly shorter hospital stay and avoids the risks of failed conservative treatment. this approach is well supported by a recent international consensus published as tokyo guidelines. with increased experience, improved skills, and new instruments, the high rates of conversion to oc, prolonged operation time, and increased morbidity, particularly cbd injury, of early lc for acute cholecystitis have been dramatically reduced. despite this acceptable level of outcome, acute cholecystitis remains the most significant risk factor for conversion and complications of lc. furthermore, the timing for the procedure is a hotly contested issue and in all probability a strong predictor of success of lc for acute gallstone disease. in this series, the overall conversion rate (5%) to oc conversion rates tend to have a wide range (0% to 39%) with an overall rate of 16%. these inconsistent results are attributed to differences in patient demographics, severity of inflammation, surgeon 's experience, and timing of early lc. it is worth mentioning that the overall conversion rate of 5% in this study is similar to the 6.7% conversion rate reported in our initial experience and probably reflects a sustained experience of the team in the management of acute cholecystitis. as expected, the most common reason for conversion among our patients was distorted anatomy at calot 's triangle due to inflammatory changes. although our rate of complications is generally acceptable (table 3), all complications, including the single cbd injury, were limited to group 2 patients who had lc 72 hours after admission. it is worth noting that we had no single case of cbd injury in our initial experience with lc performed during the first 72 hours of admission for acute cholecystitis. results of trials addressing timing of early laparoscopic cholecystectomy for acute cholecystitis lc = laparoscopic cholecystectomy ; ot = operation time ; ns = not significant ; nr = not reported. the question of relating the timing of lc for acute cholecystitis to admission or onset of symptoms has been overemphasized. in our view we do, however, concur that patient - physician factors, such as patient delay of more than 48 hours and a variable delay in diagnosis, influence surgical decisions and timing of intervention. these tend to vary considerably according to the population attitude to illness and type of health care facility. based on international experience and our own results, it is our observation that lc within this period is technically less demanding because the edema planes magnify the structures and facilitate dissection a compelling argument in favor of early lc for acute cholecystitis is the morbidity and escalating cost of prolonged waiting time for surgery following conservative treatment at index admission. somasekar and colleagues in the united kingdom found that 58% of patients were readmitted as emergencies, some with biliary pancreatitis, necessitating laboratory and x - ray investigations, thereby escalating the cost of emergency care by as much as 1544 per patient. lawrentschuk in australia found that the cost per patient for emergency readmission while on the waiting list was $ a 6129 compared with $ a 3725 for an uncomplicated elective lc. in addition, these findings do not take into account the patient suffering, loss of work hours and income, and the effect on the community as a whole. in the realm of cost - benefit analysis, lc has been evaluated, and the overall cost of the procedure is cheaper than oc, mainly because the patient 's stay in the hospital is shortened. the key question in the economics of lc is the overall hospital stay as end point. analysis of 5 recent studies including our series (table 3) shows that the total hospital stay is significantly less when lc for acute cholecystitis is performed early irrespective of the conversion. stevens and colleagues reported a mean total stay as low as 2.6 days, and this is achievable now that the role of clinical pathways and specialist - led services are available in some countries. caplan in australia prospectively studied the effect of reengineered surgical service (consisting of a perioperative unit, preadmission anesthetic assessment, day of surgery admissions, enhanced patient education, clinical pathways, and postacute care) on total costs, patient satisfaction, time off work, and pain score in 224 patients. they concluded that besides high patient satisfaction with the treatment, the cost savings to the hospital outweighed the cost of increased services in the community. mercer and colleagues listed similar clinical and economic benefits in their specialist - led service for the management of acute gallstone disease in the united kingdom. in addition, uchiyama in japan highlighted the role of clinical pathways in reducing hospital stay and cost of laparoscopic surgery. our overall mean total hospital stay of 9 days is considerably longer than that reported in the current literature. then ours is not an lc - specific facility, and lc for acute gallstone disease has to fit in with the rest of the operating room schedule. besides we also have to satisfy cultural and social demands of the population. as yet, there is no insurance - related scheme in our institution that would trigger the development and introduction of specialist - led or reengineered service with suitable clinical pathways. our future plan of reorganization is on the drawing board, and we already have some of the components of a dedicated and specialist - led unit, ie, a short - stay / outpatient facility, and preadmission anesthesia clinic. we are hopeful that in future reporting the current halfway facility would evolve into a dedicated and internationally comparable laparoscopic unit. laparoscopic cholecystectomy for acute cholecystitis performed within the same admission is safe and associated with a low conversion rate and no mortality. however, lc should be performed as early as possible, preferably within the golden period of 72 hours after admission, to decrease the morbidity rate, operation time, and total hospital stay.	objective : although many surgeons advocate early laparoscopic cholecystectomy (lc) in acute cholecystitis, debate still exists regarding its optimal timing. this study compares the outcome of lc performed within and after 72 hours of admission in patients with acute cholecystitis.methods:between january 2001 and december 2006, lc was performed in 196 consecutive patients with acute cholecystitis. laparoscopic cholecystectomy was performed within 72 hours of admission in 82 patients (group 1) and after 72 hours in 114 patients (group 2). data were collected prospectively.results:both groups were matched in terms of age, sex, body mass index, fever, white blood cell count, and ultrasound findings. the overall conversion rate was 5%. no significant difference existed in conversion rates between group 1 (2.4%) and group 2 (7%) (p=0.3). the operation time (105 versus 126 minutes, p=0.008), complications (0% versus 6%, p=0.02), and total hospital stay (5 versus 12 days, p<0.001) were significantly reduced in group 1. no deaths occurred in this study.conclusion:early lc can be performed safely in most patients with acute cholecystitis, but we recommend intervention within 72 hours of admission to minimize the complication rate and shorten the operation time and total hospital stay.
considering folkloric use of tribulus terrestris (t. terrestris) in diabetes and proven anti - hyperglycemic and anti - hyperlipidemic effects of t. terrestris in animal studies, we aimed to evaluate the efficacy of the hydro alcoholic extract of t. terrestris on the serum glucose and lipid profile of women with diabetes mellitus. ninety - eight diabetic women were randomly allocated to receive the t. terrestris (1000 mg / day) or placebo for three months. the patients were evaluated in terms of the fasting blood glucose, 2-hour postprandial glucose, glycosylated hemoglobin and lipid profile. t. terrestris showed a significant blood glucose lowering effect in diabetic women compared to placebo (p<0.05). also, the total cholesterol and low - density lipoprotein of the t. terrestris group was significantly reduced compared with placebo, while no significant effect was observed in the triglyceride and high - density lipoprotein levels.	background : considering folkloric use of tribulus terrestris (t. terrestris) in diabetes and proven anti - hyperglycemic and anti - hyperlipidemic effects of t. terrestris in animal studies, we aimed to evaluate the efficacy of the hydro alcoholic extract of t. terrestris on the serum glucose and lipid profile of women with diabetes mellitus.methods:ninety-eight diabetic women were randomly allocated to receive the t. terrestris (1000 mg / day) or placebo for three months. the patients were evaluated in terms of the fasting blood glucose, 2-hour postprandial glucose, glycosylated hemoglobin and lipid profile.results:t. terrestris showed a significant blood glucose lowering effect in diabetic women compared to placebo (p<0.05). also, the total cholesterol and low - density lipoprotein of the t. terrestris group was significantly reduced compared with placebo, while no significant effect was observed in the triglyceride and high - density lipoprotein levels.conclusion:this study showed preliminary promising hypoglycemic effect of t. terrestris in diabetic women.
mental disorders cause a substantial proportion of the burden of disease in the general population. neuropsychiatric disorders rank second in the list of disability adjusted life years (daly) in europe, preceded only by cardio - vascular diseases. they are also responsible for the largest part of causes for years lived with disability (yld), and account for 19.5% of dalys and 40% for yld,. an overview over 27 studies on the prevalence of mental disorders in europe yielded an estimate of 27% of the adult population between 18 and 65 years, who fulfilled criteria of at least one mental disorder. prevalence rates between studies ranged considerably, with highest prevalence rates for a) affective disorders with a median (md) rate of 6.9%, b) specific phobias, md = 6.4%, c) somatoform disorders, md = 6.3%, d) substance use disorders, md = 2.4%, and e) social phobias, md = 2.3%. in germany point - prevalence (four weeks) is estimated with 20%, the 12-months - prevalence with 31%, and life - time prevalence with 43%. the most frequent disorders are affective, somatoform, substance - use and panic disorders. prevalence is estimated with 26% and increases with geographical agglomeration up to 35% in metropolitan areas. standardised diagnostic procedures distinguished themselves because of their high reliability in epidemiological studies as well as in clinical settings. on the other hand, clinical diagnoses without structuring are less reliable and valid. as basis for estimates on morbidity and ensuing planning of demand, clinical diagnoses have to be regarded with greater care. persons with mental disorders call on medical treatments more often and are more frequently on sick leave. however only one third of all persons concerned, has sought out psychotherapeutic treatments, or have received psychotherapy. these high demands on health care provision are met with a differentiated system of services. in international comparison, the wide range of differentiated services of inpatient psychotherapeutic care in germany takes a leading position. it encompasses a) specialists in neurology / psychiatry who are contracted through a staturory health insurance (shi), b) shi psychotherapists, c) shi psychotherapists for children and adolescents, d) psychosomatic primary care through shi general practitioners, e) outpatient care clinics, f) psycho - social counselling centres, and g) day care centres. waiting time for an intake interview lasts about two months, waiting time for psychotherapy to begin lasts about four and a half months. in the geographical region of the former gdr (east - germany) waiting times last about ten weeks and over three months respectively. comparing the mental health care provision by regional aspects, schulz and colleagues summarise : a comparison of density of supply in different regions within germany shows, with the exception of inpatient care for children and adolescents, a striking east - west - difference to the disadvantage of east - germany. the under - supply seems to be especially striking in the outpatient provision for children and adolescents. even in the otherwise well provisioned federal state of baden - wrttemberg, only between 4% and 35% of all children and adolescents concerned found a treatment place. the aim of this study is, with regard to a posited bottleneck in supply, to paint a more precise picture of the provision of care for persons with mental disorders within a rural area in east - germany. we do not only take data from the psychotherapeutic provision of care into account, but data from the whole statutory health insurance. we assume that by analysing reimbursement claims from shi physicians, we gain insights into areas where persons with mental disorders are cared for outside psychotherapeutic provision of care. this view creates an image of the real - life provision of in an economically underdeveloped region. this parallels a population density of 72 inhabitants per square kilometre (in germany the population density is 231 inhabitants per square kilometre). about one third lives in cities with between 45,000 and 198,000 inhabitants, another third in communities with less than 2,000 inhabitants. only one mental disorders take middle and forward ranks in the burden of disease. with respect to their relative frequencies the following rank order results in mecklenburg - west pomerania : a) 29.2% of all new entries into the statutory pension funds (rank 1), b) 18.1% of all rehabilitative treatments (rank 3), c) 8.1% of all sick certificates (rank 4), d) 1.7% of mortality (rank 7), and e) 6.1% of all hospitalisations (rank 8). the density of supply with psychotherapists varies between 5,000 and 120,000 inhabitants per psychotherapist ; in the very sparsely inhabited areas the supply density ranges between 15,000 and 150,000. to address our research questions, we analysed the billing data of mecklenburg - west pomerania s association of shi physicians from the years 2006 / 2007. the exception here is ambulatory health care centres (mvz), where more than one specialised practice work as one treatment unit. they bill under one single billing account, so that the specialty of every single provider can not be identified. also the group of neurologists and psychiatrists bill their reimbursement claims under one single account so it s not possible to differentiate between neurological and psychiatric practices. and within the group of psychotherapists it s not possible to differentiate between medical and psychological psychotherapists. in analysing a treatment corresponds to a medical treatment or service that has been billed within one billing quarter (three months). a case corresponds to a person (patient), for whom one or more treatments may be billed within one quarter. following this logic this entails that with respect to cases the relative frequencies of diagnoses may vary. for example a patient with a depression, a phobic disorder and nicotine dependence would be counted with three diagnoses, when the distribution of diagnoses on the level of cases is considered. since no ordering according to importance of disorder (primary diagnosis, secondary diagnosis etc.) has been recorded within the data, we were unable to rank - order diagnoses accordingly for each case. therefore we investigated from the data from the last quarter of 2007 as a proxy for the whole period how many diagnoses and how many cases have been billed by how many treatment providers. for the last quarter in 2007 that corresponds to 564,279 treatments for 303,839 cases. the data have been provided by the association of shi physicians mecklenburg - west pomerania. ethical, legal or data - safety regards are warranted : anonymous data were provided, data can not be traced back to individuals and additional diagnostic or therapeutic means have not been taken. the basis for the relative frequencies for treatment cases it should be noted that more that one diagnosis may be valid for one case. accordingly single cases may weigh with more than single weight in the determination of relative frequencies. altogether data stem from 4,246,734 treatments from the years 2006 and 2007, that have been billed to the association of shi physicians in mecklenburg - west pomerania for an icd-10 chapter v diagnosis. of these treatments, inaccurate means a diagnostic code has been billed that does not exist in icd-10 chapter v (e.g. f45.51, or f10.17) ; incomplete means the billed diagnostic code does nt allow for unambiguous allocation to one category (e.g. f45, or f10). for our further analysis, we limit ourselves to the remaining 3,905,262 treatments. category encompasses medical treatment centres (mvz), ophtalmotologists, surgeons, anaesthesiologists, pulmologists, emergency clinics, laboratory practices, radiologists, neurosurgeons, central emergency services, oral and facial surgery, nuclear physicians and other central services. the distributions of the relative frequencies of diagnoses according to icd-10 subchapters on the basis of treatments as well as on the basis of cases are given in table 1. remarkable is that more than one third of all treatments billed for reimbursement are allocated to neurotic, stress - related and somatoform disorders (f4x.xx), a little bit less than one quarter of all claims are allocated to mood (affective) disorders (f3x.xx) and a little more than one tenth of all claims are allocated to substance use disorders (f1x.xx). here it is worth remarking that almost half of all claims were filed by general practitioners. psychotherapists (medical and psychological psychotherapists combined) claimed with only 3.1% a minor proportion of claims. for the 303,839 cases from the last quarter of 2007, claims were billed in 193,717 cases (63.8%) for a single diagnosis, in 63,941 cases (21.0%) for two diagnosis, in 26,006 cases for (8.6%) three diagnoses, and in 20,175 cases (6.6%) for more than three different icd-10 chapter v diagnoses. these were billed in 244,070 cases (80.3%) from one group of specialists, in 49,528 cases (16.3%) from two different groups, in 8,757 cases (2.9%) from three different groups, and in 1,484 cases (0.5%) from more than three different groups of specialists. the significance of alcohol and tobacco related disorders, depression, anxiety and somatoform disorders, that has been reported in large epidemiological studies, also pictures in everyday health care provision. a broad spectrum of practitioners provides health care for the patients, with the stress on general practices and family doctors who bill about half of the claims. the group of neurologists / psychiatrists cares for about one seventh of all cases. opposed to that, psychotherapeutic practices (medical as well as psychological) bill claims for only about three percent of all cases. collaborative treatment of patients with mental disorders seems to be the exception rather than the rule ; treatments for four fifth of cases are claimed by one single practitioner. these results complement the image from other german studies on health care provision of patients with mental disorders. striking is the large proportion of treatments that are claimed by general practitioners. since 80% of all treatment claims are billed by one single practitioner, we may well assume that to a large degree, patients with mental disorders are cared for by general practitioners alone. taking into account the urban - rural - divide that schulz and colleagues observed in 2008 with regard to psychotherapeutic provision of care, and against the background of long waiting times as reported by zepf and colleagues and peikert and colleagues it may be assumed that the treatments of mental disordered patients provided by general practitioners owe to at least to a certain degree a bottleneck in the provision of care by neurologists / psychiatrists and psychotherapists ; and that this shows more pronounced in a rural and remote area as mecklenburg - west pomerania. that the outpatient provision of care in rural and remote areas lacks behind that in metropolitan areas is also shown in the study by peikert and colleagues ; about one fifth of the inhabitants in east - germany live in urban areas. accordingly the provision of care in urban areas is evaluated more favourably by psychotherapists and waiting times are much shorter in bigger cities than in the other regions. in sum, this study provides insight into the epidemiology of mental disorders in germany under the actual status quo with the regard to health care provision. and these reflect on the one hand the large proportion of substance use disorders, depression, anxiety, and somatoform disorders on the burden of disease due to mental disorders. on the other hand, the study supports the picture of a suboptimal provision of mental health care outside the metropolitan areas. in order to improve the provision of care especially in rural and remote areas, it could be considered to take aspects of population density into account, when practice settlements for psychotherapists and neurologists / psychiatrists are planned. it appears also worthwhile to think about a further accentuation in basic and continuing medical education in diagnostics and treatment of mental disorders of general practitioners. but also alternative concepts to secure service provision, such as the implementation of mobile treatment facilities or actively calling on patients in their homes, may cause some relief. additionally to these ramifications, in the face of the high prevalence of mental disorders, interventions to prevent mental disorders should also be kept in mind. the results of this investigation have to be put into perspective by a number of limitations. for instance it can not be safely assumed that all diagnoses have been secured by standardised and structured diagnostic interviews. it may well be that the frequency of certain diagnoses have been under- or overestimated. it has to be seen critically that almost ten percent of all billed claims base on incorrect or incomplete diagnostic codes and had to be discarded from our analyses. furthermore our study does not provide differential insight as to what kind of treatment has been provided for which kind of disorder. for future evaluations it may be advisable to be able to differentiate the treatments provided according to the kind of disorder. taking a future perspective, utilising modern methods of spatial statistics in combination with geographical information systems may serve as a basis for empirically and geographically based service provision planning. this may lead to demand - oriented plans for service provision that is more strongly oriented towards the demands of the population serviced.	mental disorders cause a substantial amount of the burden of disease. although they are less frequent in rural areas, their provision of care is disproportionately lower. reimbursement claims in the federal state of mecklenburg - west pomerania of the years 2006/2007 serve as the basis for the descriptive distribution of subgroups on the total number of mental disorders and their outpatient care. of all claims, 35.3% were allotted to neurotic, stress - related and somatoform disorders, 24.2% to affective disorders and 12.5% to substance use disorders. claims for reimbursement were made for 44.7% by general practitioners, 15.1% by neurologists and psychiatrists, 12.6% by gynaecologists, and 8.1% by internists. psychotherapists claimed 3.1%. these results cause considerations regarding the establishment of psychotherapeutic and neurological / psychiatric practices as well as the significance of mental disorders in the training of general practitioners.
	the objective of this study was to investigate the impact of long - term pyrene exposure on molting and reproduction in the model estuarine invertebrate, the grass shrimp (palaemonetes pugio). grass shrimp were exposed to measured concentrations of 5.1, 15.0, and 63. 4 ppb (microg / l) pyrene for 6 weeks, during which time we determined molting and survivorship. at the end of the exposure, we immediately sacrificed some of the shrimp for biomarker (cyp1a and vitellin) analyses. the remaining shrimp were used to analyze fecundity and embryo survivorship during an additional 6 weeks after termination of pyrene exposure. male shrimp at the highest pyrene dose (63 ppb) experienced a significant delay in molting and in time until reproduction, and showed elevated ethoxycoumarin o - deethylase (ecod) activity immediately after the 6-week exposure period. in contrast, 63 ppb pyrene did not affect these parameters in female shrimp. females produced the same number of eggs per body weight, with high egg viability (98 - 100%) at all exposure levels, but with decreased survival for the offspring of the 63-ppb pyrene - exposed females. in addition, vitellin levels were elevated only in females at 63 ppb pyrene after the 6-week exposure. we hypothesize that the elevated vitellin binds pyrene and keeps it biologically unavailable to adult females, resulting in maternal transfer of pyrene to the embryos. this would account for the lack of effect of pyrene exposure on ecod activity, molting, and reproduction in the adult females, and for reduced survival of their offspring.imagesfigure 1figure 2figure 3
in the preceding issue of critical care corstjens and coworkers investigated three different methods of glucose measurements. while data about the beneficial effects of normoglycemia in critically ill patients are conflicting and inconsistent, there is no doubt about the importance of accurate glucose measurements to achieve glycemic control without increased risk of hypoglycemia. similar to the results from the diabetes control and complications trial, which showed increasing frequency of hypoglycemia after tight glycemic control to reduce long - term complications, there is an increase of hypoglycemic episodes in critically ill patients when strict glycemic control is established. although the measurement of glucose is one of the oldest established tests in the clinical chemistry laboratory, it is extremely complex and sometimes rather approximate due to the different fractions of the blood sample used. glucose measurement can be performed in whole blood, plasma and serum and these may be native or deproteinized, or hemolyzed in the case of capillary whole blood. furthermore, blood may be arterial, capillary or venous in origin. the simple answer is no, and moreover, the difference may depend on nutritional state, perfusion, hematocrit or albumin blood concentrations. glucose is dissolved only in the aqueous part of the drawn sample and not in its entire volume (which contains other dissolved solids such as proteins). this is the major reason for differing glucose concentrations in plasma and whole blood samples. the protein content differs in blood cells (mostly red blood cells) and plasma (or serum, or hemolysate). the water content (figure 1) of red blood cells is lower (because of a high concentration of hemoglobin) than that of an equal volume of plasma (which has a lower concentration of albumin and other plasma proteins). even with the glucose concentration being the same in plasma water and red blood cell water, the concentration of glucose per unit volume of red blood cells is lower than that per unit volume of plasma. the concentration of glucose per unit volume of whole blood is in between that for plasma and red blood cells. as the water content of whole blood is the sum of plasma water and red blood cell water, glucose concentration will strongly depend on the hematocrit of the sample (figure 1). with no changes in the protein concentration of plasma or red blood cells, a change in hematocrit from 0.4 to 0.7 will change the plasma / whole blood ratio for glucose from 1.10 to 1.38, an error of 26%. blood glucose strips retain red blood cells through a filtering process and measure glucose content in plasma in their reaction zone. this is yet another way by which hematocrit can influence the results : whole blood samples with differing amounts of red blood cells alter flow and volume of plasma entering the reaction zone. even the yellow springs instruments ' blood glucose analyzer, which is often used as a reference method, such as in the study by corstjens and colleagues, yields glucose results dependent on hematocrit when whole blood samples are used. this is due to the fact that the instrument performs a 25-fold sample dilution before analysis. in theory, the only systems that should not be affected by hematocrit are instruments using direct - reading electrodes without sample dilution, such as those used in blood gas analysis. next to the composition of the blood sample, calibration of the instrument is equally important and may often add to unknown errors. depending on the glucose standard and reference method used for calibration, the same instrument will give varying results, the most obvious being the calibration for whole blood and plasma. and just what is the standard reference method for glucose, and should there not be a reference method for each sample type whole blood, plasma and hemolysate ? the methodology of glucose analysis in routine clinical use is nowadays based on either chromogenic or electrochemical reactions of the three enzymes glucose oxidase, dehydrogenase and hexokinase. this gives rise to method - based specific interferences, such as the blood oxygen tension dependency of glucose oxidase, which is a major issue in intensive care unit patients. critically ill patients may have very low hematocrits, high or low arterial of venous oxygen tensions and may present extreme acid - base abnormalities, all of which have to be evaluated for every glucose analyzer under consideration. precision and accuracy have to be determined using standardized protocols and care has to be taken to choose the suitable reference method, which may not be the one available in the central laboratory where the study is being performed. instruments such as the continuous glucose monitoring system (cgms system gold, medtronic minimed) used in the study of corstjens and colleagues seem to find their place in the monitoring of diabetic patients but still need further evaluation of their clinical utility in critically ill patients. at the end of their discussion corstjens and colleagues state the following : " our study has too few patients and therefore too little data points under extreme conditions of ph, temperature, electrolyte disturbances and hypoglycaemia to make statements about reliability of the specific analyzers under these circumstances. " otherwise we might never get an evidence - based answer about benefit versus potential harm of intensive insulin therapy in critically ill patients. glucose concentration : water content expressed as percent volume. with a hematocrit of 0.4 and a water content for the fraction ' cells ' of approximately 70%, the total water content will be 28% of total volume (whole blood). from the 60% plasma volume, 90% will be water, thus giving a water content of 54% for the plasma portion of whole blood. the total water content of whole blood then is 82% (54% + 28%). for a hematocrit of 0.4 the plasma / whole blood ratio of water content is 0.9/0.82 = 1.10, which reflects the 10% higher glucose values in plasma compared to whole blood.	a crucial determinant for the success of intensive insulin therapy in critically ill patients is the frequent and accurate measurement of blood glucose values with immediate feedback of results. in general, therefore, this is achieved by point - of - care testing, raising the question of the best way of monitoring blood glucose. corstjens and coworkers, in the previous issue of critical care, demonstrate that, in spite of good correlation to " conventional " laboratory glucose assessment, absolute glucose levels may differ systematically. this commentary reviews the problems of glucose measurements arising from matrix effects, interferences and the use of different assays.
materials recombinant human aldh2 was expressed in and purified from escherichia coli (16). gtn (nitropohl ampoules ; g. pohl - boskamp gmbh & co, hohenlockstedt germany) was obtained from a local pharmacy as a 1 mg / ml aqueous isotonic solution, containing 49 mg / ml glucose monohydrate, and diluted in 50 mm triethanolamine / hcl (ph 7.4). [2-c]gtn (50 mci / mmol), manufactured by american radiolabeled chemicals, was supplied locally by humos diagnostica (maria enzersdorf, austria). 1,2-gdn and 1,3-gdn, used as standards in radio - thin layer chromatography, were obtained from lgc promochem (wesel, germany). naphthoic acid was purchased from abcr gmbh & co kg (karlsruhe, germany). determination of the inactivation kinetics to determine the rate of inactivation of aldh2 by gtn, the optical absorption of a 1-cm quartz cuvette (type 105.200-qs, hellma, mllheim, germany) containing substrate (acetaldehyde or p - npa) and additional compounds like nad or dtt as indicated in 50 mm potassium pi (ph 7.4), was monitored at 340 (dehydrogenase) or 400 nm (esterase) in a hewlett - packard 8452a diode array spectrophotometer. after 12 min aldh2 was added and the reaction was allowed to proceed for 23 min. subsequently, gtn (or in some instances nad) was added to start the inactivation. usually, when inactivation was complete, dtt, substrate, or both were added to reactivate the enzyme. the total volume of the samples after gtn addition was 250 l. the total time over which the reaction was monitored was 2030 min. although new conditions were always explored with a single sample for optimal time resolution, concentration dependences or variations of cofactor or reductant (see results) were usually studied with up to 6 samples per experiment with the 89073a multicell transport accessory. the temperature of the samples was kept at 20 c by a refrigerating circulating water bath (lauda rm6, bartelt gmbh, austria). the various segments of the time traces (initial activity, restored activities) were fitted linearly, except for the inactivation, which was fitted to a combination of a single - exponential and a straight line. the linear component yielded the residual activity ; the exponential component yielded the apparent inactivation rate constant, as can be derived as follows : if the uninhibited activity is ve = kee, with ke and e representing the catalytic rate constant and the enzyme concentration, respectively, inactivation will lower the activity by decreasing the catalytically competent enzyme fraction in a mono - exponential fashion with et = e0e, in which e0 is the total enzyme concentration and ki is the apparent inactivation rate constant. the activity will therefore change in time according to the equation ve(t) = ke consequently, the absorbance will display simple monoexponential behavior during inactivation according, (eq. 1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\;\;a(t)=a_{0}+{\delta}{\epsilon}\frac{v_{0}}{k_{i}}(1-e^{-k_{i}t})\;\;\end{equation}\end{document } in which a0 and correspond to the initial absorbance and the absorbance difference coefficient between product and substrate, respectively. inactivation of aldh2 by preincubation with gtn (gtn preconditioning)purified aldh2 (0.27 mg / ml) was preincubated with 1 m gtn in 50 mm napi (ph 7.5) containing 0.2 mm mgcl2 and 2 mm nad at 37 c for 10 min. dehydrogenase activity was determined as the formation of nadh from nad in the presence of acetaldehyde by monitoring the increase in absorbance at 340 nm (340 = 6.22 mm cm). esterase activity was measured by monitoring the formation of p - nitrophenolate from p - nitrophenylacetate (p - npa, 400 = 16 mm cm). the reaction mixture contained substrate (0.2 mm acetaldehyde or 0.1 mm p - npa), 2 mm nad, 10 mm mgcl2, and 1.5 g / ml aldh2 in 50 mm napi (ph 7.5) at 25 c. in a second series of experiments, preincubation with 1 or 10 m gtn was performed in 0.1 m mes (ph 6.5) containing 0.2 mm mgcl2 at 37 c for 10 min, dehydrogenation was measured at ph 9.0 (50 mm napi), and esterase activity was determined in the absence of nad (17). determination of 1,2-gdn formation by radio - thin layer chromatography the rate of the conversion of gtn to 1,2- and 1,3-gdn was determined according to a described protocol (12). purified aldh2 (4 g) was incubated with c - labeled gtn (2 m ; 50,000 dpm) at 37 c for 10 min in a final volume of 100 l of 50 mm potassium pi (ph 7.4), containing 3 mm mgcl2, 1 mm nad, 1 mm edta, 1 mm egta, and 2 mm gsh with 2 mm dtt as indicated. after incubation, reaction mixtures were filtered over ym-10 columns (millipore gmbh, vienna, austria) and washed twice with 60 l of 50 mm potassium pi (ph 7.4) to yield 200-l filtrate and 20-l residues containing the enzyme. the recovered protein was incubated again with c - labeled gtn (2 m, 50,000 dpm) at 37 c for 10 min in a final volume of 200 l of potassium pi buffer (50 mm, ph 7.4), containing 3 mm mgcl2, 1 mm nad, 1 mm edta, 1 mm egta, and 2 mm gsh with 2 mm dtt as indicated. reaction products from the filtrate of the first incubation (pre - column) and from the reaction mixture of the second incubation (post - column) were extracted twice with 1 ml of diethyl ether, separated by thin layer chromatography, and quantified by liquid scintillation counting. blank values, determined in the absence of protein under identical conditions, were subtracted. mean values s.e. were calculated from reaction rates measured as duplicates in three independent experiments. to evaluate the effect of the procedure on enzyme activity, dehydrogenase and esterase assays the filtered enzyme was washed twice with 60 l of 50 mm napi (ph 9.0 or ph 7.5 for the dehydrogenase and esterase determination, respectively). assay conditions were : 50 mm napi (ph 9.0), 0.2 mm acetaldehyde, 2 mm nad, 10 mm mgcl2, and 1.5 g / ml aldh2 for the dehydrogenase activity ; 50 mm napi (ph 7.5), 0.1 mm p - npa, 10 mm mgcl2, and 1.5 g / ml aldh2 for the esterase activity. inactivation by gtn and reactivation by thiols of aldh in mitochondria rat liver mitochondria were prepared according to a previously published method (18). rat livers were homogenized in tris buffer (250 mm sucrose, 10 mm tris, 5 mm egta, 2 mm mgcl2, ph 7.4) and centrifuged at 600 g (10 min at 4 c). the pellet was resuspended in tris buffer and centrifuged at 15,000 g for 5 min. the mitochondrial fraction (2030 mg / ml protein) was frozen at -70 c until use. mitochondria (0.8 mg / ml) were preincubated with gtn (100 m) in tris buffer for 10 min at 37 c. after preincubation, the mixture was centrifuged (15,000 g, 5 min at 4 c) and washed three times with tris buffer to remove gtn. the pellet was resuspended in tris buffer at a final concentration of 8 g / ml and mixed with 0.1 mm nad in a cuvette at 37 c. after 3 min, the dehydrogenase substrate monal 62 (3 m) was added and fluorescence was monitored (ex 310 nm, em 360 nm) in a ls50b luminescence spectrometer (perkinelmer life sciences). after another 3 min, thiol (dtt, -mercaptoethanol, gsh, cys, or lpa - h2) was added at the indicated concentrations and fluorescence was measured for 8 more min. at the end of the experiment, signal intensity was calibrated by comparison with the fluorescence of known concentrations of naphthoic acid. inhibition of dehydrogenase activity by gtn to study the inactivation of aldh2 by gtn in real time we monitored the formation of nadh from nad spectrophotometrically at 340 nm and determined how the activity of aldh2 was affected by the addition of gtn. 1a shows a typical trace recorded for the inhibition of dehydrogenase activity with acetaldehyde as the substrate. it illustrates how the linear increase of the nadh concentration is gradually reduced to a very low residual level. fitting the curve corresponding to this process to a single exponential yielded a first - order rate constant that can be equated to the apparent inactivation rate constant under this particular set of conditions (see experimental procedures). the magnitude of reactivation was not affected by the concentrations of dtt (between 0.2 and 2 mm) and acetaldehyde (up to 2 mm). for instance, at a fixed concentration of 0.43 mm acetaldehyde the inactivation rate constant increased from 2.96 0.08 10 s at 0.05 mm gtn to 21.1 0.5 10 s at 0.6 mm gtn. by contrast, inactivation slowed down at higher acetaldehyde concentrations : at a fixed concentration of 0.5 mm gtn the rate constant decreased from 23 5 10 s at 0.1 mm acetaldehyde to 2.9 0.3 10 s at 20 mm acetaldehyde. a detailed description of the effects of substrate and inhibitor concentrations on the inactivation rate is given under the supplemental materials (fig. panel a shows a time trace for the formation of nadh from nad, monitored at 340 nm. at t = 0 the cuvette contained 0.2 mm acetaldehyde and 0.2 mm nad in 50 mm potassium pi (ph 7.4). at t = 80 s, catalysis was initiated by the addition of 33 g / ml aldh2. inactivation started at t = 480 s by the addition of 0.05 mm gtn. after inactivation of the enzyme, at t = 1160 s, an attempt was made to restore activity by the addition of 1 mm dtt. the dots represent the data points, whereas the continuous lines are best fits to the data. linear fits were applied to the phases before (no catalysis (-0.10 0.06) 10 absorbance units (a.u.)/s), and after aldh2 addition (initial activity, (8.03 0.05) 10 a.u./s), and to the activity after dtt admission (restored activity (1.378 0.006) 10 a.u./s) ; for the inactivation after gtn addition a combination of a single exponential and a linear fit was applied (apparent inactivation rate constant (7.77 0.06) 10 s ; residual activity (0.133 0.007) 10 a.u./s). panel b compares the residual and restored rates of acetaldehyde dehydrogenation after addition of gtn and dtt, respectively. experimental conditions : 33 g / ml aldh2, 0.43 mm acetaldehyde, 0.4 mm nad, 0.4 mm dtt, and concentrations of gtn as indicated in 50 mm potassium pi (ph 7.4). initial dehydrogenase activities under the conditions applied here amounted to 289 13 nmol min mg, which corresponds to a turnover number of 69 3 min. panel a shows a time trace for the formation of nadh from nad, monitored at 340 nm. at t = 0 the cuvette contained 0.2 mm acetaldehyde and 0.2 mm nad in 50 mm potassium pi (ph 7.4). at t = 80 s, catalysis was initiated by the addition of 33 g / ml aldh2. inactivation started at t = 480 s by the addition of 0.05 mm gtn. after inactivation of the enzyme, at t = 1160 s, an attempt was made to restore activity by the addition of 1 mm dtt. the dots represent the data points, whereas the continuous lines are best fits to the data. linear fits were applied to the phases before (no catalysis (-0.10 0.06) 10 absorbance units (a.u.)/s), and after aldh2 addition (initial activity, (8.03 0.05) 10 a.u./s), and to the activity after dtt admission (restored activity (1.378 0.006) 10 a.u./s) ; for the inactivation after gtn addition a combination of a single exponential and a linear fit was applied (apparent inactivation rate constant (7.77 0.06) 10 s ; residual activity (0.133 0.007) 10 a.u./s). panel b compares the residual and restored rates of acetaldehyde dehydrogenation after addition of gtn and dtt, respectively. experimental conditions : 33 g / ml aldh2, 0.43 mm acetaldehyde, 0.4 mm nad, 0.4 mm dtt, and concentrations of gtn as indicated in 50 mm potassium pi (ph 7.4). initial dehydrogenase activities under the conditions applied here amounted to 289 13 nmol min mg, which corresponds to a turnover number of 69 3 min. the residual activity and the extent of restoration in the presence of dtt diminished when the concentration of gtn was increased (fig. reactivation was not appreciably improved by extra dtt : at 0.032 mm acetaldehyde and 0.1 mm gtn, reactivation amounted to 8 2 and 10 1% with 0.2 and 1.0 mm dtt, respectively. similarly, the substrate concentration had little effect on reactivation (supplementary fig. s1c) and addition of extra substrate after inactivation did not affect the restored activity either. panel a, restoration by different thiols of aldh2-catalyzed dehydrogenation after inactivation by gtn. plotted are the relative residual activities (vresidual / vinitial) after inactivation by 0.1 mm gtn and the relative restored activities (vrestored / vinitial) after addition of 1 mm dtt, lpa - h2, or gsh. initial conditions : 25 g / ml aldh2, 0.45 mm acetaldehyde, and 0.4 mm nad in 0.2 m potassium pi (ph 7.4). panel b, protection by dtt of dehydrogenase activity against gtn - mediated inactivation. the figure shows representative time traces of the aldh2-catalyzed formation of nadh, as monitored at 340 nm before and after addition of gtn in the presence or absence of dtt. experimental conditions : 33 g / ml aldh2, 0.45 mm acetaldehyde, 0.4 mm nad, and 0.1 mm gtn with or without 1 mm dtt in 50 mm potassium pi (ph 7.4). panel a, restoration by different thiols of aldh2-catalyzed dehydrogenation after inactivation by gtn. plotted are the relative residual activities (vresidual / vinitial) after inactivation by 0.1 mm gtn and the relative restored activities (vrestored / vinitial) after addition of 1 mm dtt, lpa - h2, or gsh. initial conditions : 25 g / ml aldh2, 0.45 mm acetaldehyde, and 0.4 mm nad in 0.2 m potassium pi (ph 7.4). panel b, protection by dtt of dehydrogenase activity against gtn - mediated inactivation. the figure shows representative time traces of the aldh2-catalyzed formation of nadh, as monitored at 340 nm before and after addition of gtn in the presence or absence of dtt. experimental conditions : 33 g / ml aldh2, 0.45 mm acetaldehyde, 0.4 mm nad, and 0.1 mm gtn with or without 1 mm dtt in 50 mm potassium pi (ph 7.4). as illustrated by fig. 2a, lpa - h2 restored gtn - inactivated dehydrogenation to about the same level as dtt (from 6 1 to 20 3% of the initial activity with lpa - h2 compared with 26 1% with dtt). gsh, on the other hand, had no effect (7 2%). 1b and 2a), it largely prevented inactivation if added before gtn (fig. dtt slowed down inactivation by 0.2 mm gtn (7.4 0.6)-fold3 with an ec50 of 10 1 m (supplementary fig. the results indicate that dtt strongly protects against inactivation, but can not completely abolish it. whereas gsh, cys, and ascorbate did not prevent inactivation, lpa - h2 lowered the inactivation rate constant to a similar extent as dtt (results not shown). inhibition of the esterase activity of aldh2 by gtn in addition to the nad - dependent dehydrogenation of aldehydes, aldh is known to catalyze the hydrolysis of certain esters in a reaction that does not require nad (20). when the effect of gtn on this activity was examined under standard conditions, i.e. in the absence of nad, gtn hardly affected the rate of hydrolysis of p - npa ; however, subsequent addition of nad did cause inactivation (fig. esterase inactivation also occurred when gtn was added in the presence of nad (fig. the rate of inactivation was independent of the order in which gtn and nad were added. the rate of inactivation increased when the gtn concentration was raised, and decreased at higher concentrations of p - npa. at a fixed concentration of 0.5 mm p - npa the inactivation rate constant increased from 2.8 0.3 10 s at 0.05 mm gtn to 11.7 0.8 10 s at 1.0 mm gtn ; at a concentration of 0.1 mm gtn the inactivation rate constant decreased from 12.6 0.9 10 s to 1.5 0.5 10 s, when the p - npa concentration was raised from 0.05 to 1.0 mm. the residual esterase activity after inactivation by gtn was affected by the concentration of p - npa : the relative activity (vresidual / vinitial) increased from 0.024 0.002 to 0.12 0.03 when the substrate concentration was raised from 0.2 to 1.0 mm (supplementary fig. s4a). on the other hand, the activity was not affected by the gtn concentration (supplementary fig. thiol - induced reactivation of p - npa hydrolysis could not be determined, because of the ability of thiols (not just dtt but also gsh) to hydrolyze p - npa in the absence of aldh (results not shown). in line with previous reports (21), nad enhanced esterase activity, and an intermediate stimulation was observed in the presence of nadh (supplementary fig. the apparent rate constant for gtn - induced inactivation increased by an order of magnitude in the presence of nad, and again the effect of nadh was intermediate (fig. although the presence of nad(h) is no prerequisite for the reduction of gtn by aldh2, it is conceivable that the enzyme uses nadh as a facultative electron donor. however, we observed no absorbance changes at 340 nm upon incubating aldh2 with gtn and nad or nadh, which rules out the participation of either nucleotide as a redox agent in the decomposition of gtn. the figure shows the formation of p - nitrophenol from p - npa, as monitored at 400 nm. panel a shows that, in the absence of nad, gtn did not significantly affect esterase activity. however, inactivation set in immediately after the addition of nad. experimental conditions were : p - npa (0.2 mm) in 50 mm potassium pi (ph 7.4) was present at the start of the reaction ; aldh2 (33 g / ml), gtn (0.1 mm), dtt (0.2 mm), and nad (0.2 mm) were added as indicated. 1 yielded (1.0 0.5) 10 absorbance units (a.u.)/s for the uncatalyzed reaction, (1.07 0.02) 10 a.u./s for the initial rate, (1.083 0.008) 10 a.u./s and (1.15 0.01) 10 a.u./s after addition of gtn and ddt, respectively, (1.051 0.008) 10 s for the apparent inactivation rate constant after addition of nad, and (7.2 0.1) 10 a.u./s for the residual activity. experimental conditions : p - npa (0.2 mm) and nad (0.2 mm) in 50 mm potassium pi (ph 7.4) were present at the start of the reaction ; aldh2 (33 g / ml) and gtn (0.1 mm) were added as indicated. curve fitting yielded the following results : (0.048 0.009) 10 a.u./s for the uncatalyzed reaction, (3.65 0.08) 10 a.u./s for the initial activity, (2.6 0.1) 10 s for the apparent inactivation rate constant, and (0.0567 0.0008) 10 a.u./s for the residual activity. the figure shows the formation of p - nitrophenol from p - npa, as monitored at 400 nm. panel a shows that, in the absence of nad, gtn did not significantly affect esterase activity. however, inactivation set in immediately after the addition of nad. experimental conditions were : p - npa (0.2 mm) in 50 mm potassium pi (ph 7.4) was present at the start of the reaction ; aldh2 (33 g / ml), gtn (0.1 mm), dtt (0.2 mm), and nad (0.2 mm) were added as indicated. 1 yielded (1.0 0.5) 10 absorbance units (a.u.)/s for the uncatalyzed reaction, (1.07 0.02) 10 a.u./s for the initial rate, (1.083 0.008) 10 a.u./s and (1.15 0.01) 10 a.u./s after addition of gtn and ddt, respectively, (1.051 0.008) 10 s for the apparent inactivation rate constant after addition of nad, and (7.2 0.1) 10 a.u./s for the residual activity. experimental conditions : p - npa (0.2 mm) and nad (0.2 mm) in 50 mm potassium pi (ph 7.4) were present at the start of the reaction ; aldh2 (33 g / ml) and gtn (0.1 mm) were added as indicated. curve fitting yielded the following results : (0.048 0.009) 10 a.u./s for the uncatalyzed reaction, (3.65 0.08) 10 a.u./s for the initial activity, (2.6 0.1) 10 s for the apparent inactivation rate constant, and (0.0567 0.0008) 10 a.u./s for the residual activity. activities of aldh2 after gtn preconditioning in the experiments described so far, we studied the inactivation of aldh in real time by monitoring the dehydrogenase and esterase activities in the presence of gtn. although this procedure enables the determination of the inactivation kinetics, the presence of gtn after inactivation may compromise subsequent attempts to restore enzyme activity. moreover, because of the inevitable presence of the substrate during preincubation, fairly high gtn concentrations were required. therefore, in a different procedure, referred to as gtn preconditioning, we preincubated the enzyme in the presence of gtn followed by determination of the activity (dehydrogenase or esterase) after 200-fold dilution of the preincubation mixture. in this way inactivation takes place in the absence of substrate, allowing application of much lower gtn concentrations and determination of activities in the virtual absence of gtn. figure 4.effect of nad and nadh on the rate of inactivation by gtn of ester hydrolysis. the figure shows the effects of nad and nadh (0.2 mm) on the apparent inactivation rate constant in the absence and presence of 0.4 mm dtt. (n = 3). further conditions : 33 g / ml aldh2, 0.2 mm p - npa, and 0.1 mm gtn in 50 mm potassium pi (ph 7.4). effect of nad and nadh on the rate of inactivation by gtn of ester hydrolysis. the figure shows the effects of nad and nadh (0.2 mm) on the apparent inactivation rate constant in the absence and presence of 0.4 mm dtt. (n = 3). further conditions : 33 g / ml aldh2, 0.2 mm p - npa, and 0.1 mm gtn in 50 mm potassium pi (ph 7.4). as is shown in fig. 5, preincubation with 1 m gtn at ph 7.5 resulted in complete inactivation of acetaldehyde dehydrogenation, whereas the enzyme remained active after preincubation in the absence of gtn. part of the dehydrogenase activity that was lost after preincubation with gtn was recovered in the presence of dtt. partial reactivation occurred in the presence of lpa - h2 as well, although to a smaller degree. comparable results were obtained when preincubation took place at ph 6.5 and when standard assay conditions were applied for both reactions (ph 9.0 with nad for the dehydrogenase reaction, and ph 7.5 without nad for the esterase reaction) (see supplemental fig. time dependence of irreversible aldh2 inactivation to further elucidate the mechanism of irreversible inactivation, we performed a series of experiments of the type shown in fig. 1a and varied the time of dtt addition, leaving all other conditions equal. the results, which are described in greater detail under supplemental materials (fig. s7 and accompanying text), showed that most of the inactivation took place within the same time span in which nad reduction was inhibited by gtn in the absence of dtt, although a slow additional inactivation was also apparent. the rapid irreversible inactivation was exacerbated when the gtn concentration was increased, whereas the slower process was not affected. the figure shows the effects of preincubation of aldh2 at ph 7.5 with 1 m gtn on the subsequent determination of dehydrogenase activity in the presence of nad at ph 7.4 in the absence and presence of 2 mm dtt and lpa - h2. experimental conditions for preincubation were : 0.27 mg / ml aldh2, 1 m gtn, 0.2 mm mgcl2, 2 mm nad, 50 mm napi (ph 7.5) for 10 min at 37 c. assay conditions were : 1.5 g / ml aldh2, 2 mm nad, 10 mm mgcl2, and 0.2 mm acetaldehyde in 50 mm napi (ph 7.5) at 25 c. the figure shows the effects of preincubation of aldh2 at ph 7.5 with 1 m gtn on the subsequent determination of dehydrogenase activity in the presence of nad at ph 7.4 in the absence and presence of 2 mm dtt and lpa - h2. experimental conditions for preincubation were : 0.27 mg / ml aldh2, 1 m gtn, 0.2 mm mgcl2, 2 mm nad, 50 mm napi (ph 7.5) for 10 min at 37 c. assay conditions were : 1.5 g / ml aldh2, 2 mm nad, 10 mm mgcl2, and 0.2 mm acetaldehyde in 50 mm napi (ph 7.5) at 25 c. we also determined the effect of incubation time in experiments of the type described in fig. 5, in which we preincubated the enzyme in the presence and absence of gtn (0.18 mm) and dtt (2 mm) for different lengths of time, and then measured the dehydrogenase activity after 15-fold dilution. the results, presented in fig. 6, show that in the absence of dtt the enzyme tends to lose activity over time (34 2% in 1 h). the same trend was observed in the presence of gtn, but in that case approximately half of the activity was additionally lost within the first 15 min. as a result, 13 4% of the activity remained after 1 h of preincubation. dtt offered moderate protection against the gradual inactivation in the absence of gtn (20 3% activity loss after 1 h). in the combined presence of dtt and gtn, inactivation and reactivation of aldh2-catalyzed gtn reduction to assess how gtn affects its own reduction by mitochondrial aldh we measured gtn reductase activity, removed low molecular weight ingredients from the assay mixture by gel filtration, and repeated the activity assay with the recovered enzyme. before gel filtration, 1,2-gdn formation was always observed, but omission of dtt from the reaction mixture reduced pre - column activity by 85 2% (fig. the activity in the absence of dtt corresponded to approximately one turnover (280 nm 1,2-gdn were formed by 332 nm aldh), in line with the proposal that reduction of gtn results in oxidation of active site cysteine residues. after gel filtration, the recovered enzyme showed no activity in the absence of dtt, but produced a greater than stoichiometric amount of 1,2-gdn in its presence, in agreement with the proposed role of dtt in regeneration of active site thiols. fig. 7 also shows that the recovered enzyme was significantly less active than before treatment, suggesting partially irreversible inactivation. in parallel experiments, the same procedure (enzymatic assay, enzyme recovery, repeated assay) did not affect dehydrogenase and esterase activities (dehydrogenation : 0.39 0.05 and 0.37 0.07 mol / mg / min before and after enzyme recovery, respectively ; ester hydrolysis : 0.219 0.003 and 0.223 0.004 nmol / mg / min before and after enzyme recovery, respectively), which rules out that partial inactivation of gtn reduction was an artifact. the enzyme was preincubated for various times, after which dehydrogenase activities were determined by monitoring the linear absorbance increase at 340 nm over a period of 5 min. preincubation was performed in the absence (open symbols) and presence (filled symbols) of dtt, and in the absence (circles) or presence (squares) of gtn. preincubation conditions were : 0.48 mg / ml aldh2, 1 mm nad, and 0.18 mm gtn and 0.2 mm dtt as indicated in 300 mm potassium pi (ph 7.4). assay conditions were : 19 g / ml aldh2, 1 mm acetaldehyde, 1 mm nad, and 2 mm dtt in 50 mm potassium pi (ph 7.4). the enzyme was preincubated for various times, after which dehydrogenase activities were determined by monitoring the linear absorbance increase at 340 nm over a period of 5 min. preincubation was performed in the absence (open symbols) and presence (filled symbols) of dtt, and in the absence (circles) or presence (squares) of gtn. preincubation conditions were : 0.48 mg / ml aldh2, 1 mm nad, and 0.18 mm gtn and 0.2 mm dtt as indicated in 300 mm potassium pi (ph 7.4). assay conditions were : 19 g / ml aldh2, 1 mm acetaldehyde, 1 mm nad, and 2 mm dtt in 50 mm potassium pi (ph 7.4). figure 7.effect of preincubation of aldh2 with gtn on the aldh2-catalyzed reduction of gtn. gtn reduction was determined in the absence or presence of dtt (pre - column activity). subsequently, the enzyme was rapidly recovered from the assay mixture by ym-10 chromatography, and the gtn reductase activity was measured again in the absence or presence of thiols (post - column activity). experimental conditions were : 4 g of aldh2, 2 m [c]gtn (50 000 dpm), 3 mm mgcl2, 1 mm nad, 1 mm edta, 1 mm egta, and 2 mm gsh with 2 mm dtt as indicated in 50 mm potassium pi (ph 7.4) for 10 min at 37 c. gtn reduction was determined in the absence or presence of dtt (pre - column activity). subsequently, the enzyme was rapidly recovered from the assay mixture by ym-10 chromatography, and the gtn reductase activity was measured again in the absence or presence of thiols (post - column activity). experimental conditions were : 4 g of aldh2, 2 m [c]gtn (50 000 dpm), 3 mm mgcl2, 1 mm nad, 1 mm edta, 1 mm egta, and 2 mm gsh with 2 mm dtt as indicated in 50 mm potassium pi (ph 7.4) for 10 min at 37 c. the figure depicts the effect of preincubation with gtn (0.1 mm, 10 min, 37 c) on the dehydrogenation of monal 62 in the absence and presence of various thiols. experimental conditions were : 8.1 g / ml rat liver mitochondria, 3 m monal 62, 0.1 mm nad, and dtt (2 mm), -me (50 mm), or lpa - h2 (0.1 mm) as indicated, in 10 mm tris (ph 7.4), 250 mm sucrose, 5 mm egta, and 2 mm mgcl2. inhibition by gtn and reactivation by thiols of aldh in intact mitochondria. the figure depicts the effect of preincubation with gtn (0.1 mm, 10 min, 37 c) on the dehydrogenation of monal 62 in the absence and presence of various thiols. experimental conditions were : 8.1 g / ml rat liver mitochondria, 3 m monal 62, 0.1 mm nad, and dtt (2 mm), -me (50 mm), or lpa - h2 (0.1 mm) as indicated, in 10 mm tris (ph 7.4), 250 mm sucrose, 5 mm egta, and 2 mm mgcl2. inactivation by gtn and reactivation by thiols of aldh in intact mitochondria to probe the inactivation by gtn and reactivation by thiols in a physiologically more relevant setting, we investigated the inhibition of aldh by gtn and its reactivation by thiols in isolated rat liver mitochondria. dehydrogenase activity was determined as the conversion of the aldehyde monal 62 to naphthoic acid by fluorescence spectroscopy in the presence of intact mitochondria preincubated with 100 m gtn (37 c, 10 min). as illustrated in fig. 8, preincubation with gtn caused a loss of activity of 84 2% that was partly (51 5%) restored by 2 mm dtt. lpa - h2 (2 mm) and -mercaptoethanol (-me) at the very high concentration of 50 mm reactivated the enzyme to comparable levels. at a concentration of 2 mm, -me, cys, and gsh caused no or marginal reactivation ; at a physiologically relevant concentration of 0.1 mm, lpa - h2 was ineffective as well (supplemental fig. s8a). these results are in good agreement with the observations for the purified enzyme and with a previous report on aldh inactivation in intact mitochondria (30). raising the gtn concentration gradually increased the extent of inactivation from 20 10% at 1 m to 81 4% at 100 m gtn with an ic50 of 2.7 0.5 m (supplemental fig. dtt (2 mm) and lpa - h2 (10 mm) restored activity to 80 and 60% of the original activity with 1 and 100 m gtn, respectively. these results suggest that partially irreversible aldh inactivation occurs in intact mitochondria as well. inactivation of mitochondrial aldh by gtn in agreement with previous reports (10, 22, 23), we found that gtn caused time - dependent inactivation of both the dehydrogenase and esterase activities of aldh2. the inactivation rates exhibited a tendency toward saturation at high gtn and low substrate concentrations, and we could fit the curves of kobs versus [gtn ] and 1/[acald ] or 1/[p - npa ] to hyperbolic functions. the data also show that any contribution from reversible competitive inhibition can be neglected (see supplemental materials for a discussion of this issue). rapid - equilibrium binding of substrate and inhibitor, followed by a slow inactivation step, is described by an equation of the form, (eq. 2)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\;\;k_{{\mathrm{obs}}}=\frac{k_{i}{\times}[i]}{[i]+k_{i}{\times}(1+[{\mathrm{s}}]/k_{s})}\end{equation}\end{document } in which [i ] and [s ] are the concentrations of inhibitor and substrate, ki and ks are the respective dissociation constants, and ki is the inactivation rate constant. this model predicts the observed hyperbolic dependence of kobs on the gtn concentration and the reciprocal substrate concentration. however, our results suggest an apparent dissociation constant (ks) for acetaldehyde some 23 orders of magnitude above the published km. in fact, analysis of the data suggests values for the apparent dissociation constants for acetaldehyde, p - npa, and gtn in the order of 10 m (see supplementary materials). similar discrepancies have been reported for the mutual inhibition of esterase and dehydrogenase activities (21, 24). interestingly, inhibition of gtn reduction by aldh2 also required more than 1000-fold higher concentrations of acetaldehyde than might have been expected (9). this was ascribed to the absence of nad, which has been reported to increase the substrate affinity of mitochondrial aldh (20). because our results suggest that the phenomenon persists in the presence of nad, an alternative explanation seems to be required. it has been debated whether the dehydrogenase and esterase reactions take place at the same site. this matter appears settled now, with convincing evidence in favor of a single active site, although some of the earlier data that suggested a 2-site model remain unexplained (21, 2428). to accommodate the disparate observations, we propose that all substrates react in the same active site pocket, but that the unnatural esterase substrates and gtn can occupy any of a number of poorly defined low - affinity positions, whereas the dehydrogenase substrates can and must be bound to a strictly defined high - affinity site to enable hydride transfer (supplementary materials scheme s1). somewhat surprisingly, nad profoundly affected the rate of inactivation of the esterase reaction by gtn. in the presence of dtt, a substantial decrease of the dissociation constant for gtn in the presence of nad seems to be an implausible explanation.4 it is more likely that nad binding exposes a target to gtn that is poorly accessible in the absence of the cofactor. protection and restoration of the dehydrogenase reaction by thiols the inhibition of mitochondrial aldh by isosorbide dinitrate is completely reversed by -me (23). inhibition of yeast aldh by no was almost completely prevented and largely reversed by dtt, whereas -me and gsh were less efficient (29). mitochondrial aldh was partially protected against gtn - induced inactivation by dtt and lpa - h2, but not by gsh (30). it has been demonstrated that the conversion of gtn to 1,2-gdn by mitochondrial aldh requires the presence of a reducing agent (3). the highest activity was observed in the presence of dtt, whereas lpa - h2 was about half as effective ; activity was low in the presence of cys and -me and virtually absent in the presence of gsh. in line with these reports, we found that dtt and lpa - h2, but not cys or gsh, protected dehydrogenation against gtn - induced inhibition. accordingly, inactivation of aldh2 seems to be due to the oxidation and subsequent disulfide formation of an essential cysteine residue. the poorer performance of gsh compared with dtt has been ascribed to a combination of the lower redox potential of dtt and the presence of two thiol groups per molecule, which gives dtt a kinetic advantage over gsh (and -me) in achieving complete reduction of the disulfide bridge (29). when dtt was added to the enzyme after complete inactivation by gtn, the thiol could only rescue a minor fraction of dehydrogenase activity, suggesting that at that time most of the enzyme was no longer present as a disulfide. both residual and thiol - restored activities diminished when the gtn concentration was increased. this might suggest that the magnitude of residual and restored activity is determined by competition between gtn and substrate ; however, the acetaldehyde concentration hardly affected either activity. conceivably, the gtn concentration could also modulate restored activity by competition with dtt, because the gtn to dtt ratio might affect the steady - state levels of the inactive and active aldh2 species in a reaction cycle involving oxidation / inactivation by gtn and reduction / reactivation by dtt. however, the dtt concentration also did not affect restored activity. we propose that, in addition to the dtt - reversible oxidation of the active site cysteine by gtn, the oxidized cysteinyl and/or some other active site residues may engage in a secondary reaction with gtn to yield an enzyme species that does not support dehydrogenation and is not reduced by dtt. scheme 1 shows a simple model for gtn - induced inactivation that accommodates most of the observations, particularly the observations that (i) irreversible inhibition in the absence of dtt is only partial and (ii) the extent of irreversible inhibition increases with the gtn concentration. accordingly, partitioning between reversible and irreversible inhibition in the absence of a reductant will depend on the ratio of the rates of disulfide formation (krev) and irreversible inactivation (kirr[gtn ]), the latter of which depends on the gtn concentration. in the presence of a reductant (e.g. dtt), reversible inactivation will be abolished by regeneration of the active enzyme state (esh) from the disulfide species (es - s), and irreversible inhibition will be slowed down by the dtt - induced reduction of the oxidized thiol species (es). the model correctly predicts that the fraction of aldh2 that is irreversibly inactivated by gtn in the absence of dtt will depend on the gtn concentration but not on the concentrations of dtt and acetaldehyde applied to reactivate the enzyme. scheme 1.irreversible inactivation of aldh - catalyzed gtn reduction. under low turnover conditions ([gtn ], [dtt ] < < km, km), the enzyme cycles between the reduced thiol / thiolate, oxidized thiyl, and oxidized disulfide states. at higher concentrations, gtn and the thiol reductant (here represented by dtt) compete for the oxidized thiyl state, resulting in direct regeneration of the reduced enzyme with dtt, or in irreversible inactivation with gtn (esh, es-, es - s, and ei represent the reduced (thiol / thiolate), oxidized thiyl, oxidized disulfide, and irreversibly inactivated enzyme states ; kox, krev, kirr, kred, and kred stand for the rate constants for gtn - induced thiolate oxidation, dtt - reversible disulfide formation, gtn - induced dtt - irreversible inactivation, and dtt - exacted reduction of the es - s and es enzyme states, respectively). note that because of the branching of the reaction after thiol oxidation, irreversible inactivation in the absence of dtt will only be partial, with the extent depending on the concentration of gtn. inactivation will be slower in the presence of dtt but, on account of the continuous recycling of the disulfide to the active reduced thiol, all of the enzyme should eventually be inactivated irreversibly. irreversible inactivation of aldh - catalyzed gtn reduction. under low turnover conditions ([gtn ], [dtt ] < < km, km), the enzyme cycles between the reduced thiol / thiolate, oxidized thiyl, and oxidized disulfide states. at higher concentrations, gtn and the thiol reductant (here represented by dtt) compete for the oxidized thiyl state, resulting in direct regeneration of the reduced enzyme with dtt, or in irreversible inactivation with gtn (esh, es-, es - s, and ei represent the reduced (thiol / thiolate), oxidized thiyl, oxidized disulfide, and irreversibly inactivated enzyme states ; kox, krev, kirr, kred, and kred stand for the rate constants for gtn - induced thiolate oxidation, dtt - reversible disulfide formation, gtn - induced dtt - irreversible inactivation, and dtt - exacted reduction of the es - s and es enzyme states, respectively). note that because of the branching of the reaction after thiol oxidation, irreversible inactivation in the absence of dtt will only be partial, with the extent depending on the concentration of gtn. inactivation will be slower in the presence of dtt but, on account of the continuous recycling of the disulfide to the active reduced thiol, all of the enzyme should eventually be inactivated irreversibly. as a rather trivial alternative explanation for the dependence of the extent of irreversible inactivation on the concentration of gtn, one could assume that gtn or one of its metabolites is inactivating the enzyme in a totally unrelated reaction. although such a reaction would be expected to affect the rate rather than the extent of irreversible inactivation, it might explain the observations of fig. 1, provided the reaction is slow and the time of gtn and dtt additions did not vary much between experiments. to investigate that possibility we performed the experiments illustrated by figs. 6 and supplemental s7, in which we varied the time between gtn and dtt addition. the results indicated that most of the activity was lost within the same time frame in which gtn inhibited the enzyme (in the absence of dtt), in agreement with the mechanism - based model of inactivation presented in scheme 1. although a slower reaction was also apparent, it did not depend on the gtn concentration and also occurred in the absence of gtn, suggesting that it represents unspecific deterioration of the enzyme upon prolonged incubation. in the preincubation experiments (fig. 6) we made the observation that, although dtt protected the enzyme against inactivation in the absence of gtn, it exacerbated the inactivation in its presence. although paradoxical at first sight, this observation is in line with expectations for the mechanism - based model of irreversible inactivation proposed by us. in the absence of dtt, the enzyme undergoes only one turnover in the presence of gtn, and mechanism - based inactivation will be halted after that. in the presence of dtt, multiple turnovers with gtn will occur, which will result in complete inactivation, even if dtt diminishes the fraction of irreversibly inactivated enzyme per turnover. taken together, these results provide strong support for mechanism - based inactivation as the origin of irreversible inactivation. the strictly kinetic nature of the present study precludes conclusions about the site or identity of the structural changes underlying the inactivation processes, although the literature may offer some clues. a secondary reaction, in which gtn or one of its metabolites reacts with an oxidized or nitrated thiol before generation of the disulfide can occur, might result in irreversible formation of thiol sulfinate and sulfonate products (6, 14). one candidate inactivating agent is nitroxyl (no), which has been proposed to be a product of gtn metabolism (31), and which was reported to cause partially irreversible inhibition of aldh, presumably by the formation of sulfinamides (32). gtn can stimulate sgc in the presence of purified aldh2, and this stimulation is mediated by no even though the proposed mechanism for aldh - supported decomposition of gtn predicts nitrite as the only product (12). formation of no in the secondary reaction occurring at higher gtn concentrations might explain those observations. we are currently carrying out mass spectrometric studies with wild - type aldh2 and several active site mutants to this end. physiological implications nitrate tolerance is a complex and almost certainly multifactorial phenomenon, with potential contributions from impaired bioactivation, oxidative stress, sgc desensitization, and physiological counter - regulation (3, 6, 14, 15). the role of impaired gtn bioactivation depends on the physiologically relevant mechanism of gtn biotransformation. because decomposition of gtn by aldh2 results in oxidation of the active site thiol that must subsequently be reduced to sustain catalysis, the proposed involvement of aldh2 in the bioactivation of gtn revived the early proposal that ascribed nitrate tolerance to thiol depletion (34). however, whereas the mechanism of bioactivation involves reversible thiol oxidation, an irreversible or slowly reversible process must underlie nitrate tolerance. the irreversible inhibition reported in the present study may, therefore, contribute to nitrate tolerance in vivo. in the absence of dtt the rate of irreversible inactivation can be estimated from the observed inactivation rates (reversible + irreversible) and the relative activity after reactivation. s1 yields an apparent inhibition rate constant of 3 10 m s with a limit of 0.040.06 s. according to figs. 1 and 2 a large fraction of the inactivation (70%) will be irreversible, suggesting that the rate constant for irreversible inactivation is of similar magnitude. for gtn concentrations in the physiological range (0.010.1 m) this would result in lifetimes for active aldh2 between 6 and 60 h. for the inactivation in the presence of dtt a rate constant of 4 10 s can be derived (fig. 2), but this rate corresponds to saturating concentrations of gtn and dtt and can not be easily extrapolated to physiologically relevant levels. moreover, the present model does not account for the small but significant fraction of irreversible inhibition (7%) that seems to be refractory to dtt (fig. s2). a crucial question regarding the physiological significance of irreversible aldh2 inactivation concerns the applicability of the observations to gtn reduction. the results presented in fig. 7 suggest that gtn reduction behaves like aldehyde dehydrogenation in this respect. in the absence of dtt the enzyme turned over only once and after recovery no more gtn was reduced, in line with the hypothesis that the reaction with gtn results in oxidation of the active site thiol. in the same time frame the enzyme underwent multiple turnovers (5.35 times) in the presence of dtt. however, activity diminished by 33 and 51% after preincubation with 2 m gtn in the absence and presence of dtt, respectively. considering that, during preincubation in the presence of dtt, aldh2 underwent 5.35 turnovers on average, the 51% inhibition after recovery corresponds to a loss of active enzyme of 12.5% per turnover. comparison with the 33% inhibition during one turnover in the absence of dtt indicates that dtt provides partial protection against irreversible inactivation by gtn. the central observation of the present paper is that in addition to the previously noticed thiol - reversible inhibition, there is an irreversible aspect to the inactivation of aldh2 by gtn. this irreversible component, which affects dehydrogenase activity as well as gtn reductase activity, may contribute to the phenomenon of nitrate tolerance. in line with this hypothesis, thiols are often unable to reverse nitrate tolerance completely (14, 30). in summary, the present study allows the following conclusions : (i) in the absence of dtt, gtn oxidizes the active site thiol of mitochondrial aldh resulting in complete inactivation of dehydrogenation, ester hydrolysis, and gtn reduction ; (ii) the rate of inactivation is affected by the concentrations of substrate and inhibitor, indicative of competitive binding, but the apparent dissociation constants (in the order of 10 m) are much higher than reported km values for the respective reactions, which suggests that competition takes place outside of the high - affinity binding site ; (iii) dehydrogenase activity is only restored to a modest extent by dtt, indicating that a large fraction of inactivation is irreversible ; (iv) gtn reduction is irreversibly inactivated in a similar fashion as dehydrogenase activity, suggesting a role for irreversible aldh2 inactivation in the development of nitrate tolerance.	mitochondrial aldehyde dehydrogenase (aldh2) may be involved in the biotransformation of glyceryl trinitrate (gtn), and the inactivation of aldh2 by gtn may contribute to the phenomenon of nitrate tolerance. we studied the gtn - induced inactivation of aldh2 by uv / visible absorption spectroscopy. dehydrogenation of acetaldehyde and hydrolysis of p - nitrophenylacetate (p - npa) were both inhibited by gtn. the rate of inhibition increased with the gtn concentration and decreased with the substrate concentration, indicative of competition between gtn and the substrates. inactivation of p - npa hydrolysis was greatly enhanced in the presence of nad+, and, to a lesser extent, in the presence of nadh. in the presence of dithiothreitol (dtt) inactivation of aldh2 was much slower. dihydrolipoic acid (lpa - h2) was less effective than dtt, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. when dtt was added after complete inactivation, dehydrogenase reactivation was quite modest (16%). the restored dehydrogenase activity correlated inversely with the gtn concentration but was hardly affected by the concentrations of acetaldehyde or dtt. partial reactivation of dehydrogenation was also accomplished by lpa - h2 but not by gsh. we conclude that, in addition to the previously documented reversible inhibition by gtn that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to aldh inactivation. importantly, aldh2-catalyzed gtn reduction was partly inactivated by preincubation with gtn, suggesting that the inactivation of gtn reduction is also partly irreversible. these observations are consistent with a significant role for irreversible inactivation of aldh2 in the development of nitrate tolerance.
birds were caught by mist - nets, banded (ringed), and examined for ticks at the cernek bird ringing station (4136n, 3605e) in the kizilirmak delta in turkey, an internationally important wetland area for birds (4). we conducted the study during the spring and autumn migration seasons in 2010 and 2011 and in spring 2012. the identified ticks were placed in tubes with steel beads and homogenized at the maximum speed (50 hz) for 10 min in tissuelyser lt device (qiagen, hilden, germany). rna was isolated according to the manufacturer s recommendations by using high pure viral nucleic acid kit (roche applied science, mannheim, germany), but as a small modification, the homogenized tick mixture was kept at 37c for 1 h. in accordance with the manufacturer s recommendations, we obtained viral cdna using the revertaid first strand cdna synthesis kit (thermo scientific, vilnius, lithuania). real - time pcr was performed by using the combination of primer pairs and the faststart taqman probe master kit (roche applied science), as described by yapar. (6), for each tick sample. cdna from patient samples, which previously had been determined as positive, were used as the positive control sample. cchfv small (s) segment (encoding for the nucleocapsid protein) specific primer pairs (f3 : 3-gaatgtgcatgggttagctc-5 and r2 : 3-gacatcacaatttcaccagg-5) and same pcr conditions defined by schwarz. (7) were used in the pcr. sequence analysis was performed on the 260-bp pcr product, when positivity was detected, by using the primers of f3 and r2 in the abi 310 genetic analyzer (applied biosystems, foster city, ca, usa). our sequences and genbank sequences were aligned in mega 5.1 (http://www.megasoftware.net), and the phylogenetic tree was drawn on the basis of the 260 bp of the s segment of the cchfv genome. to compare the sequences and phylogenetic analysis, we used the maximum - likelihood method. we found attached ticks on 65 (0.5%) of the 13,377 captured and banded birds, which represented 17 species. a total of 188 ticks collected on these birds belonged to ixodes, hyalomma, haemaphysalis, and rhipicephalus genera (table). (nymphs) (127 [0.8% ]) on great reed warblers (acrocephalus arundinaceus) (9.7% of banded birds) and on european robins (erithacus rubecula) (0.5% of banded birds), respectively. the samsun cchfv partial sequences of the s segments obtained in this study have been deposited in genbank under the accession nos. kf727976 and kf727977. the cchfv sequences obtained in the present study belong to genotype 4 (figure 1). phylogenetic tree based on the 260 bp of the small segment of the cchfv genome. the multiple sequence alignment was obtained by using mega 5.1 (http://www.megasoftware.net), and the phylogenetic tree was constructed by the maximum - likelihood method using 1,000 bootstrap replicates of the sequence data. the tree is drawn to scale with branch length in the same unit as those of the evolutionary distance used to infer the phylogenetic tree. cchfv, crimean - congo hemorrhagic fever ; s., south ; c., central. (nymphs) on european robins, which migrate across turkey twice a year en route from their breeding sites to their wintering sites (figure 2) and back. the probability of cchfv transportation by ticks among different regions and countries is high during migration of both bird species. because these birds stop several times during migration (9), cchf in europe possibly could increase, especially at the stopover sites in southern europe, which provide suitable ecologic environments. a) great reed warbler (acrocephalus arundinaceus) eastern migration routes (red lines), breeding grounds (yellow) and wintering areas (purple). bodies of water are blue, and nonbreeding / nonwintering areas are light green.. b) european robin (erithacus rubecula) eastern migration routes (red lines), resident grounds (green), breeding grounds (yellow), and wintering areas (purple). bodies of water are blue, and nonbreeding / nonwintering areas are light green. although hyalomma ticks are the most commonly encountered ticks that carry cchfv in turkey, the virus also was detected in ixodid ticks, such as rhipicephalus spp. and haemaphysalis spp. picked up from humans and animals (10). also, albayrak. (11) detected cchfv in i. ricinus ticks. because the 2 sequences detected showed similarity with cchfv genotype 4, which was widespread in turkey (1), whether the ticks were infected in turkey or infected earlier during bird migration is impossible to say. other studies have shown that cchfv could be transported by ticks on birds (1214). by itself, transportation of infected ticks by birds might not be sufficient to cause the epidemics in turkey, but along with this, climate changes, environmental changes, increased number of sensitive animals, and tick and animal movements might play a role in spreading cchf (1). in ecologically important regions, such as the kizilirmak delta, where resident and migratory birds are mixed, different microorganisms or ticks can be transferred among birds, and then carried by birds for long distances. therefore, knowing migration routes and what pathogens birds are infected with may help predict future epidemics in various countries and provide highlight the geographic regions where diseases could be detected (15).	we investigated migratory birds role in spreading crimean - congo hemorrhagic fever virus (cchfv) through attached ticks. we detected cchfv rna in ticks on migratory birds in turkey. two isolates showed similarity with cchfv genotype 4, suggesting a role for ticks in cchfv epidemics in turkey and spread of cchfv by birds.
permission was obtained from the patient included in this report. in june 2014, a 25-year - old woman presented to the outpatient clinic with a 6-month history of progressive left hip pain. physical examination revealed mild pain with motion in the hip joint and slightly limited range of motion. radiographs showed a large osteolytic lesion with well - defined margins in the proximal metaphysis of the femur, partially surrounded by sclerotic cortical bone (fig. 1). magnetic resonance imaging (mri) showed a well - demarcated intra - osseous lesion measuring 584242 mm. t1-weighted images demonstrated a lesion with heterogeneously distributed low- and high - signal intensities, while t2-weighted images revealed similar findings, partially surrounded by areas of low signal intensity, concordant with reactive bone sclerosis. fat - suppressed images showed an area of low signal intensity at the center of the lesion. a radionuclide bone scan demonstrated a lesion with peripherally increased uptake of tc-99 m methyl diphosphonate (mdp) in the metaphysis of the femur in the area of radiographic abnormality (fig. these findings reflected benign bone tumor, such as intraosseous lipoma or liposclerosing myxofibrous tumor, rather than fibrous dysplasia, giant cell tumor, aneurysmal bone cyst, or simple bone cyst. because the lesion was suspected to be a benign bone tumor with progressive pain leading to impending fracture, surgery was performed in august 2014. curettage of the lesion and bone grafting were performed with a femoral head allograft, which was stored in our bone bank. prophylactic internal fixation using the zimmer natural nail system (zimmer, warsaw, in, usa) was performed to prevent pathologic fracture (fig. macroscopically, the pathologic specimen had abundant tan - brown soft tissue and multiple bone fragments. these histiocytes showed abundant foamy cytoplasm, and were positive for cd68 but negative for cd1a and s100 (fig. after surgery, we evaluated the lipidemic status of the patient including her family history. she had no relevant history of elevated laboratory results, but her mother did have a history of hyperlipidemia. laboratory tests revealed the following results : fasting blood glucose, 95 g / dl ; hba1c, 5.4% ; total cholesterol, 157 mg / dl ; triglycerides, 63 mg / dl ; high - density lipoprotein cholesterol, 44 mg / dl ; and low - density lipoprotein cholesterol, 101 mg / dl. immunoelectrophoretic fractionation of lipoprotein revealed normal values for alpha, pre - beta, beta, and chylomicrons. implant removal and bone grafting with allograft bone chip for new bone formation were performed to remove foreign body sensation. there was no sign of local recurrence and the patient could successfully perform normal activities at the last follow - up (fig. pix is an extremely rare, benign bone tumor that usually occurs in the appendicular skeleton, such as the calcaneus, rather than the axial skeleton. the lesion in this patient showed a sharply defined lytic lesion with a narrow zone of transition, and partially or completely sclerotic margins. these characteristics also are seen in intraosseous lipoma, liposclerosing myxofibrous tumor, simple bone cyst, aneurysmal bone cyst, non - ossifying fibroma, chondroblastoma, giant cell tumor, and fibrous dysplasia. the location of the lesion made it difficult to include pix in the differential diagnosis preoperatively. pix can exhibit an aggressive appearance, with an expansile border or cortical disruption, and may mimic malignancy, such as multiple myeloma or metastatic bone tumor3). mri is typically characterized by a circumscribed area of heterogeneous signal intensity on t1- and t2-weighted images, and associated loss of signal when fat is suppressed, which is hypothesized to result from the cholesterol - laden histiocytes seen on histopathology5). the typical histopathologic appearance of pix is a lesion consisting of numerous foamy cells and non - foamy mononuclear or multinucleated giant cells, and, occasionally, touton - type giant cells6). abundant cholesterol clefts with giant cells at the focal area and cholesterol clefts between bone spicules also can be seen in the lesion7). in our case, we could see numerous foamy cells and multinucleated giant cells, but touton - type giant cells and cholesterol clefts between bone spicules were not observed. giant cell tumor and intra - osseous lipoma might be considered in the differential diagnosis. giant cell tumors usually show a lytic lesion in the epiphysis, rather than in the metaphysis, and sometimes grow to the articular surface. the key histopathologic appearance is multinucleated giant cells with up to 100 nuclei that have prominent nucleoli8). intraosseous lipomas are usually located in the long bones and have radiologic findings similar to pix. however, a well - defined lesion with sclerotic borders and mineralization of the central lesion are often pathognomonic. computed tomography and mri also can be diagnostic because they show high composition of fat content in the lesion9). however, we suggest that this lesion is highly associated with familial factors because her mother had a history of hyperlipidemia. further study is needed to investigate the specific genes associated with xanthoma in patients with a family history of hyperlipidemia. when pix is accompanied by progressive pain or aggressive appearance, the optimal treatment typically consists of curettage of the lesion and bone grafting10). the necessity of prophylactic internal fixation depends on the location and extent of the lesion. because the pelvis, proximal femur, and tibia are particularly prone to pathologic fracture, internal fixation may be indicated. in this patient, although the lesion did not exhibit an aggressive appearance, it showed extensive osteolysis with thin cortical bone accompanied by progressive pain in the left proximal femur. thus, internal fixation was performed to prevent pathologic fracture. in conclusion, we report a rare case of pix of the proximal femur in a patient without hyperlipidemia. this diagnosis can be difficult to make preoperatively using clinical examination, laboratory findings, and imaging studies. the variable clinical and radiologic characteristics of pix reinforce the necessity of bone biopsy to confirm diagnosis.	we report the case of a 25-year - old woman presenting with left hip pain. a lesion was found in the proximal femoral metaphysis. benign bone tumor, such as intraosseous lipoma or liposclerosing myxofibrous tumor, was suspected based on simple radiographs and magnetic resonance images. curettage of the lesion and bone grafting was performed. histologic findings reflected primary intraosseous xanthoma of the proximal femur. laboratory tests revealed the patient to be normolipidemic, while immunoelectrophoretic fractionation of lipoproteins revealed normal values for alpha, pre - beta, beta, and chylomicrons. at the one - year follow - up, there was no evidence of local recurrence. this is the first reported case of primary intraosseous xanthoma of the proximal femur in a normolipidemic patient.
, the result can be destruction of the bone itself.1 ciprofloxacin is a broad - spectrum fluoroquinolone antibacterial agent used in the treatment of osteomyelitis caused by pathogens like staphylococcus aureus and pseudomonas aeruginosa.2 a high concentration of the drug is required for 5 weeks to several months to cure the bone infection. thus, local drug delivery would be advantageous and reduce the side effects that occur due to high dosage of the drug.3 compared with metal, metal alloy, and ceramic bone - substitute materials, organic / inorganic composite structures are best suited to orthopedic biomaterials as their properties closely mimic those of natural bone.4 a novel method for the preparation of ceramic / polymer composites is required to combine the advantages of the bioactive, biodegradable, and biological properties of scaffolds for bone regeneration. the development of organic / inorganic composites as orthopedic materials has attracted much attention during the last few years. natural bone is composed of inorganic compound (hydroxyapatite [hap ] 65% on the nanoscale) and organic compounds (collagen matrix 35%). hence, to mimic natural bone and to maintain biocompatibility, a polymer / ceramic material would be ideal for bone drug delivery. it is attractive for use in synthetic bone - tissue scaffolds as a means of more closely mimicking natural tissue composition.3 the polymer chosen for drug delivery should be biodegradable and biocompatible. polycaprolactone (pcl) is a biodegradable, bio - resorbable polymer used for drug formulations and medical purposes. it is an ideal polymer for the application of drug or biomolecule delivery, as it has a relatively low degradation rate that extends over 1 year. the us food and drug administration has approved pcl as it is ideal for a number of medical and drug - delivery devices.5 ahola investigated in vitro bone filling composite materials that release ciprofloxacin to kill any remaining bacteria and that contain bio - ceramic to help the bone to heal.6 kim developed an hap porous scaffold coated with hap and pcl composite and entrapped the antibiotic drug tetracycline hydrochloride within the coating layer.5 they found that the release rate of the drug was sustained for prolonged periods and was highly dependent on the degree of coating dissolution, suggesting the possibility of controlled drug release from the porous scaffold with hap - pcl coating.5 in the work presented here, a composite film of nanoscale hap, prepared from eggshell, and pcl was prepared and loaded with ciprofloxacin for drug delivery to bones. the phase composition and morphology of the developed nano - composite were studied by x - ray diffraction (xrd), transmission electron microscopy (tem), and fourier - transform infrared (ftir) spectroscopy. the drug - release study was done in vitro and its absorbance value was measured using a uv - visible spectrophotometer. the degradation of the composite film was studied by immersing it in phosphate - buffered saline (pbs) for 7 days. cytotoxicity studies were performed in a fibroblast cell line, and cell proliferation was examined in an osteoblast cell line. apart from standard calcium salts, hap can also be produced from some natural sources of calcium like sea corals,7 eggshell,8 sea shells,9 and also from body fluids.10 the utilization of calcium from a natural source minimizes the chances of impurities like silica being present in the finished product. moreover, the cost of production can be reduced, as there will be no need to purify. an important aspect in the development of methods for the synthesis of hap is the use of raw materials from unconventional sources. the world s egg production was estimated to be approximately 6.3710 tons in 2010.11 taking into account that the shell comprises about 11% of the weight of each egg, the amount of eggshell produced in the world in the last year ascends to about 710 tons.12 though eggshells are occasionally used as a fertilizer due to their high calcium and nitrogen content, most are discarded as waste. by utilizing this organic waste, the cost of a high - quality calcium source for preparation of hap is reduced, and, at the same time, the recycling of a waste product of human daily activity can also be taken care of, underscoring the importance of using eggshell as a calcium source.13,14 eggshell is composed of calcium carbonate, which accounts for around 94% of the total weight ; calcium phosphate (1%) ; organic matter (4%) ; and magnesium carbonate (1%).15 hap from eggshell was synthesized via a precipitation and low - combustion method that was lengthy, complicated, and required ph control and adjustment. the eggshells were cleaned manually with de - ionized water then boiled in water for 30 minutes. the uncrushed and washed eggshells were placed in a porcelain crucible and calcined in a kanthal wire heating furnace for 2 hours to decompose organic matter and to convert the calcium carbonate to calcium oxide. caco3cao+co2(1) a stoichiometry amount of calcined eggshell (1 m) was dispersed in 50 ml of distilled water. after dispersing in distilled water cao+h2oca(oh)2(exo)(2) under rigorous stirring, reagent - grade orthophosphoric acid solution (0.6 m) was added in drops at a controlled rate to the suspension at room temperature. initially, the ph of the solution was found to be 12, but at the end of the addition of orthophosphoric acid, it had decreased to 8.5 and a precipitate had formed. the solution was then stirred for another 30 minutes without heating and then left for another 10 hours for complete precipitation occur. finally, after drying at 80c for 3 hours, the precipitate was calcined at 900c for 2 hours. initially, ciprofloxacin was dissolved in dichloromethane for 1 hour, then hap powder, prepared from eggshell, was added to the solution and the mixture stirred for 6 hours at room temperature. during this process, the drug concentration was adjusted to 75% w / w with respect to hap - pcl.16 lastly, pcl pellets were added and the suspension was stirred for 12 hours to prepare the drug - loaded hap - pcl solution (figure 1). the pcl film is prepared by dissolving 1.5 % w / v pcl pellets into dichloromethane. then the mixture is poured into a glass petri dish and dried under ambient conditions. the mixture was poured into a glass petri dish and dried under ambient conditions for 24 hours to obtain the composite film.17,18 as a medium for in vitro degradation tests, pbs (ph 7.4) was used. the pcl film and drug - loaded film were weighed and incubated in 30 ml of pbs at 37c for a period of 7 days. the medium was refreshed every 1 hour for the first 5 hours, and then every 24 hours thereafter. at predetermined periods of time, the samples were taken out, blotted on a filter paper, and weighed using a balance. the water uptake was obtained by determining the weight difference between the sample initially and the wet sample (wwet / winitial). the material degradation was obtained by determining the weight difference between the sample initially and the sample after drying ; [1 (wdry / winitial)].19 in vitro drug - release studies were conducted to estimate the rate of drug release from the drug - loaded film and to find the concentration of the drug released at particular times. pbs (ph 7.4) was used as the medium for the in vitro drug - release tests. the drug - loaded film was immersed in a glass vessel containing medium and incubated at 37c and ph 7.4 for periods of up to 7 days. the drug - release studies were performed by directly immersing the films into the beaker containing distilled water. at frequent time intervals, 3 ml of sample was extracted and analyzed with a uv - visible spectrophotometer.20 diffusion studies were carried out using dialysis membrane. the film was placed to one end of the dialysis tube, then the dialysis tube was placed in a beaker containing 100 ml distilled water / pbs so that the film was dipped in the water / pbs. 5 ml of sample is withdrawn from the beaker and the concentration of drug in each sample is estimated using uv - visible spectrophotometer at 271 nm. an equal volume of fresh distilled water / pbs is replaced after withdrawal of each sample. although biologically acceptable ingredients were present in the final form of the composite, the problem of non - biocompatibility could have arisen due to the presence of trace amounts of solvents and/or monomers released from the polymer, or due to processing factors. thus, the biocompatibilities of the nanocomposite scaffolds were studied by in vitro cell culture by employing the fibroblast cell line nih-3t3 and osteoblast cell line mg-63.21,22 cell growth on the surface, and cell migration into the pores, of the composite, if any, and cell morphology and attachment all indicated the biocompatibility and suitability of the material for in vivo use. cellular responses (eg, cell proliferation) of the nanoscale hap filled pcl composites were assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay using the fibroblast cell line nih-3t3 and osteoblast cell line mg-63 obtained from the national center for cell science, pune, india. cells were cultured in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum, containing l - glutamine, penicillin, and streptomycin. cells were trypsinized and resuspended in fresh media before being seeded onto the sample surfaces in a 24-well plate at a density of 110 well. then 1 ml of mccoy s 5a medium (with l - glutamine, without phenol red or sodium bicarbonate) supplemented with 5% fetal calf serum and 5% bovine calf serum was added to each well. the cells were maintained at 37c in an atmosphere of 5% co2 and 95% air, and the culture medium was changed every day without disturbing the cell monolayer. mtt assay (sigma - aldrich co, st louis, mo, usa) was performed to assess cell proliferation on the sample surface. an mtt solution was prepared by adding 5 mg of mtt in 1 ml pbs (5 mg / ml) then filter - sterilized. the mtt was diluted (50 l into 450 l) in serum - free, phenol red - free dulbecco s modified eagle s medium. the diluted mtt solution (500 l) was then added to each sample in 24-well plates. after 2 hours of incubation, 500 l of solubilization solution, containing 10% triton x-100, 0.1 n hcl, and isopropanol, was added to dissolve the formed purple formazan crystals from the mtt that had been reduced by the active mitochondria of viable cells. a volume of 100 l of the resulting supernatant was transferred into a 96-well plate and read at 570 nm at a bench - top microplate reader (make). data are presented as mean standard deviation.23 cells were cultured (100,000 cells / disc) in a 6.3 cm petri dish, as shown in figure 2. apart from standard calcium salts, hap can also be produced from some natural sources of calcium like sea corals,7 eggshell,8 sea shells,9 and also from body fluids.10 the utilization of calcium from a natural source minimizes the chances of impurities like silica being present in the finished product. moreover, the cost of production can be reduced, as there will be no need to purify. an important aspect in the development of methods for the synthesis of hap is the use of raw materials from unconventional sources. the world s egg production was estimated to be approximately 6.3710 tons in 2010.11 taking into account that the shell comprises about 11% of the weight of each egg, the amount of eggshell produced in the world in the last year ascends to about 710 tons.12 though eggshells are occasionally used as a fertilizer due to their high calcium and nitrogen content, most are discarded as waste. by utilizing this organic waste, the cost of a high - quality calcium source for preparation of hap is reduced, and, at the same time, the recycling of a waste product of human daily activity can also be taken care of, underscoring the importance of using eggshell as a calcium source.13,14 eggshell is composed of calcium carbonate, which accounts for around 94% of the total weight ; calcium phosphate (1%) ; organic matter (4%) ; and magnesium carbonate (1%).15 hap from eggshell was synthesized via a precipitation and low - combustion method that was lengthy, complicated, and required ph control and adjustment. the eggshells were cleaned manually with de - ionized water then boiled in water for 30 minutes. the uncrushed and washed eggshells were placed in a porcelain crucible and calcined in a kanthal wire heating furnace for 2 hours to decompose organic matter and to convert the calcium carbonate to calcium oxide. caco3cao+co2(1) a stoichiometry amount of calcined eggshell (1 m) was dispersed in 50 ml of distilled water. after dispersing in distilled water cao+h2oca(oh)2(exo)(2) under rigorous stirring, reagent - grade orthophosphoric acid solution (0.6 m) was added in drops at a controlled rate to the suspension at room temperature. initially, the ph of the solution was found to be 12, but at the end of the addition of orthophosphoric acid, it had decreased to 8.5 and a precipitate had formed. the solution was then stirred for another 30 minutes without heating and then left for another 10 hours for complete precipitation occur. finally, after drying at 80c for 3 hours, the precipitate was calcined at 900c for 2 hours. initially, ciprofloxacin was dissolved in dichloromethane for 1 hour, then hap powder, prepared from eggshell, was added to the solution and the mixture stirred for 6 hours at room temperature. during this process, the drug concentration was adjusted to 75% w / w with respect to hap - pcl.16 lastly, pcl pellets were added and the suspension was stirred for 12 hours to prepare the drug - loaded hap - pcl solution (figure 1). the pcl film is prepared by dissolving 1.5 % w / v pcl pellets into dichloromethane. then the mixture is poured into a glass petri dish and dried under ambient conditions. the mixture was poured into a glass petri dish and dried under ambient conditions for 24 hours to obtain the composite film.17,18 as a medium for in vitro degradation tests, pbs (ph 7.4) was used. the pcl film and drug - loaded film were weighed and incubated in 30 ml of pbs at 37c for a period of 7 days. the medium was refreshed every 1 hour for the first 5 hours, and then every 24 hours thereafter. at predetermined periods of time, the samples were taken out, blotted on a filter paper, and weighed using a balance. the water uptake was obtained by determining the weight difference between the sample initially and the wet sample (wwet / winitial). the material degradation was obtained by determining the weight difference between the sample initially and the sample after drying ; [1 (wdry / winitial)].19 in vitro drug - release studies were conducted to estimate the rate of drug release from the drug - loaded film and to find the concentration of the drug released at particular times. pbs (ph 7.4) was used as the medium for the in vitro drug - release tests. the drug - loaded film was immersed in a glass vessel containing medium and incubated at 37c and ph 7.4 for periods of up to 7 days. the drug - release studies were performed by directly immersing the films into the beaker containing distilled water. at frequent time intervals, 3 ml of sample was extracted and analyzed with a uv - visible spectrophotometer.20 diffusion studies were carried out using dialysis membrane. the film was placed to one end of the dialysis tube, then the dialysis tube was placed in a beaker containing 100 ml distilled water / pbs so that the film was dipped in the water / pbs. the beaker was then placed on a magnetic stirrer. 5 ml of sample is withdrawn from the beaker and the concentration of drug in each sample is estimated using uv - visible spectrophotometer at 271 nm. an equal volume of fresh distilled water / pbs is replaced after withdrawal of each sample. although biologically acceptable ingredients were present in the final form of the composite, the problem of non - biocompatibility could have arisen due to the presence of trace amounts of solvents and/or monomers released from the polymer, or due to processing factors. thus, the biocompatibilities of the nanocomposite scaffolds were studied by in vitro cell culture by employing the fibroblast cell line nih-3t3 and osteoblast cell line mg-63.21,22 cell growth on the surface, and cell migration into the pores, of the composite, if any, and cell morphology and attachment all indicated the biocompatibility and suitability of the material for in vivo use. cellular responses (eg, cell proliferation) of the nanoscale hap filled pcl composites were assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay using the fibroblast cell line nih-3t3 and osteoblast cell line mg-63 obtained from the national center for cell science, pune, india. cells were cultured in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum, containing l - glutamine, penicillin, and streptomycin. cells were trypsinized and resuspended in fresh media before being seeded onto the sample surfaces in a 24-well plate at a density of 110 well. then 1 ml of mccoy s 5a medium (with l - glutamine, without phenol red or sodium bicarbonate) supplemented with 5% fetal calf serum and 5% bovine calf serum was added to each well. the cells were maintained at 37c in an atmosphere of 5% co2 and 95% air, and the culture medium was changed every day without disturbing the cell monolayer. mtt assay (sigma - aldrich co, st louis, mo, usa) was performed to assess cell proliferation on the sample surface. an mtt solution was prepared by adding 5 mg of mtt in 1 ml pbs (5 mg / ml) then filter - sterilized. the mtt was diluted (50 l into 450 l) in serum - free, phenol red - free dulbecco s modified eagle s medium. the diluted mtt solution (500 l) was then added to each sample in 24-well plates. after 2 hours of incubation, 500 l of solubilization solution, containing 10% triton x-100, 0.1 n hcl, and isopropanol, was added to dissolve the formed purple formazan crystals from the mtt that had been reduced by the active mitochondria of viable cells. a volume of 100 l of the resulting supernatant was transferred into a 96-well plate and read at 570 nm at a bench - top microplate reader (make). data are presented as mean standard deviation.23 cells were cultured (100,000 cells / disc) in a 6.3 cm petri dish, as shown in figure 2. figure 3 shows the xrd pattern of hap recorded using a siemens d5000 diffractometer (siemens ag, berlin and munich, germany) with cuk radiation (=0.154 nm). the diffractometer was operated at 40 kv and 30 ma at a 2 theta range of 490 employing a step size of 0.1 and a 0.60-second exposure. the peaks of hap appearing at 26.2, 32.2, 49.6, and 64.0 (2) correspond to the 002, 211, 123, and 304 reflection planes. the xrd pattern of the hap powder obtained is in good in agreement with the xrd pattern of a hap standard available from the joint committee on powder diffraction standards (jcpds ; standard number 84 - 1998). the xrd pattern possesses a strong peak at around 32.2 corresponding to the 211 plane of hap s crystalline structure. no other characteristic peaks corresponding to other calcium phosphate phases or impurities were observed. from the peaks of the xrd diffraction pattern, the prepared hap samples are in hexagonal space group. the structure of the ciprofloxacin loaded hap - pcl nano - composite film was analyzed using ftir spectroscopy, as shown in figure 4. the first indication of the formation of hap is the strong, complex, broad ftir band centered around 1,0001,100 cm due to the asymmetric stretching mode of vibration of the po4 group. as a major peak of the phosphate group, the n3 vibration peak could be identified in the region between 1,100 and 960 cm for both powders, which was due to the asymmetric stretching of p - o in po 34. the presence of the peak in the region 1,4001,450 cm is due to absorbed carbon dioxide. the crystalline powder generated two characteristic stretching modes of o - h bands at about 3,497 cm which are noticed in all ftir spectra of hap. for ciprofloxacin, one prominent characteristic peak was found between 3,500 and 3,450 cm, which was attributed to the stretching vibration of the oh group and intermolecular hydrogen bonding (figure 4). another band at 3,0002,950 cm represents the alkenes and aromatic c - h stretching, mainly = c - h. the peak at 2,900 cm was assigned to the c - h stretching vibration of the cyclopropyl group. the 1,9501,450 cm region exhibited ftir absorption from a wide variety of double - bonded functional groups. the peak at 1,6501,600 cm was assigned to quinolones. the band at the 1,4501,400 cm represents c - o and that at 1,3001,250 cm suggests the bending vibration of the o - h group, proving the presence of carboxylic acid. the ftir spectrum of the nanocomposite film shows all the characteristic peaks of hap and ciprofloxacin along with those of pcl 2,972, 2,868, and 1,723 cm, which were assigned to the vibration of the ch2 and c = o bonds, respectively. a jem2100 microscope operating at 100 kv is employed to study the morphologies of the prepared samples. the sample for tem was prepared by dispersing a small amount of the sample in ethanol and sonication for 30 minutes. the results from energy - dispersive x - ray spectroscopy (edax)-coupled tem show that the obtained samples had stoichiometric hap and exhibited characteristic nanorods and spherical nanoparticles. the tem image in figure 6a demonstrates that the hap was in the submicron to nanosize range, with all particles in the longest dimensions of 50100 nm. the larger crystals may have been formed due to the lower number of crystal nucleation sites because of the rapid addition of ca in po. the edax results (figure 6b) confirm the existence of the elements such as ca, p, and o. the atomic ratio was in agreement with that of calcium - rich hap. in vitro degradation study was carried out for pure pcl film and the ciprofloxacin - loaded hap - pcl film for 7 days. figure 7a shows the weight loss (in grams) versus immersion time (hours) of both films. the pure pcl film had quite a low dissolution rate after an initial sharp weight loss. the ciprofloxacin - loaded hap - pcl nanocomposite film had a large weight loss due to its fast drug release. in the beginning, so, due to the absorbed water, we had negative weight loss in the beginning. figure 7b shows the water absorption capacity of the films with and without the addition of hap and ciprofloxacin. for the drug - loaded hap - pcl nanocomposite film, the water absorption showed an initial sharp increase within a short period (12 hours), then a maximum and subsequent drop, and further slowing of the rate. for pure pcl, after a initial sharp increase, a steady and gradual increase with time was observed. the composite film had higher water absorption than the pure pcl, and increasing the hap content increased the water absorption for all periods. the absorbance values of the drug - loaded composite film placed in the dialysis membrane was measured from filtrate dilutions of pbs. the maximum wavelength (max), obtained by scanning all samples from 200 to 400 nm, was found to be 272 nm. the drug release was studied for a prolonged period and the absorbance value was found to be constant. figure 8 shows the drug - release kinetics, which were burst in the initial period and sustained later. the same procedure was repeated at 37c and the results showed a large amount of drug was released compared with the amount of drug released at room temperature. the control cells (figure 9) grew on the surface of a cultivation dish, and during 24 hours of incubating, a monolayer was formed. figure 10 shows the significant morphology and cell number changes that occurred within 96 hours of the extracts influence. the effects of the composite on nih-3t3 cell viability measured by mtt assay is shown in figure 11. mtt is a yellow, water - soluble tetrazolium dye, which is reduced by live cells to a purple formazan product insoluble in aqueous solutions. the cell viability of the prepared ciprofloxacin - loaded hap - pcl nanocomposite film was measured with the help of fibroblast cells. this implies that the test materials did not show any detectable toxic zone around or under the specimen. the cell proliferation rate depends on various chemical and physical aspects of a material, such as composition, roughness, porosity, morphology, and surface energy. the maximum cell density (110 cells in an area of 6.3 cm) thus, from the cytotoxicity results it can be said that the hap - pcl composite film is less cytotoxic than pure pcl film. this implies that the presence of a more bioactive / resorbable phase lowers the cytotoxicity. from figure 12, it can be seen that the differentiation rate of mg-63 cells on the composite film was much higher than on pure pcl film, indicating that the composite film could accelerate the differentiation of mg-63 cells. figure 3 shows the xrd pattern of hap recorded using a siemens d5000 diffractometer (siemens ag, berlin and munich, germany) with cuk radiation (=0.154 nm). the diffractometer was operated at 40 kv and 30 ma at a 2 theta range of 490 employing a step size of 0.1 and a 0.60-second exposure. the peaks of hap appearing at 26.2, 32.2, 49.6, and 64.0 (2) correspond to the 002, 211, 123, and 304 reflection planes. the xrd pattern of the hap powder obtained is in good in agreement with the xrd pattern of a hap standard available from the joint committee on powder diffraction standards (jcpds ; standard number 84 - 1998). the xrd pattern possesses a strong peak at around 32.2 corresponding to the 211 plane of hap s crystalline structure. no other characteristic peaks corresponding to other calcium phosphate phases or impurities were observed. from the peaks of the xrd diffraction pattern, the prepared hap samples are in hexagonal space group. the structure of the ciprofloxacin loaded hap - pcl nano - composite film was analyzed using ftir spectroscopy, as shown in figure 4. the first indication of the formation of hap is the strong, complex, broad ftir band centered around 1,0001,100 cm due to the asymmetric stretching mode of vibration of the po4 group. as a major peak of the phosphate group, the n3 vibration peak could be identified in the region between 1,100 and 960 cm for both powders, which was due to the asymmetric stretching of p - o in po 34. the presence of the peak in the region 1,4001,450 cm is due to absorbed carbon dioxide. the crystalline powder generated two characteristic stretching modes of o - h bands at about 3,497 cm which are noticed in all ftir spectra of hap. for ciprofloxacin, one prominent characteristic peak was found between 3,500 and 3,450 cm, which was attributed to the stretching vibration of the oh group and intermolecular hydrogen bonding (figure 4). another band at 3,0002,950 cm represents the alkenes and aromatic c - h stretching, mainly = c - h. the peak at 2,900 cm was assigned to the c - h stretching vibration of the cyclopropyl group. the 1,9501,450 cm region exhibited ftir absorption from a wide variety of double - bonded functional groups. the peak at 1,6501,600 cm was assigned to quinolones. the band at the 1,4501,400 cm represents c - o and that at 1,3001,250 cm suggests the bending vibration of the o - h group, proving the presence of carboxylic acid. the ftir spectrum of the nanocomposite film shows all the characteristic peaks of hap and ciprofloxacin along with those of pcl 2,972, 2,868, and 1,723 cm, which were assigned to the vibration of the ch2 and c = o bonds, respectively. a jem2100 microscope operating at 100 kv is employed to study the morphologies of the prepared samples. the sample for tem was prepared by dispersing a small amount of the sample in ethanol and sonication for 30 minutes. the results from energy - dispersive x - ray spectroscopy (edax)-coupled tem show that the obtained samples had stoichiometric hap and exhibited characteristic nanorods and spherical nanoparticles. the tem image in figure 6a demonstrates that the hap was in the submicron to nanosize range, with all particles in the longest dimensions of 50100 nm. the larger crystals may have been formed due to the lower number of crystal nucleation sites because of the rapid addition of ca in po. the edax results (figure 6b) confirm the existence of the elements such as ca, p, and o. the atomic ratio was in agreement with that of calcium - rich hap. in vitro degradation study was carried out for pure pcl film and the ciprofloxacin - loaded hap - pcl film for 7 days. figure 7a shows the weight loss (in grams) versus immersion time (hours) of both films. the pure pcl film had quite a low dissolution rate after an initial sharp weight loss. the ciprofloxacin - loaded hap - pcl nanocomposite film had a large weight loss due to its fast drug release. in the beginning, so, due to the absorbed water, we had negative weight loss in the beginning. figure 7b shows the water absorption capacity of the films with and without the addition of hap and ciprofloxacin. for the drug - loaded hap - pcl nanocomposite film, the water absorption showed an initial sharp increase within a short period (12 hours), then a maximum and subsequent drop, and further slowing of the rate. for pure pcl, after a initial sharp increase, a steady and gradual increase with time was observed. the composite film had higher water absorption than the pure pcl, and increasing the hap content increased the water absorption for all periods. the absorbance values of the drug - loaded composite film placed in the dialysis membrane was measured from filtrate dilutions of pbs. the maximum wavelength (max), obtained by scanning all samples from 200 to 400 nm, the drug release was studied for a prolonged period and the absorbance value was found to be constant. figure 8 shows the drug - release kinetics, which were burst in the initial period and sustained later. the same procedure was repeated at 37c and the results showed a large amount of drug was released compared with the amount of drug released at room temperature. the control cells (figure 9) grew on the surface of a cultivation dish, and during 24 hours of incubating, a monolayer was formed. figure 10 shows the significant morphology and cell number changes that occurred within 96 hours of the extracts influence. the effects of the composite on nih-3t3 cell viability measured by mtt assay is shown in figure 11. mtt is a yellow, water - soluble tetrazolium dye, which is reduced by live cells to a purple formazan product insoluble in aqueous solutions. the cell viability of the prepared ciprofloxacin - loaded hap - pcl nanocomposite film was measured with the help of fibroblast cells. this implies that the test materials did not show any detectable toxic zone around or under the specimen. the cell proliferation rate depends on various chemical and physical aspects of a material, such as composition, roughness, porosity, morphology, and surface energy. the maximum cell density (110 cells in an area of 6.3 cm) was observed after 24 hours of culture. thus, from the cytotoxicity results it can be said that the hap - pcl composite film is less cytotoxic than pure pcl film. this implies that the presence of a more bioactive / resorbable phase lowers the cytotoxicity. from figure 12, it can be seen that the differentiation rate of mg-63 cells on the composite film was much higher than on pure pcl film, indicating that the composite film could accelerate the differentiation of mg-63 cells. a key issue is that it is very complex replacing damaged bone, as fast as possible, with a new mature bone. in this work, an organic inorganic composite material suitable for bone - tissue engineering based on hap and pcl was studied. the nanoscale hap obtained from eggshell had good agreement with natural hap. the nanoscale hap and pcl were blended together in an organic solvent to form a composite film. furthermore, the in vitro tests revealed that the processed scaffold has the potential for use in drug - delivery systems. the biodegradation and cytotoxicity studies prove that the prepared ciprofloxacin - loaded hap - pcl nanocomposite film would be suitable for sustainable bone implants, as it had good interaction with the natural tissue and cells and degradation of the polymer helps with drug release and in avoiding a second surgery.	introductionin recent years there has been a steep increase in the number of orthopedic patients for many reasons. one major reason is osteomyelitis, caused by pyrogenic bacteria, with progressive infection of the bone or bone marrow and surrounding tissues. so antibiotics must be introduced during bone implantation to avoid prolonged infection.aimthe objective of the study reported here was to prepare a composite film of nanocrystalline hydroxyapatite (hap) and polycaprolactone (pcl) polymer loaded with ciprofloxacin, a frequently used antibiotic agent for bone infections.methodsnanocrystalline hap was synthesized by precipitation method using the precursor obtained from eggshell. the nanocomposite film (hap - pcl - ciprofloxacin) was prepared by solvent evaporation. drug - release and biodegradation studies were undertaken by immersing the composite film in phosphate - buffered saline solution, while a cytotoxicity test was performed using the fibroblast cell line nih-3t3 and osteoblast cell line mg-63.resultsthe pure pcl film had quite a low dissolution rate after an initial sharp weight loss, whereas the ciprofloxacin - loaded hap - pcl nanocomposite film had a large weight loss due to its fast drug release. the composite film had higher water absorption than the pure pcl, and increasing the concentration of the hap increased the water absorption. the in vitro cell - line study showed a good biocompatibility and bioactivity of the developed nanocomposite film.conclusionthe prepared film will act as a sustainable bone implant in addition to controlled drug delivery.
about 178,000 people develop cancer annually, with an age - standardized rate of 321.3 per 100 thousand in 2009. this cancer incidence rate is less than the 335.0 per 100 thousand rate in the us (2008), but higher than the 280.0 per 100 thousand in the uk (2008) and 247.3 per 100 thousand in japan (2008). cancer patients ' 5-year survival rate between 2005 and 2009 was 62.0%, which showing how far the state of the art has advanced globally. the five leading primary cancer sites in 2009 were stomach, colon, lung, liver, and prostate among males, whereas the most common cancer sites among females were thyroid, breast, colon, stomach, and lung. according to recent research findings, infection and smoking are the most important contributing factors in korea. the smoking rate in korea is still high, 46.9% in men and 7.1% in women in 2009. the fractions of all cancers attributable to infection were 25.1% and 16.8% for cancer incidence in men and women, and 25.8% and 22.7% of cancer mortality in men and women, respectively. over 97% of infection - related cancers, such as stomach cancer and liver cancer, were attributable to infection with h. pylori, hepatitis b virus (hbv), hepatitis c virus and human papilloma virus. occupation is also an important attributing factor at 7.8% [5 - 7 ], compared with france, which reported 2.4% as the attributable fraction (af) of occupation to cancer. drinking alcohol, reproductive factors, obesity, and physical inactivity are minor factors, as each is less than 2%. afs of radiation and environmental pollution were not calculated because of insufficient data in korea. but there are reliable data supporting the considerable environmental burden of diseases, including cancer. the world health organization (who) reported the surprising news that korea ranked in 50th place in the world in the burden of disease for environmental risk in 2007. by another method, yoon suggested that korea ranked in 25th place in the world in the burden of disease for environmental risk in 2007. the prevalence of people exposed to environmental tobacco smoke (ets) in korea is still high, at 44.9% in males and 34.2% in females in 2009. many people have been exposed to asbestos in the vicinity of asbestos textile industries and mines in korea, even though the korean government completely prohibited the use of asbestos in 2009. especially near highways and high traffic areas, people are exposed to traffic - related air pollutants, including diesel exhaust particles (dep). dep was classified as a definite human carcinogen (group 1) by the international agency for research on cancer (iarc) last year based on sufficient epidemiological evidence [13 - 15 ]. taking environmental carcinogens, such as ets, asbestos, and dep, into consideration, afs due to environmental exposure in korea may be higher than 0.2%, which was the estimated af% in france. recently in korea, some issues related to carcinogen exposure have received much media attention, i.e., the first compensation case of breast cancer in a laborer exposed to shift work, radiation and organic solvents, the announcement of iarc 's decision on the carcinogenicity of radiofrequency radiation exposure by mobile phone use and dep, as well as the fukushima nuclear power plant accident in a neighboring country. with its rapidly aging population, reducing the cancer burden at the national level has become one of the major public health issues in korea. the government formulated its first 10-year plan for cancer control in 1996. in 2000, the national cancer center was established and the cancer control division was set up within the ministry of health and welfare. korea 's major national cancer control programs included anti - smoking campaigns, hbv vaccination, cancer registration and networking, reinforcement of research and development activities for cancer control, education and training for cancer control and prevention, management of the national cancer information center, a mass screening program for five common cancers, caring for cancer patients at home, financial support for cancer patients and designation of regional cancer centers. the second 10-year plan for cancer control was initiated in 2006 to make up for flaws in the first plan and establish an efficient 10-year national cancer plan for a cancer control management system. the goals for the second plan are as follows : intensifying cancer prevention by stricter management of cancer risk factors, early screening of all people for cancer, extension of support for cancer patients, reinforcing security for cancer patients, strengthening support for rehabilitation and palliative medicine for cancer patients, establishment of infrastructure for active national cancer management, development of diagnostic and therapeutic technology, enforcement of education and public relations to make it touching to people, and a systemic cancer registry and management. the second 10-year plan for cancer control provides a framework to prepare a great shift in cancer policy from primarily treatment oriented to precautionary health promotion. in spite of the national cancer control plans, the increasing number of cancer patients in korea poses devastating social and economic consequences for households, communities, and countries as well. therefore at this point, we need to steer the main direction of cancer control policy toward carcinogen management and primary prevention of cancer. it is urgently needed to define the carcinogenicity of suspect substances and monitor their exposures. the korean toxic chemicals control act (tcca) was implemented in 1991 by the ministry of environment (moe) for the general management and control of industrial chemicals in korea. the purpose of this act is to prevent any risk caused by chemicals to human health. the national institute of environmental research is responsible for new chemical notifications under the act. the korea chemicals management association of moe is responsible for accepting declarations on details of other chemicals and applications for confirmation certificates. for example, radioactive substances prescribed by the atomic safety act ; medicines, non - pharmaceutical drugs, and cosmetics by the pharmaceutical affairs act ; technical ingredients and agrochemicals by the agrochemicals control act ; fertilizers by the fertilizer control act ; foods and food additives by the food sanitation act ; explosives by the control of firearms, swords, explosives, etc. carcinogens are prescribed by many authorities, such as the ministry of health and welfare, moe, ministry of labor, ministry of science and technology and ministry of agriculture, according to their properties and legislation in korea (table 1). the tcca covers new chemicals, toxic chemicals, observational chemicals and restricted or banned chemicals. but internationally, the who resolution on cancer control (wha58.22) provides a strong impetus for countries to develop programs aimed at reducing cancer incidence and mortality. the iarc was established in may, 1965 through a resolution of the xviiith world health assembly as an extension of the who. europe and the us have carried out the task of defining carcinogens and classifying them with criteria. hazard information including usages and amount in circulations have been received from companies for all chemicals with more than 1 ton in circulation in the registration, evaluation, authorization and restriction of chemicals (reach), eu. based on lists of dangerous substances, assessments of carcinogens and rankings are being done in the eu [24 - 26 ]. the us national toxicology program (ntp) determines carcinogens based on authoritative data from the iarc combined with its own animal experimentation data. the ntp prepares the report on carcinogens (rog) on behalf of the secretary, health and human services. the 12th roc, the latest edition, was published on june 10, 2011. the roc is a congressionally mandated, science - based, public health report that identifies agents, substances, mixtures, or exposures (collectively called " substances ") in our environment that may potentially put people in the us at increased risk for cancer. the ntp 's report is limited because it evaluates few agents and does not distinguish between probable and possible human carcinogens on its b - list [27 - 29 ]. many countries are making international collaborative networks with the iarc and who to share their carcinogen lists (table 2) [30 - 33 ]. the korean government is also trying to classify carcinogens by introducing a globally harmonized system of classification and labeling of chemicals (ghs). the moe and labor has provided information about ghs material safety data sheets (msds) on 43,000 chemicals since 2009. the moe is trying to build a chemical registration and assessment system based on ghs and the iarc 's carcinogen list. however, there are no integrated regulatory laws and management systems guarding against carcinogen exposure in korea. many separate laws have been put into effect to manage carcinogen exposure (table 1). yet some carcinogens are still not regulated by the toxic chemicals control law in korea (table 3). in these circumstances, it is inevitable there is a chance of exposure to some definite and probable human carcinogens in certain workers and citizens. there remain several dead zones where carcinogen exposures can not be controlled properly in korea. for example, crystalline silica, controlled by the industrial safety and health act, is a hazardous substance for which occupational exposure limits are established. yet even though crystalline silica is a definite human carcinogen, there is no regulation by the tcca. besides crystalline silica, certain other definite human carcinogens, including 1,3-butadiene and beryllium, are not regulated by the tcca in korea (table 3). korean governmental bodies need to establish a national action plan on management of carcinogenic risk in korea. the importance of primary prevention has been recognized, but it has not been actively carried out because of limited budgets and difficulties in proving its effects in a short period of time. current cancer control programs are focused on management of existing cancers. under this system, all natural cycles, including etiologies, exposures, diseases, and aftereffects were not taken into comprehensive consideration. effective cancer prevention strategies were not established in the current system. a national carcinogen list in korea should be established soon and should be based on the ghs. to deal with some carcinogens in our environment that may potentially increase risk for cancer, we can start our own ntp, an interagency program to evaluate agents of public health concern by developing and applying tools of modern toxicology and molecular biology. this list may include three categories of substances and processes regarded as carcinogenic, primarily by iarc, and to a lesser extent, the government 's annual report on carcinogens from an ntp - like program. if we can have national carcinogen lists, the korean government could reach the first milestone on the way toward its final goal of primary prevention of cancer.	cancer has been the leading cause of death in korea for the last 30 years. cancer patients ' 5-year survival rate between 2005 and 2009 was 62.0%, representing a highly advanced standard of care, as much as developed countries in the eu and the us. the korean government formulated its first 10-year plan for cancer control in 1996 and has been carrying out a second 10-year plan for cancer control since 2006. but despite the korean government 's efforts, the cancer burden in korea continues to increase. many separate laws have gone into effect concerning the management of carcinogen exposure. however, there are no integrated regulatory laws or management systems against carcinogen exposure in korea. dead zones remain where carcinogen exposure can not be controlled properly in korea. in this paper, we suggest the need to establish a national carcinogen list based on international harmonization as a prerequisite for a paradigm shift in cancer control policy from treatment to primary prevention.
a 64-year - old healthy man underwent phacoemulsification with intraocular lens implant on the left eye. preoperative fundus examination was normal and his best - corrected visual acuity in the left eye was 6/24 because of nuclear sclerosis. the patient was administered a single uneventful peribulbar injection consisting of 5 ml of lidocaine 2% and epinephrine 1:200,000 using a 25-gauge 25 mm peribulbar needle through the temporal portion of the inferior lid. the patient had no complaints and his visual acuity had improved to 6/6. however, on examination, a left afferent pupillary defect was noted. dilated ophthalmoscopy of the left eye revealed several large confluent peripapillary patches of retinal whitening mimicking soft exudates (purtscher flecken) [fig. fundus fluorescein angiography revealed multiple hypofluroscent areas in the peripapillary region corresponding to the areas of the soft exudates [fig. 2 ]. swept - source optical coherence tomography of the left eye revealed edema of the inner retinal layers with hyperreflectivity of the nerve fiber layers corresponding to the areas of retinal whitening [fig. 3 ]. as the patient was symptomless, no treatment was initiated for the treatment of the retinopathy. the patient underwent a comprehensive medical screening including liver function tests, pancreatic enzyme assay, carotid doppler, and transthoracic echocardiography to rule out other causes of purtscher - like retinopathy. subsequent follow - ups at 2 and 4 weeks revealed gradual disappearance of the cotton - wool spots [fig. 1b and c ], resolution of the peripapillary scotoma and decrease in the afferent papillary defect. (c) follow - up at 4 weeks fundus fluorescein angiography of the left eye (postoperative day 4) revealed blocked fluorescence in the sites corresponding to the retinal and preretinal hemorrhages, capillary dropout in the areas of purtscher flecken and cotton - wool spots optical coherence tomography of the left eye (postoperative day 4) revealed edema of the inner retinal layers with hyperreflectivity of the nerve fiber layers corresponding to the areas of retinal whitening unilateral purtscher 's retinopathy has been reported following facial trauma, periorbital steroid injection, and retrobulbar anesthesia. there have been two previous case reports of purtscher - like retinopathy after retrobulbar anesthesia. to the best of our knowledge, peribulbar anesthesia is known to be safer, but it has still shown to have the tendency to cause potentially dangerous complications including central retinal artery occlusion through the remote effects of the anesthetic agent, amount injected, speed of injection, and postinjection mechanical compression. the pathogenesis of purtscher 's retinopathy is still a subject of debate with several theories being forwarded since the initial mechanical theory proposed by purtscher. the most currently accepted theory is microembolization, causing arteriolar precapillary occlusion and microvascular infarct of retinal nerve fiber layer, forming cotton - wool spots. although bilateral purtscher - like retinopathy has been linked to the activation of complement and formation of leukoemboli, it is difficult to correlate this systemic mechanism to a local event. in this unilateral case, which was triggered by a local event of peribulbar anesthetic injection, the peripapillary distribution of the cotton - wool spots suggests an infarction of the peripapillary arterioles in an area of no collaterals. it could be hypothesized that the sudden increase in orbital volume might have increased the hydrostatic pressure and reproduced the conditions of a valsalva maneuver. however, this theory is challenged by the fact that considering the significantly larger volume of the extraconal space in comparison to intraconal space, 5 ml of anesthetic agent is unlikely to produce significant hydrostatic pressure to produce vascular occlusion. another possibility is that the infarction might have been caused by the accidental embolization of the central retinal artery or a posterior ciliary artery by either some residual air bubbles in the syringe or an orbital fat embolus mobilized by the needle. most likely, the infarction may have been caused by the vasospastic effects of adrenaline accentuated by the intrinsic vasoconstrictive properties of lidocaine. reported a decrease in retinal blood flow velocity by 1015%, 15 min, respectively, following peribulbar anesthesia without a vasoconstrictive agent like adrenaline. we report this case to inform ophthalmic surgeons and anesthetists that although peribulbar anesthesia avoids direct optic - nerve injury, indirect injury may occur from vasospasm in response to the injection.	purtscher and purtscher - like retinopathy is a distinctive retinal syndrome characterized by ischemic retinal whitening in a peripapillary pattern. we report a case of purtscher - like retinopathy in a healthy 64-year - old man after a routine peribulbar anesthetic injection for cataract surgery. although peribulbar anesthesia is considered to be a safer alternative to retrobulbar anesthesia, it has been associated with unusual but grave complications including central retinal artery occlusion.
yttrium-90 (y) microsphere radioembolization is an emerging treatment for metastatic hepatic malignancies. in this procedure, 2060 micron - sized -radiation - emitting particles are administered to liver tumors using a transcatheter approach to deliver high - dose localized internal radiation. this therapy takes advantage of the predominant hepatic arterial flow to liver tumors ; cancerous tissue derives 80100% of its blood supply from the hepatic arterial system, while normal liver gains 7580% of its perfusion from the portal vein. radioactive microspheres delivered from a catheter in the hepatic artery are preferentially deposited in malignant tissue at a 3:1 to 20:1 ratio compared to normal liver, and result in high tumor response rates and extended survival. while the use of y radioembolization for treatment of hepatic metastases from colorectal, neuroendocrine, and breast cancer has been broadly investigated, application of this therapy toward management of lung cancer hepatic metastases has not been extensively documented. herein, we present 2 cases of effective y radioembolization for treatment of chemorefractory lung cancer hepatic metastases, and highlight the potential coadjuvant value of this procedure in patients with stage 4 lung cancer and liver - dominant metastatic disease. institutional review board approval is not required for small retrospective case studies at the authors hospital. a 68-year - old woman with a 50-pack - year smoking history presented with a complaint of dyspnea and cough. she underwent a chest radiograph followed by computed tomography (ct), which revealed a 4.8 2.8 cm mass in the upper lobe of the left lung (fig. a bronchoscopy was performed 3 months after presentation for tissue diagnosis, but both bronchoalveolar lavage and transbronchial needle aspirate were negative for malignancy. the patient was then referred to interventional radiology (ir) for ct - guided transthoracic biopsy, which yielded a diagnosis of well - differentiated squamous cell carcinoma. work - up with whole - body positron emission tomography (pet) and brain magnetic resonance imaging demonstrated stage 3b (t4n2m0) disease. as such, the patient was not a candidate for surgical resection, and was placed on chemoradiation treatment. she was given cisplatin and etoposide for 1 cycle, and was treated with a 66-gy radiation dose using 2-field 3-dimensional conformal radiotherapy. the patient was subsequently followed with ct imaging ; a scan performed 2 months after therapy showed size reduction of the primary tumor to 4.6 2.2 cm, but also demonstrated a new 2.0 1.9 cm lesion in the right hepatic lobe suspicious for metastasis. she was started on second - line chemotherapy 2 months later with carboplatin auc6 and paclitaxel. follow - up ct 4 months later showed smaller size of the lung mass (fig. 1a), but increased size of the hepatic metastasis to 4.3 4.3 cm (fig. the patient was therefore started on third - line chemotherapy with gemcitabine for 4 cycles. ct scan 2 months after completing chemotherapy showed no decrease in the size of the hepatic metastasis and revealed a new 0.9 cm satellite lesion. at this point, the patient was referred to ir for transcatheter treatment of her lung cancer hepatic metastases. given good baseline performance status, with eastern cooperative oncology group (ecog) status 1, as well as preserved liver function, the patient was a good candidate for y microsphere therapy. in preparation for treatment, m macroaggregated albumin (tc - maa) scan for lung shunt fraction (lsf) calculation. 1c), and the hepatic vascular anatomy was found to be amenable for y therapy. 5.4 mci of tc - maa was administered from the right hepatic artery for lsf analysis. finally, the femoral vascular access was removed and hemostasis was accomplished by manual compression. tc - maa scan demonstrated a hepatopulmonary shunt fraction of 17.7%, below the 20% threshold for safe treatment. the radioembolization procedure was scheduled for 1 week later. on the day of the treatment procedure, an approximately 120-gy radiation dose was administered via injection of 3 gbq of y - labeled glass microspheres (therasphere ; mds nordion, ottawa, ont., canada). completion angiography was performed, all vascular devices were removed, and access site hemostasis was achieved by manual compression. after an uneventful post - procedure recovery, the patient was seen 1 month later. ct scan at that time demonstrated complete necrosis of the right hepatic lesion (fig. 1d), and pet ct scan performed 2 months after treatment showed no evidence for residual viable tumor (fig. at present, the patient is alive with persistent complete response in the treated liver lesion 28 months after initial diagnosis, 21 months after progression to stage 4 disease, and 11 months after y therapy. of note, she has since undergone 2 additional successful hepatic radioembolization procedures to treat new small metastases that developed since the time of initial liver therapy. a 53-year - old woman with a history of stage 1b (t2n0m0) left lung squamous cell carcinoma status post lobectomy and adjuvant chemotherapy with carboplatin and paclitaxel 2 years prior presented for evaluation of a solid mass on the left tongue undersurface. biopsy demonstrated poorly differentiated squamous cell carcinoma, and the patient underwent wide local excision of the floor of mouth and left tongue, bilateral neck dissection, and flap reconstruction. one month after surgery, the patient was placed on cisplatin chemoradiation therapy during which time she received a total dose of 66 gy. upon completion of adjuvant therapy, she subsequently underwent pet ct imaging surveillance, and 11 months later was found to have a new 2.8 2.4 cm left hepatic lobe mass. ultrasound - guided biopsy was positive for metastatic squamous cell carcinoma consistent with the patient 's lung primary tumor. the patient was placed on a second round of chemotherapy using cisplatin, 5-fluorouracil, and cetuximab. she was also referred to ir for concomitant transcatheter treatment of her lung cancer hepatic metastasis ; given good performance status (ecog 0) and preserved liver function, y radioembolization was selected. the patient underwent mapping arteriogram in preparation for y radioembolization, performed from a conventional femoral artery approach. left hepatic artery arteriogram demonstrated a hypervascular left hepatic lobe tumor (fig. 2b), and the hepatic vascular anatomy was found to be amenable for y radioembolization therapy. 5.4 mci of tc - maa was then administered from the proper hepatic artery for lsf analysis. finally, the femoral vascular access was removed and hemostasis was accomplished by manual compression. the radioembolization procedure was scheduled for 1 month later. on the day of the treatment procedure, an approximately 120-gy radiation dose was administered via injection of 1.57 gbq of y - labeled glass microspheres (therasphere ; mds nordion). completion angiography was performed, all vascular devices were removed, and access site hemostasis was achieved by manual compression. following y administration, correct tumor targeting the patient experienced an uneventful post - procedure recovery and is currently continuing with her 6-cycle chemotherapy regimen. pet ct performed 2 months after y treatment showed complete tumor response without evidence for residual tumor (fig. at present, the patient is alive without active disease 16 months after initial diagnosis, 4 months after progression to stage 4 disease, and 2 months after y radioembolization therapy. a 68-year - old woman with a 50-pack - year smoking history presented with a complaint of dyspnea and cough. she underwent a chest radiograph followed by computed tomography (ct), which revealed a 4.8 2.8 cm mass in the upper lobe of the left lung (fig. a bronchoscopy was performed 3 months after presentation for tissue diagnosis, but both bronchoalveolar lavage and transbronchial needle aspirate were negative for malignancy. the patient was then referred to interventional radiology (ir) for ct - guided transthoracic biopsy, which yielded a diagnosis of well - differentiated squamous cell carcinoma. work - up with whole - body positron emission tomography (pet) and brain magnetic resonance imaging demonstrated stage 3b (t4n2m0) disease. as such, the patient was not a candidate for surgical resection, and was placed on chemoradiation treatment. she was given cisplatin and etoposide for 1 cycle, and was treated with a 66-gy radiation dose using 2-field 3-dimensional conformal radiotherapy. the patient was subsequently followed with ct imaging ; a scan performed 2 months after therapy showed size reduction of the primary tumor to 4.6 2.2 cm, but also demonstrated a new 2.0 1.9 cm lesion in the right hepatic lobe suspicious for metastasis. she was started on second - line chemotherapy 2 months later with carboplatin auc6 and paclitaxel. follow - up ct 4 months later showed smaller size of the lung mass (fig. 1a), but increased size of the hepatic metastasis to 4.3 4.3 cm (fig. the patient was therefore started on third - line chemotherapy with gemcitabine for 4 cycles. ct scan 2 months after completing chemotherapy showed no decrease in the size of the hepatic metastasis and revealed a new 0.9 cm satellite lesion. at this point, the patient was referred to ir for transcatheter treatment of her lung cancer hepatic metastases. given good baseline performance status, with eastern cooperative oncology group (ecog) status 1, as well as preserved liver function, the patient was a good candidate for y microsphere therapy. in preparation for treatment, m macroaggregated albumin (tc - maa) scan for lung shunt fraction (lsf) calculation. 1c), and the hepatic vascular anatomy was found to be amenable for y therapy. 5.4 mci of tc - maa was administered from the right hepatic artery for lsf analysis. finally, the femoral vascular access was removed and hemostasis was accomplished by manual compression. tc - maa scan demonstrated a hepatopulmonary shunt fraction of 17.7%, below the 20% threshold for safe treatment. on the day of the treatment procedure, a 5-french catheter and coaxial 3-french microcatheter were advanced into the right hepatic artery. an approximately 120-gy radiation dose was administered via injection of 3 gbq of y - labeled glass microspheres (therasphere ; mds nordion, ottawa, ont., completion angiography was performed, all vascular devices were removed, and access site hemostasis was achieved by manual compression. after an uneventful post - procedure recovery, the patient was seen 1 month later. ct scan at that time demonstrated complete necrosis of the right hepatic lesion (fig. 1d), and pet ct scan performed 2 months after treatment showed no evidence for residual viable tumor (fig at present, the patient is alive with persistent complete response in the treated liver lesion 28 months after initial diagnosis, 21 months after progression to stage 4 disease, and 11 months after y therapy. of note, she has since undergone 2 additional successful hepatic radioembolization procedures to treat new small metastases that developed since the time of initial liver therapy. a 53-year - old woman with a history of stage 1b (t2n0m0) left lung squamous cell carcinoma status post lobectomy and adjuvant chemotherapy with carboplatin and paclitaxel 2 years prior presented for evaluation of a solid mass on the left tongue undersurface. biopsy demonstrated poorly differentiated squamous cell carcinoma, and the patient underwent wide local excision of the floor of mouth and left tongue, bilateral neck dissection, and flap reconstruction. one month after surgery, the patient was placed on cisplatin chemoradiation therapy during which time she received a total dose of 66 gy. upon completion of adjuvant therapy, she subsequently underwent pet ct imaging surveillance, and 11 months later was found to have a new 2.8 2.4 cm left hepatic lobe mass. ultrasound - guided biopsy was positive for metastatic squamous cell carcinoma consistent with the patient 's lung primary tumor. the patient was placed on a second round of chemotherapy using cisplatin, 5-fluorouracil, and cetuximab. she was also referred to ir for concomitant transcatheter treatment of her lung cancer hepatic metastasis ; given good performance status (ecog 0) and preserved liver function, y radioembolization was selected. the patient underwent mapping arteriogram in preparation for y radioembolization, performed from a conventional femoral artery approach. left hepatic artery arteriogram demonstrated a hypervascular left hepatic lobe tumor (fig. 2b), and the hepatic vascular anatomy was found to be amenable for y radioembolization therapy. 5.4 mci of tc - maa was then administered from the proper hepatic artery for lsf analysis. finally, the femoral vascular access was removed and hemostasis was accomplished by manual compression. the radioembolization procedure was scheduled for 1 month later. on the day of the treatment procedure, a 5-french catheter and coaxial 3-french microcatheter were advanced into the left hepatic artery. an approximately 120-gy radiation dose was administered via injection of 1.57 gbq of y - labeled glass microspheres (therasphere ; mds nordion). completion angiography was performed, all vascular devices were removed, and access site hemostasis was achieved by manual compression. following y administration, correct tumor targeting was confirmed by bremsstrahlung imaging (fig. the patient experienced an uneventful post - procedure recovery and is currently continuing with her 6-cycle chemotherapy regimen. pet ct performed 2 months after y treatment showed complete tumor response without evidence for residual tumor (fig. at present, the patient is alive without active disease 16 months after initial diagnosis, 4 months after progression to stage 4 disease, and 2 months after y radioembolization therapy. lung cancer is the most common malignancy worldwide and a significant cause of global morbidity and mortality. an estimated 1,600,000 new cases and 1,380,000 deaths occurred internationally in 2008, and a projected 226,000 new cases and 160,000 deaths are expected in the united states in 2012. liver metastases complicate approximately 6% of lung cancer cases at the time of initial diagnosis, and hepatic tumor spread occurs in up to 36% of patients over the course of their disease. 10% 5-year survival rates, and is most commonly treated with palliative systemic chemotherapy. new treatment approaches for this challenging disease stage are necessary to improve discouraging survival outcomes, and a role for targeted transcatheter therapy in the treatment of lung cancer liver metastases has been recently suggested in small clinical studies [9, 16 ]. radioembolization is a minimally invasive targeted therapy that selectively delivers internal -radiation via the arteries that supply tumors. radioembolization describes the mode of action of this procedure, in that embolic particles act as a vehicle for delivery and administration of radiation. y is a pure -emitting isotope with a half - life of 64.2 h, after which it decays into stable zirconium. the average energy of -emission is 0.9367 mev, and the mean tissue penetration is 2.5 mm. one gbq (27 mci) of y provides a 50-gy dose to 1 kg of liver tissue ; 120-gy tumoricidal doses are typically administered during treatment procedures. glass (therasphere ; mds nordion) and resin (sir - sphere ; sirtex medical, lane cove, n.s.w. because y radioembolization targets tumors through a transarterial route, a planning angiographic study is required to assess visceral anatomy, identify anatomic variants, isolate the hepatic circulation, and determine tumor location for appropriate catheter placement. both the planning and treatment procedures may be performed on an outpatient basis. in the present case series, we effectively devitalized 2 large lung cancer metastatic nodules using y radioembolization. in both cases, the liver nodules represented the dominant site of active disease and were refractory to systemic chemotherapy, showing enlargement or viability despite standard of care pharmacologic treatment. both patients showed no pet active disease in the index lesion at 10 and 2 months after treatment, indicating robust and durable complete response to therapy and significant tumor cytoreduction using the targeted treatment approach. while patients in each case were administered a treatment dose of 120 gy, tumor hypervascularity and preferential blood flow dictates that each lesion likely received a several thousand gy dose, far exceeding 120-gy tumoricidal levels, while normal liver parenchyma contracted low doses approximating 1020 gy, minimizing risk for radiation hepatitis. in this manner, internal y radiotherapy allows for higher cytotoxic radiation doses than traditional external beam therapy by making use of internal targeted delivery. to date, the application of y radioembolization therapy toward treatment of lung cancer hepatic metastases has been specifically described in one small clinical series. in 2008, murthy. reported on 6 patients with liver - dominant lung cancer spread who underwent y treatment. disease control was achieved in 3 of 6 (50%) patients, while 3 of 6 (50%) patients developed progressive disease. these suboptimal outcomes may be explained on the basis of advanced disease among treated patients, of which two - thirds had multifocal bilobar liver metastases at the time of y therapy. in contrast, our patients were treated earlier in the disease course in the setting of solitary liver metastases that could be specifically targeted with an extraordinarily high radiation dose. our encouraging outcomes suggest need for further investigations to assess the role of early y radioembolization in combination with systemic chemotherapy for lung cancer liver metastases ; this treatment approach has precedent in similar ongoing trials for cancers such as metastatic colorectal carcinoma. the effect of non - target pulmonary radiation is an important consideration in patients with lung cancer liver metastases who undergo y radioembolization, particularly those with a history of lobectomy or prior radiation, in order to avoid untoward effects on pulmonary function. current standard of care with y treatment requires performance of a tc - maa scan in order to calculate a lsf prior to radioembolization ; non - target pulmonary deposition of microspheres may occur due to systemic passage of particles via small intrahepatic peri - tumoral arteriovenous communications. while the incidence of radiation pneumonitis is well below 1% if standard dosimetry models are used [20, 21 ], single - session doses do not exceed 30 gy, and total doses do not surpass 50 gy, high lsfs resulting in greater lung doses may necessitate y dose reduction to avoid radiation pneumonitis. for comparison, patients receiving external beam radiation treatment for lung cancer are usually given approximately 60-gy doses, but the lung volume irradiated with > 20 gy is minimized to decrease risk of pneumonitis. in summary, we describe 2 cases of lung cancer liver metastases successfully treated with y radioembolization. in both cases, tumors were effectively devitalized and both patients showed excellent clinical outcome. while our clinical observations are made on the basis of a small sample size and should not be overstated, the promising results herein affirm the utility of y radioembolization for targeted treatment of liver metastases, and underscore the need for future investigation into such treatment strategies for advanced lung cancer.	because stage 4 lung cancer is associated with dismal 5-year survival rates, new treatment approaches targeting extrapulmonary disease are necessary. yttrium-90 microsphere radioembolization is an emerging treatment for metastatic hepatic malignancies that results in high tumor response rates and extended patient survival. to date, application of this therapy toward management of lung cancer hepatic metastases has not been extensively described. herein, we present 2 cases of effective yttrium-90 radioembolization for treatment of lung cancer hepatic metastases, and emphasize the potential coadjuvant value of this procedure in patients with advanced - stage lung cancer and liver - dominant metastatic disease.
older adults with abdominal pain have a six- to eight - fold increase in mortality compared to younger patients [1, 2 ]. intramural colonic hematomas manifest rarely as complications following blunt traumas or accompanying diseases with bleeding tendency. a 72-year - old male with cerebral palsy was admitted following development of symptomatic pulmonary embolism for the initiation of therapeutic anticoagulation including heparin drip and oral coumadin. prior to initiation of therapeutic anticoagulation, he had a computed tomography (ct) scan angiogram of his chest, abdomen and pelvis. a pulmonary embolism and large stool burden were noted on the ct scan and tap water enemas were initiated following the start of the heparin drip and coumadin initiation (fig. a complete blood count demonstrated an acute drop in hemoglobin from 12 to 6, partial thromboplastin time 100 and international normalized ratio was 3.5. the patient was transfused 2 units packed red blood cells, but his hemoglobin did not increase. on exam there was also some new mucoid blood drainage and abdominal tenderness with distension. a repeat ct scan angiogram demonstrated a 15 6 3 cm abdominal intramural hematoma (fig. given the increasing abdominal pain and concern for perforation as well as lack of response to blood products, an exploratory laparotomy was performed. at laparotomy a 30 15 cm abdominal recto - sigmoid hematoma was discovered distending the previously capacious bowel wall without perforation (fig. large clamps were used to transect the distal bowel at the recto - sigmoid junction and the area of transection was oversewn. it is likely that dilated colon allowed for a large amount of hematoma to accumulate. the patient recovered post - operatively and was discharged following return of bowel function. following the operation on postoperative day 1 coagulation was resumed without any additional issues. figure 1:ct abdomen and pelvis with intravenous contrast. admission imaging demonstrating large colonic stool burden in the axial (a), coronal (b) and sagittal orientation (c). ct abdomen and pelvis following enema demonstrating large colonic wall hematoma axial (d), coronal (e) and sagittal orientation (f). figure 2:intraoperative photograph of the large intramural hematoma of the sigmoid colon descending into the recto - sigmoid junction. admission imaging demonstrating large colonic stool burden in the axial (a), coronal (b) and sagittal orientation (c). ct abdomen and pelvis following enema demonstrating large colonic wall hematoma axial (d), coronal (e) and sagittal orientation (f). intraoperative photograph of the large intramural hematoma of the sigmoid colon descending into the recto - sigmoid junction. intramural hematomas of the alimentary tract are uncommon, occurring most frequently in the setting of blunt abdominal trauma (duodenum most common, colon least common), and in 1536% are spontaneous in patients with underlying hematologic disease or on anticoagulant therapy. the incidence of all bleeding complications during anticoagulation therapy ranges from 5 of 48%, but gastrointestinal hemorrhage occurs in only 2 to 4% of patients. small bowel hematoma has an incidence of 1 case per 2500 anticoagulated patients per year, while large bowel hematomas have an even lower incidence in the literature. gastrointestinal complications following anticoagulation can present with bleeding into the intestinal lumen, usually related to some underlying mucosal abnormality, retroperitoneal hemorrhage or bleeding into intra - abdominal organs ; less common are intramural hematomas. rectal hematoma and subsequent perforation have been described in patients following enemas and radiographic contrast enemas due to both instrumentation and hydrostatic pressure. one study demonstrated that the incidence of iatrogenic large bowel perforations ranges from 0.1 to 0.9% following colonoscopy and barium enemas. the differential diagnosis of abdominal pain for a patient on anticoagulation is broad including rectus sheath hematoma, retroperitoneal hematoma, intestinal / mesenteric hematoma, bleeding diverticula and rectal trauma from enema or foreign object. the use of ct as the first imaging modality in the evaluation of patients with acute abdominal complaints especially in the elderly population. ct characteristics of colonic hematoma include circumferential wall thickening, intramural hyper - density, luminal narrowing and intestinal obstruction. additional adjuncts for diagnosis consist of rigid proctoscopy which demonstrate colonic intramural hematomas as round shaped, dark reddish submucosal masses often obstructing the luminal space. for patients with intramural hematomas conservative management includes removing the anticoagulation therapy, correcting coagulopathy, intestinal decompression and avoiding oral intake while awaiting spontaneous resorption of the hematoma. the challenge is to distinguish patients with self - limiting intramural hematoma from those with more severe pathology. conventional physical exam signs are inadequate to distinguish more ill patients as even a simple hematoma may also present with high fevers, leukocytosis and rebound tenderness. if the intramural hematoma causes obstruction or if the cause of the bowel obstruction is unknown, surgical intervention for colonic hematoma is warranted. drainage of the hematoma may increase the risk of serious infection as it introduces bacteria to a previously sterile hematoma. a review of the literature demonstrates a shift in the management of colonic hematomas over the past 50 years. cases prior to 1969 were most commonly treated with surgical intervention. however, following 2010 they were managed more frequently conservatively. treatment of colonic hematomas depends on symptoms and clinical findings [11, 14 ]. overall bowel obstruction in a patient on anticoagulation with acute abdominal pain results in operative exploration in 67% (42 of 51 patients). for those reasons indications for immediate surgical intervention include active and persistent intra - abdominal bleeding, intestinal wall ischemia with or without bowel perforation and peritonitis. many case series and reports have demonstrated that patients with distal colonic hematomas required diverting colostomies 80% of the time. endoscopic drainage requires colonic wall viability but could result in relief of the tamponade effect of the hematoma. selective mesenteric embolization can be performed in patients with an active blush into the colonic wall with an 85% success rate, and an overall 20% of complication as a result of the procedure. recto - sigmoid hematomas should be considered as a diagnosis for a patient with a dropping hemoglobin and abdominal pain in the setting of an enema on therapeutic anticoagulation. if obvious signs of clinical deterioration such as free air and failure to respond to correction of coagulation are identified, then operative intervention should commence. the authors declare that there is no conflict of interests regarding the publication of this paper.	causes of colonic and recto - sigmoid hematomas are multifactorial. patients can present with a combination of dropping hemoglobin, bowel obstruction and perforation. computed tomography imaging can provide clues to a diagnosis of intramural hematoma. we present a case of rectal hematoma and a review of current management literature. a 72-year - old male on therapeutic anticoagulation for a pulmonary embolism, was administered an enema resulting in severe abdominal pain unresponsive to blood transfusion. a sigmoid colectomy with end colostomy was performed. although rare, colonic and recto - sigmoid hematomas should be considered as a possible diagnosis for adults with abdominal pain on anticoagulant therapy.
the family of mps one binder proteins (mobs) is highly conserved in eukaryotes. mobs represent signal transducers in essential intracellular signaling pathways through their regulatory interactions with serine / threonine protein kinases of the ndr / lats family [13 ]. in budding and fission yeast, mob1p and mob2p are crucial for mitotic exit and cell morphogenesis as regulators of the yeast ndr / lats kinases dbf2p, cbk1p, sid2p and orb6p, respectively [46 ]. in drosophila, three different mob proteins are expressed by independent genes, with dmob1 (aka mats) functioning as a core component of the hippo tumor suppressor pathway as regulator of the fly lats kinase warts [79 ]. drosophila mob2 (dmob2) contributes to neuromuscular junction and photoreceptor morphology and the biological function(s) of dmob3 is currently unknown, although all three dmobs can genetically interact with the fly ndr kinase tricornered. mammalian genomes contain at least six different mob genes termed mob1a, mob1b, mob2, mob3a, mob3b and mob3c. mob1a / b likewise to dmob1 functions as a regulator of lats kinases in mammalian hippo signaling, although current evidence proposes that the interaction of mob1 with ndr kinases is likely to also play a role in hippo signaling. in contrast to mob1, mob2 interacts specifically with ndr, but not with lats kinases in mammalian cells [1315 ]. mob3a / b / c neither form a complex with ndr nor lats kinases, but instead associate with the pro - apoptotic kinase mst1 (aka stk4). taken together, throughout the eukaryotic kingdom mobs can play diverse roles as regulators of members of the ndr / lats kinase family and apparently also other protein kinases in specific settings. in this article we will focus on discussing recent discoveries regarding roles of endogenous mob2 in cell cycle progression and the dna damage response (ddr) in the context ndr kinase signaling. up to recently, mammalian ndr kinases were the only reported binding partners of mob2. more precisely, biochemical experiments showed that mob2 competes with mob1 for ndr binding, with the mob1/ndr complex corresponding to increased ndr kinase activity and the mob2/ndr complex being associated with diminished ndr activity. in other words, mob2 binding to ndr can block the activation of ndr kinases. however, the biological significance of mob2/ndr complex formation is currently unknown. actually, no clearly defined physiologically relevant functions of endogenous mob2 were known until recently. intriguingly, a genome wide screen for novel putative ddr factors identified mob2 (also termed hcca2 and hmob3) as one of many potential candidates. considering that the ddr is essential to maintain genome stability and functions as a barrier for ageing and tumorigenesis, we investigated whether mob2 is indeed a ddr protein. intriguingly, we initially found that mob2 knockdown, but not mob2 overexpression, caused a cell proliferation defect associated with a g1/s cell cycle arrest in untransformed human cells. by profiling an array of cell cycle markers we discovered that mob2-depleted cells displayed a significant activation of the p53 and p21/cip1 cell cycle regulators, which was functionally relevant, since co - knockdown of p53 or p21 together with mob2 did not result in the activation of the g1/s cell cycle checkpoint, consequently restoring cell proliferation once we had established that mob2 knockdown causes the activation of a p53/p21-dependent g1/s cell cycle checkpoint, we wondered how this activation occurred. considering that endogenous mob2 is potentially linked to the ddr and that activation of the p53/p21 pathway can occur upon activation of ddr signaling [2022 ], we studied the levels of ddr signaling and endogenous dna damage in mob2-depleted cells. these investigations revealed that mob2 knockdown causes the accumulation of dna damage, and consequently activation of the ddr kinases atm and chk2, in the absence of exogenously induced dna damage. next, we aimed to consolidate these findings by studying the response of mob2 knockdown cells to exogenously induced dna damage. this showed that endogenous mob2 is needed to promote cell survival and g1/s cell cycle arrest upon exposure to dna damaging agents such as ionizing radiation (ir) or the topoisomerase ii poison doxorubicin. moreover, we discovered that mob2 is required to support ir - induced ddr signaling through the ddr kinase atm. collectively, these observations uncovered endogenous mob2 as a novel ddr factor that plays a role in ddr signaling, cell survival and cell cycle checkpoints upon exposure to dna damage. however, we still had not understood how mob2 may function as ddr protein on a molecular level. in this regard, using a yeast two - hybrid screen we had identified rad50 as a novel binding partner of mob2, which potentially was important, since rad50 is a central component of the essential mre11-rad50-nbs1 (mrn) dna damage sensor complex, which in turn is crucial for the sequestering / activation of the ddr kinase atm at dna lesions [2325 ]. therefore, we examined this potential interaction in more detail, revealing that mob2/rad50 complex formation can be detected using exogenous and endogenous proteins. moreover, we found that mob2 supports the recruitment of mrn and activated atm to dna damaged chromatin, suggesting that mob2-depleted cells display a defective ddr due to impaired functionality of the mrn. although we could map the binding sites of mob2 on rad50 to two functionally relevant domains of rad50, we did not succeed in identifying mob2 variants carrying single point mutations that block mob2/rad50 complex formation [gomez v and hergovich a, unpublished observation ], which would have enabled us to investigate the functional significance of the mob2/rad50 interaction in more detail. therefore, our study could not conclusively establish that the interaction of mob2 with rad50 is functionally essential for the roles of mob2 in ddr signaling, cell survival and cell cycle checkpoints upon exposure to dna damage. in this regard, it is noteworthy that in the genome wide screen performed by elledge. the knockdown of mrn components did not result in ddr defects (as judged by sensitivity to mitomycin c and a defective g2/m dna damage checkpoint) as observed in mob2-depleted transformed human cells. thus, the link of mob2 to the mrn does not appear to be relevant in all ddr settings, suggesting that additional mechanisms should be considered. in general, we have only begun to appreciate the cell biological functions of endogenous mob2 in processes that are relevant to human health and disease. in particular regarding the link of mob2 with the ddr, quite a few key questions are yet to be understood. for example, we have yet to comprehend which types of dna damage repair mechanism(s) is dependent on normal mob2 levels. maybe the expression / localization status of mob2 has the potential to be clinically exploited for the prediction and/or prognosis of responses to dna damaging agents (i.e. radiotherapy, dna damaging chemotherapeutics, targeted ddr inhibition, and others). furthermore, we have yet to obtain a clear mechanistic understanding of how mob2 can function as a ddr protein and how mob2 is regulated in a context - dependent manner. nevertheless, our previous biochemical characterization of mob2 in complex with the ndr1/2 kinases may be of help to lead some of the way. however, based on our own experiments we already speculated that mob2 apparently functions as cell cycle / ddr regulator independently of ndr1/2 kinase signaling. more specifically, we observed that knockdown of ndr1 (aka stk38) or ndr2 (stk38l) in untransformed human cells did not trigger a p53/p21-dependent g1/s cell cycle arrest as observed in mob2-depleted cells. overexpression of hyperactive ndr1-pif also did not cause an obvious cell cycle / proliferation defect. however, we believe that further investigations are still required to completely rule out that ndr1/2 signaling is not linked to cell cycle / ddr processes through mob2, since different reports have recently linked the ndr1/2 kinases to cell cycle and ddr signaling. in addition, compensatory mechanisms may occur upon selective ndr1 or ndr2 manipulations. in mammals, the ndr1/2 protein kinases have been linked to cell biological processes such as cell cycle progression, the ddr, apoptosis, stress signaling and autophagy, with important roles in embryogenesis, immunology and neurobiology. as mentioned above, in particular the connections of ndr1/2 with the cell cycle and ddr are intriguing with respect to mob2 (figure 1). on the one hand, ndr1/2 are linked to the regulation of g1/s cell cycle progression by controlling protein levels of c - myc and p21/cip1 [2932 ]. the role of ndr1/2 in the g1/s cell cycle progression is further supported by cyclin d1 and can have a role in opposing a tgf-mediated cell cycle arrest. thus, various tissue culture cell experiments support the notion that ndr1/2 can play diverse roles in the regulation of cell cycle progression. on the other hand, ndr1 possibly has a function in nucleotide excision repair, a specific type of dna damage repair. in addition, ndr1 potentially is involved in the dna damage induced g2/m cell cycle checkpoint by phosphorylating the cdc25a phosphatase, although this phosphorylation of cdc25a is also mediated by the ddr kinase chk1, hence warranting future investigations into this possible link of ndr1 and the dna damage induced g2/m cell cycle checkpoint. taken together, current evidence suggests that the ndr1/2 kinase pathway is linked to the regulation of certain aspects of cell cycle progression and signal transduction in response to dna damage (figure 1). nevertheless, in spite of the involvement of ndr1/2 in the cell cycle and ddr in a similar fashion as observed for mob2, it is currently unknown whether the mob2 and ndr1/2 pathways are functionally connected, as suggested by our recent biochemical evidence. in this regard, mob2 may act upstream of ndr1/2 by functioning as inhibitor of mob1-mediated ndr1/2 signaling. however, simply based on our current lack of evidence, we should not exclude the possibility that ndr1/2 may play a role upstream of mob2, in which case it would be very informative to understand the involvement of the ndr1/2 kinase activity, in addition to the regulation of mob2 by ndr1/2 (or vice versa) through direct protein - protein interactions. in this regard, experimenters should also keep in mind that single ndr1 or ndr2 knockdown compared to co - depletion of ndr1 and ndr2 may result in different phenotypes due to possible compensatory mechanisms. more specifically, we found that single ndr1 or ndr2 knockout mice are viable and fertile due to compensatory tissue specific up regulation of ndr2 or ndr1, respectively. in contrast these findings collectively suggest that ndr1 and ndr2 can compensate for each other in a context- and tissue - specific fashion. in case mob2 functions negatively upstream of ndr1/2 in ddr signaling, one would expect that mob2 knockdown or hyperactivation of ndr1/2 result in similar phenotypes, which does not seem to be the case. if ndr1/2 were to act upstream of mob2 in ddr signaling, one would predict that positive regulators of ndr1/2, such as mob1, may also contribute to the ddr. interestingly, this seems to be the case, since mob1a or mob1b knockdown appears to be sufficient to cause spontaneous dna double - strand break formation in human cells, proposing that the regulation of ndr1/2 by mob1 might also play a role in the ddr. however, whether ndr1/2 can function upstream of mob2 in cell cycle and/or ddr signaling is yet to be established experimentally. the possible involvement of mob1 in ndr1/2-mob2 signaling is also of purely speculative nature at the moment, in particular when considering that mob1 can associate with different kinases of the hippo core cassette such as lats1/2 and mst1/2. in this context, we also would like to emphasis the fact that currently the molecular (structural) regulation of ndr1/2 kinases by mob1 vs. mob2 is incompletely understood. possibly, mob2 is part of positive and/or negative feedback loops that serve to amplify and/or dampen cell cycle and/or ddr signaling, respectively. certainly, these speculative points illustrate the need for more intensified experimental efforts to understand mob2 as a novel ddr protein on the structural, molecular, cellular and organismal level in the context of human biology in health and disease.	this article is the authors opinion of the roles of the signal transducer mps one binder 2 (mob2) in the control of cell cycle progression and the dna damage response (ddr). we recently found that endogenous mob2 is required to prevent the accumulation of endogenous dna damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. in this regard, it is noteworthy that mob2 has been linked biochemically to the regulation of the ndr1/2 (aka stk38/stk38l) protein kinases, which themselves have functions at different steps of the cell cycle. therefore, we are speculating in this article about the possible connections of mob2 with ndr1/2 kinases in cell cycle and ddr signaling.
cardio - pulmonary fitness is related to the ability to perform large muscle, dynamic, moderate - to - high exercises for a prolonged period. the performance of such exercises depends on the functional state of the cardiovascular, respiratory, and skeletal muscle systems. a variety of tests have been used to check cardio - pulmonary fitness and some of these tests are quite popular, such as, the cooper 12-minute test. the procedure of this test involves the participants running with maximal effort for 12 minutes exactly. the advantages of cooper test are that the whole team can participate together, and compete against each other. the test is relatively easy to carry out, and does not involve too much time or equipment. it can also be used easily for training ; and, any improvements can be easily appreciated. the disadvantages include that it is an indirect test, the results of which are not very accurate. also, it is suitable only for well trained individuals due to the fact it requires maximal effort for the entire length of the test. another disadvantage is that the test does not give actual maximum oxygen consumption values ; it only gives a classification of poor to excellent. hence, it has been suggested that this test is best for comparing athlete 's individual scores against each other to monitor improvements, rather than comparing the scores of the whole team. in the laboratory, maximum oxygen consumption is the gold standard in the assessment of cardio - pulmonary fitness. normally, heart rate is controlled by autonomic nervous system activity. heart rate variability (hrv) hrv demonstrates a relationship between autonomic nervous system function (sympathetic or vagal activity) and cardiovascular system. on the other hand, hrv has been recognized as a predictor of cardiovascular events, both in symptomatic and asymptomatic population.[36 ] also, it was found that reduced hrv predicts hypertension and hyperglycemia in the future. the simplest variable is the standard deviation of normal - to - normal qrs intervals (sdnn) in a continuous electrogram (ecg) recording. traditionally, hrv is recorded over a longer period (24 hour) ; however, some investigators have reported that ultra - short hrv (2 - 15 minutes) is strongly correlated with the 24-hour hrv. the aim of this study was to assess the cardio - pulmonary fitness by ultra - short hrv. study population was divided into 3 groups : group-1 (n = 40) consisted of military sports man who volunteered to participate in this study. group-2 (n = 40) were healthy age - matched sedentary male subjects who had a normal bmi [bmi = 19 - 25 kg / m). group-3 (n = 40) consisted of healthy age - matched obese male subjects [bmi > 29 the subjects of the research were all free of cardiovascular and pulmonary disease, alcohol use, and diabetes mellitus ; and, were not taking any kind of medication, and did not present with any abnormal electrocardiographic patterns., all the subjects rested in a supine position for a minimum of 15 minutes in a room with a constant temperature of 25 degree c. for the holter recording, standard digital devices were used. after holter recording, bruce protocol treadmill test was used. at timed stages during the run, the slope of the treadmill is increased as detailed in table 1. maximum oxygen consumption (vo2max), which has been shown to be an objective criterion of cardiopulmonary fitness, was calculated by foster equation : bruce protocol treadmill test vo2max (ml / kg / min) = 14.8 [1.379 t ] + [0.451 t ] [0.012 t ] t is the total time of the test expressed in minutes and fractions of a minute. comparisons between groups were made using student 's t - test and mann - whitney u test as appropriate. study population was divided into 3 groups : group-1 (n = 40) consisted of military sports man who volunteered to participate in this study. group-2 (n = 40) were healthy age - matched sedentary male subjects who had a normal bmi [bmi = 19 - 25 kg / m). group-3 (n = 40) consisted of healthy age - matched obese male subjects [bmi > 29 the subjects of the research were all free of cardiovascular and pulmonary disease, alcohol use, and diabetes mellitus ; and, were not taking any kind of medication, and did not present with any abnormal electrocardiographic patterns. before the test, all the subjects rested in a supine position for a minimum of 15 minutes in a room with a constant temperature of 25 degree c. for the holter recording, standard digital devices were used. after holter recording, bruce protocol treadmill test was used. at timed stages during the run, the slope of the treadmill is increased as detailed in table 1. maximum oxygen consumption (vo2max), which has been shown to be an objective criterion of cardiopulmonary fitness, was calculated by foster equation : bruce protocol treadmill test vo2max (ml / kg / min) = 14.8 [1.379 t ] + [0.451 t ] [0.012 t ] t is the total time of the test expressed in minutes and fractions of a minute. comparisons between groups were made using student 's t - test and mann - whitney u test as appropriate. the mean age was identical between 3 groups as might be expected from the matching. baseline characteristics of the 3 groups involved in the study relationship between sdnn and estimated vo2 max are demonstrated in table 3. relation between standard deviation of normal - to - normal qrs intervals (sdnn) and maximum oxygen consumption (vo2max) of the three groups involved in the study when the study population was divided into quartiles of sdnn (first quartile : 60 and 100 and 140 msec), progressive increase was found in vo2max ; and, sdnn was significantly linked with estimated vo2max values [figure 1 ]. maximum oxygen consumption (vo2max) of study population compared by quartiles of standard deviation of normal - to - normal qrs intervals (sdnn). when the study population was divided into quartiles of sdnn (first quartile : 60 and 100 and 140 msec), progressive increase was found in vo2max. the mean age was identical between 3 groups as might be expected from the matching. relation between standard deviation of normal - to - normal qrs intervals (sdnn) and maximum oxygen consumption (vo2max) of the three groups involved in the study when the study population was divided into quartiles of sdnn (first quartile : 60 and 100 and 140 msec), progressive increase was found in vo2max ; and, sdnn was significantly linked with estimated vo2max values [figure 1 ]. maximum oxygen consumption (vo2max) of study population compared by quartiles of standard deviation of normal - to - normal qrs intervals (sdnn). when the study population was divided into quartiles of sdnn (first quartile : 60 and 100 and 140 msec), progressive increase was found in vo2max. previously, 24 hour was determined as an optimal cut point for hrv recording and analysis. bigger. found that measures of rr variability calculated from short (2 to 15 minutes) ecg recordings are remarkably similar to those calculated over 24 hours. hrv has been recognized as a predictor of cardiovascular events both in symptomatic and asymptomatic population.[36 ] it was also used in risk stratification for sudden cardiac death and diabetic autonomic neuropathy. they used hrv as a tool for noninvasive testing of autonomic nervous system activity with exercise training. they showed that regular exercises result in a significant improvement of all hrv indices. in the present study, we have analyzed cardio - pulmonary fitness (vo2max) and ultra - short hrv (sdnn). we showed that sdnn is a simple cardio - pulmonary fitness test which just requires 15 minutes, and involves no exercise such as in the treadmill or cycle test. previously, 24 hour was determined as an optimal cut point for hrv recording and analysis. bigger. found that measures of rr variability calculated from short (2 to 15 minutes) ecg recordings are remarkably similar to those calculated over 24 hours. hrv has been recognized as a predictor of cardiovascular events both in symptomatic and asymptomatic population.[36 ] it was also used in risk stratification for sudden cardiac death and diabetic autonomic neuropathy. they used hrv as a tool for noninvasive testing of autonomic nervous system activity with exercise training. they showed that regular exercises result in a significant improvement of all hrv indices. in the present study, we have analyzed cardio - pulmonary fitness (vo2max) and ultra - short hrv (sdnn). we showed that sdnn is a simple cardio - pulmonary fitness test which just requires 15 minutes, and involves no exercise such as in the treadmill or cycle test. in conclusion, the results of this study demonstrate that exercise training improves cardio - respiratory autonomic function (and increase heart rate variability). improvement in cardio - respiratory autonomic function seems to translate into a lower rate of long term mortality. ultra - short hrv is a simple cardio - pulmonary fitness test which just requires 15 minutes, and involves no exercise such as in the treadmill or cycle test.	objectives : it is known that exercise induces cardio - respiratory autonomic modulation. the aim of this study was to assess the cardio - pulmonary fitness by ultra - short heart rate variability.materials and methods : study population was divided into 3 groups : group-1 (n = 40) consisted of military sports man. group-2 (n = 40) were healthy age - matched sedentary male subjects with normal body mass index [bmi = 19 - 25 kg / m2). group-3 (n = 40) were healthy age - matched obese male subjects [bmi > 29 kg / m2). standard deviation of normal - to - normal qrs intervals (sdnn) was recorded over 15 minutes. bruce protocol treadmill test was used ; and, maximum oxygen consumption (vo2max) was calculated.results:when the study population was divided into quartiles of sdnn (first quartile : 60 and 100 and 140 msec), progressive increase was found in vo2max ; and, sdnn was significantly linked with estimated vo2max.conclusion:in conclusion, the results of this study demonstrate that exercise training improves cardio - respiratory autonomic function (and increases heart rate variability). improvement in cardio - respiratory autonomic function seems to translate into a lower rate of long term mortality. ultra - short heart rate variability is a simple cardio - pulmonary fitness test which just requires 15 minutes, and involves no exercise such as in the treadmill or cycle test.
a 60-year - old female presented with a history of serosanginous discharge and bleeding (small amount) per vaginum for the last 4 months. she was postmenopausal for the last 10 years with p5 + 1 + 0 + 5. local examination revealed hard infiltrative growth destroying both cervical lips and involving the vaginal wall. the upper third of the lateral and upper half of the anterior and posterior walls of the vagina were involved. hemogram (hb = 11.4 gm%, tlc = 7600/l) and renal function tests (urea = 24 mg / dl and creatinine = 0.81 mg / dl) were normal. ultrasound of the abdomen and pelvis showed no intra - abdominal / pelvic collection or pyometra. with carcinoma cervix iii b (figo clinical staging), she was planned for external radiotherapy (46gy in 23 fractions over 4.5 weeks). but after receiving 18gy/9 fractions, she presented with pain in the abdomen and vomiting. contrast - enhanced ct showed multiple pelvic and intra - abdominal collections, a dilated endometrial cavity with breach at the uterine fundus and its continuation with large pelvic collection, suggestive of perforated pyometra. sagittal and coronal reformats in multi - detector ct depicted the site and size of the uterine breach. the patient was managed with pigtail drainage of the larger pelvic and subhepatic collections, and with appropriate antibiotic cover (cefoperozone, sulbactam and amikacin). she improved clinically and follow - up ultrasound after 1 week showed near complete resolution of the abscesses, and the catheter was taken out (fig. 1 and 2). figure 1contrast - enhanced axial ct images showing dilated endometrial cavity with site of the breach at uterine fundus (arrow). pelvic collections () are seen with the abdominal / mesenteric extension and resultant intra - abdominal collections (). figure 2contrast - enhanced coronal and sagittal reformatted ct image depicting dilated endometrial cavity with site and size of the breach at uterine fundus (arrow). contrast - enhanced axial ct images showing dilated endometrial cavity with site of the breach at uterine fundus (arrow). pelvic collections () are seen with the abdominal / mesenteric extension and resultant intra - abdominal collections (). contrast - enhanced coronal and sagittal reformatted ct image depicting dilated endometrial cavity with site and size of the breach at uterine fundus (arrow). pyometra is an uncommon condition occurring mainly in elderly postmenopausal females, and results when natural drainage of the uterine cavity is compromised. the common causes of pyometra include malignant condition of the genital tract and sequele of their treatment, benign conditions such as infection and congenital cervical anomalies. the benign or malignant conditions cause accumulation of secretions and gradual enlargement of the uterus, leading to thinned uterine walls which may be sloughed with spontaneous uterine rupture and causing generalized peritonitis. spontaneous uterine perforation is rare and extensive review of the english literature has revealed only 26 such reported cases to date. furthermore, spontaneous rupture of pyometra in cervical cancer presenting as generalized peritonitis is extremely rare and only four cases have been described. we reviewed all the cases of spontaneous uterine perforation in cervical cancer and the findings are summarized in table 1. no.agesymptomsprovisional diagnosisperforation sitehistologytreatment867ap, gbgpfundussquamous cell carcinomaaspiration and drainage234apgp, ppleft corneal regionsquamous cell carcinomadrainage and pl272apgpfundussquamous cell carcinomadrainage and pl160ap, fgpfundussquamous cell carcinomatah with bsopresent case60ap, f, vgpfundusadeno - carcinomapigtail drainageap, abdominal pain ; bso, bilateral salpingo - oophorectomy ; f, fever ; gb, genital bleeding ; gp, generalized peritonitis ; pl, peritoneal lavage ; pp, perforated pyometra ; tah, total abdominal hysterectomy ; v, vomiting. cases of spontaneous uterine perforation in cervical cancer ap, abdominal pain ; bso, bilateral salpingo - oophorectomy ; f, fever ; gb, genital bleeding ; gp, generalized peritonitis ; pl, peritoneal lavage ; pp, perforated pyometra ; tah, total abdominal hysterectomy ; v, vomiting. the most common perforation site was the uterine fundus and in only one case it was the uterine cornual region. ct features of perforated pyometra have been described in only one case in which ct revealed the diagnosis and surgical intervention was performed. in our case, the patient presented with features of generalized peritonitis, ct showed perforated pyometra at the uterine fundus with multiple pelvic and intra - abdominal collections. sagittal and coronal reformats in multi - detector ct are very helpful in depicting the site and size of uterine breach, demonstrating the resultant intra - abdominal collections and staging of cervical cancer. the site of uterine breach can be missed on axial images as it is usually seen in fundus ; hence, in elderly postmenopausal females with peritonitis sagittal and coronal reformats of uterus should be carefully evaluated. sonography plays a limited role in the diagnosis of ruptured pyometra because of its inability to demonstrate the uterine breach and the limited sonographic window available due to perforation. treatment of ruptured pyometra in cervical cancer patients varies depending on the clinical condition of the patient and the preoperative diagnosis. in most cases drainage of the collections was carried out, except for one case in which total abdominal hysterectomy and bilateral salpingoophorectomy were peformed. the index case was managed by putting percutaneous pigtail catheters in pelvic and subhepatic collections. spontaneous rupture of pyometra in cervical cancer is very rare and should be considered in elderly postmenopausal women with cervical cancer presenting with an acute abdomen. multi - detector ct with sagittal and coronal reformatted images could play an important role in the diagnosis of ruptured pyometra.	abstractpyometra is an uncommon condition with an incidence of less than 1% in gynaecologic patients. spontaneous rupture of pyometra in cervical cancer presenting as generalized peritonitis is very rare. only four cases have been described in the english literature to the best of our knowledge and from a pubmed search. the index case is an elderly postmenopausal female who was diagnosed with cervical cancer, started on radiotherapy and presented with features of generalized peritonitis. contrast - enhanced ct revealed uterine perforation at the fundus with multiple abdominal and pelvic collections. a brief review of all the cases of ruptured pyometra in cervical cancer in the literature and a discussion of the role of imaging is presented.
laboratory information is a key driver of evidence - based clinical care and medical decision analysis. timely (turnaround - time based) availability of this information at the point of service is crucial in an increasingly complex medical environment. furthermore, the ability to mine for data in order to monitor the health of condition - specific populations and to meet the call for quality reporting relies on a sophisticated repository system that is accessible and flexible, yet secure and in compliance with regulatory requirements. a well - integrated laboratory information system (lis) is part of the bedrock of a successfully implemented electronic medical record (emr). laboratory data are said to account for the bulk (around 70%) of data stored in any given emr. such laboratory data populating an emr include both quantitative results (i.e. numerical values) and qualitative information (e.g. formatted pathology reports that contain predominantly text). optimal use of laboratory data in the emr requires careful consideration of electronic laboratory result formatting and display in the emr. the meaningful configuration of laboratory data is critical to the success of the burgeoning emr technology. with laboratory data included in the emr, users can begin to benefit from decision support tools that capitalize on this data (e.g. lab - drug - related e - alerts). in its current state, emrs can display laboratory data in the form of tables, spreadsheets, group listings, or graphs. this allows users to rapidly search, view, sort and pool laboratory information to support trend analyses and their clinical decision making process. however, to best serve the user, electronic laboratory data need to be flexible and customizable. our aim was to determine all the various ways in which laboratory data get utilized by clinicians in our health system 's emr. all the laboratory data from our different liss (sunquest version 6.2, copath plus version 3.1, mediware hcll) are transmitted electronically in hl7 and/or rtf format to the enterprise emr (millennium v2007.13, cerner corporation). the enhanced view version of the emr provides both a table of contents menu and a labeled (tab (electronic folder)) layout that together facilitate viewing of all result types. currently, there are discrete menu bars within the emr for all results (including vital signs, radiology, pharmacy information, and laboratory results), laboratory results only, microbiology results, and anatomical pathology reports. to account for the complexity of the medical record, there is a drop down menu that provides nearly three dozen custom flowsheets based on specialty and/or administrative needs. not only are data presented for discursive viewing, but also they are linked to a defined set of rules and internal feeds that generate decision support alerts, trigger automated health maintenance reminders, and can be used to populate clinical notes and emr - generated letters. the authors were involved in the customization and maintenance of several tools within their enterprise emr over a 5-year period. during this time period, the aforementioned electronic menus, tabs, flowsheets and subsections within the emr were explored to determine how clinicians utilize laboratory data. the results of the authors ' experience from observations, training, user feedback, and management of issues related to laboratory data in the emr are described. all the laboratory data from our different liss (sunquest version 6.2, copath plus version 3.1, mediware hcll) are transmitted electronically in hl7 and/or rtf format to the enterprise emr (millennium v2007.13, cerner corporation). the enhanced view version of the emr provides both a table of contents menu and a labeled (tab (electronic folder)) layout that together facilitate viewing of all result types. currently, there are discrete menu bars within the emr for all results (including vital signs, radiology, pharmacy information, and laboratory results), laboratory results only, microbiology results, and anatomical pathology reports. to account for the complexity of the medical record, there is a drop down menu that provides nearly three dozen custom flowsheets based on specialty and/or administrative needs. not only are data presented for discursive viewing, but also they are linked to a defined set of rules and internal feeds that generate decision support alerts, trigger automated health maintenance reminders, and can be used to populate clinical notes and emr - generated letters. the authors were involved in the customization and maintenance of several tools within their enterprise emr over a 5-year period. during this time period, the aforementioned electronic menus, tabs, flowsheets and subsections within the emr were explored to determine how clinicians utilize laboratory data. the results of the authors ' experience from observations, training, user feedback, and management of issues related to laboratory data in the emr are described. both clinical and anatomical pathology laboratory data in the emr were utilized by clinicians in five distinct ways [table 1 ], viz., within flowsheets, their personal inbox (emr messaging), with decision support tools, in the health maintenance tool, and when incorporating laboratory data into their clinical notes and letters. laboratory data presented as a flowsheet is similar to a spreadsheet with columns and rows. data within small preset cells (that do not autosize to fit the result) may cause results to be hidden or truncated. hidden data require users to perform an additional right - hand click to display this information. nevertheless, this is often the default format of many emrs. default settings frequently require customization for each user, which means training of the users is required. depending on the number of results and time range selected, users may need to scroll down and across the screen in order to view all results. data may be missed when rapidly scanning large tables. looking over many blank cells without results in this format can be easily sorted (e.g. by specified date range). by arranging laboratory data into a list or group of tests, users can rapidly view all the results obtained for a given day on their patient. specialty flowsheets (e.g. diabetes, heart failure, hiv infection view) can be customized to group relevant laboratory results [figure 1 ] to assist in disease - specific management. for example, physicians providing care in the acute hemodialysis unit select the renal flowsheet for immediate viewing of salient results. data can then be selected for graphical display of trends across custom timeframes. in the ambulatory setting, ready access to condition - specific flowsheets assists in the surveillance of preventive and maintenance measures necessary both for optimal patient care and quality reporting. for example, the hiv infection flowsheet displays relevant routine laboratory results (e.g. complete blood count, metabolic panel), related results (annual std screening, hepatitis antibody status) and hiv - specific results (t - cell subsets, hiv rna viral load assays). printing laboratory results in draft format from flowsheets may fail to incorporate pertinent data (e.g. reference ranges). screenshot showing portion of an hiv flowsheet in the emr with laboratory data in a table format for laboratory tests that are ordered by health care providers in the ambulatory setting, results not only post to the flowsheet in the patient 's chart, but also get routed using the emr messaging system to the ordering provider 's inbox. however, this requires that the correct provider is identified to be notified. within the inbox, these results are stratified into three categories : normal, abnormal, and critical. for example, each result needs to be individually opened in order to see if it is normal or abnormal. the critical results folder is highlighted in red and duplicates direct telephone alerting to the ordering clinician. the delivery of results to the inbox allows the clinician to track those results that were recently ordered. the results arrive as they are statused and can be acted upon in a timely fashion as determined by the ordering clinician. results within an inbox can be endorsed, deleted, forwarded, and/or set up to be handled by a proxy. laboratory data in our emr have also been used in the design of rules and alerts that are fundamental to clinical decision support. specifically, certain laboratory results have been associated with medication order entry so as to generate drug - related alerts that interrupt workflow. for example, ordering a potentially nephrotoxic drug in the emr triggers the automated scanning of laboratory results ' tables for creatinine values. if appropriate (e.g. there is an abnormal creatinine value), an interruptive alert is sent to the clinician recommending a drug dose reduction. on the other hand, the posting of a high potassium result yields a non - intrusive message that is sent to the ordering clinician 's inbox if he / she ordered a medication known to raise the risk of hyperkalemia. also, when laboratory values undergo post hoc modification or updating in the lis, an alert message is sent to the provider 's emr inbox notifying him / her of this changed result [figure 2 ]. although critical laboratory test results are electronically reported to providers ' inboxes, as a redundant mechanism, laboratory personnel still make telephone calls and/or page providers to verbally communicate such critical results. screenshot of a user 's emr inbox messages showing an automated e - alert informing them that a potassium result was modified from 11 to 3.5 mmol / l an added advantage of an enterprise emr is that data can be accessed as well as fed to a variety of care enhancement tools. in our emr laboratory information gets posted not only to flowsheets, but also to a health maintenance tool that is customized and based upon the latest clinical practice guidelines. for example, pap test results, lipid panel findings, and glucose test results all populate this health maintenance tool [figure 3 ] once they are transmitted to the emr. this assists the clinicians in their efforts to perform comprehensive preventative care in a timely fashion. moreover, the health maintenance tool is used to assist in the monitoring and tracking of disease - specific testing. we have linked results of biannual hemoglobin a1c and annual urine evaluation for microalbumin, low - density lipoprotein (ldl), and creatinine to the diabetes mellitus module within the health maintenance tool. similar modules have been designed to effectively manage patients with heart failure, cerebro and peripheral vascular disease, and coronary artery disease. screenshot of the emr health maintenance view populated with pending expectations for the user to act on we found that many clinicians incorporate laboratory results, often without the accompanying data elements (units, reference ranges, comments), into their clinical notes. for example, the author of an inpatient progress note can select relevant values (e.g. white blood cell count, blood uea nitrogen (bun)/creatinine) to populate their note. an electronic consultation request within the emr can also be generated to include laboratory values that are germane to the question at hand, and thus expedite the consultant 's ability to discern the details of the clinical case. moreover, laboratory data were also included within emr - generated letters to patients [figure 4 ]. letter templates that reside in the emr foster quick, yet complete, communication with patients, and they include laboratory results. while numerical results (e.g. a lipid panel) get automatically entered into such letter templates, they are associated with clear explanations from the clinician along with additional advice and treatment targets. in addition, these ad hoc letters have served as laboratory requisitions for follow - up testing when required. in such cases, clinicians simply included the specific test requested and an associated diagnostic code to facilitate billing. example of an emr - generated letter by a physician to his / her patient including laboratory results and interpretation comments for the patient laboratory data presented as a flowsheet is similar to a spreadsheet with columns and rows. data within small preset cells (that do not autosize to fit the result) may cause results to be hidden or truncated. hidden data require users to perform an additional right - hand click to display this information. nevertheless, this is often the default format of many emrs. default settings frequently require customization for each user, which means training of the users is required. depending on the number of results and time range selected, users may need to scroll down and across the screen in order to view all results. results in this format can be easily sorted (e.g. by specified date range). by arranging laboratory data into a list or group of tests, users can rapidly view all the results obtained for a given day on their patient. specialty flowsheets (e.g. diabetes, heart failure, hiv infection view) can be customized to group relevant laboratory results [figure 1 ] to assist in disease - specific management. for example, physicians providing care in the acute hemodialysis unit select the renal flowsheet for immediate viewing of salient results. data can then be selected for graphical display of trends across custom timeframes. in the ambulatory setting, ready access to condition - specific flowsheets assists in the surveillance of preventive and maintenance measures necessary both for optimal patient care and quality reporting. for example, the hiv infection flowsheet displays relevant routine laboratory results (e.g. complete blood count, metabolic panel), related results (annual std screening, hepatitis antibody status) and hiv - specific results (t - cell subsets, hiv rna viral load assays). printing laboratory results in draft format from flowsheets may fail to incorporate pertinent data (e.g. reference ranges). screenshot showing portion of an hiv flowsheet in the emr with laboratory data in a table format for laboratory tests that are ordered by health care providers in the ambulatory setting, results not only post to the flowsheet in the patient 's chart, but also get routed using the emr messaging system to the ordering provider 's inbox. however, this requires that the correct provider is identified to be notified. within the inbox, these results are stratified into three categories : normal, abnormal, and critical. for example, each result needs to be individually opened in order to see if it is normal or abnormal. the critical results folder is highlighted in red and duplicates direct telephone alerting to the ordering clinician. the delivery of results to the inbox allows the clinician to track those results that were recently ordered. the results arrive as they are statused and can be acted upon in a timely fashion as determined by the ordering clinician. results within an inbox can be endorsed, deleted, forwarded, and/or set up to be handled by a proxy. laboratory data in our emr have also been used in the design of rules and alerts that are fundamental to clinical decision support. specifically, certain laboratory results have been associated with medication order entry so as to generate drug - related alerts that interrupt workflow. for example, ordering a potentially nephrotoxic drug in the emr triggers the automated scanning of laboratory results ' tables for creatinine values. if appropriate (e.g. there is an abnormal creatinine value), an interruptive alert is sent to the clinician recommending a drug dose reduction. on the other hand, the posting of a high potassium result yields a non - intrusive message that is sent to the ordering clinician 's inbox if he / she ordered a medication known to raise the risk of hyperkalemia. also, when laboratory values undergo post hoc modification or updating in the lis, an alert message is sent to the provider 's emr inbox notifying him / her of this changed result [figure 2 ]. although critical laboratory test results are electronically reported to providers ' inboxes, as a redundant mechanism, laboratory personnel still make telephone calls and/or page providers to verbally communicate such critical results. screenshot of a user 's emr inbox messages showing an automated e - alert informing them that a potassium result was modified from 11 to 3.5 mmol / l an added advantage of an enterprise emr is that data can be accessed as well as fed to a variety of care enhancement tools. in our emr laboratory information gets posted not only to flowsheets, but also to a health maintenance tool that is customized and based upon the latest clinical practice guidelines. for example, pap test results, lipid panel findings, and glucose test results all populate this health maintenance tool [figure 3 ] once they are transmitted to the emr. this assists the clinicians in their efforts to perform comprehensive preventative care in a timely fashion. moreover, the health maintenance tool is used to assist in the monitoring and tracking of disease - specific testing. we have linked results of biannual hemoglobin a1c and annual urine evaluation for microalbumin, low - density lipoprotein (ldl), and creatinine to the diabetes mellitus module within the health maintenance tool. similar modules have been designed to effectively manage patients with heart failure, cerebro and peripheral vascular disease, and coronary artery disease. screenshot of the emr health maintenance view populated with pending expectations for the user to act on we found that many clinicians incorporate laboratory results, often without the accompanying data elements (units, reference ranges, comments), into their clinical notes. for example, the author of an inpatient progress note can select relevant values (e.g. white blood cell count, blood uea nitrogen (bun)/creatinine) to populate their note. an electronic consultation request within the emr can also be generated to include laboratory values that are germane to the question at hand, and thus expedite the consultant 's ability to discern the details of the clinical case. moreover, laboratory data were also included within emr - generated letters to patients [figure 4 ]. letter templates that reside in the emr foster quick, yet complete, communication with patients, and they include laboratory results. while numerical results (e.g. a lipid panel) get automatically entered into such letter templates, they are associated with clear explanations from the clinician along with additional advice and treatment targets. in addition, these ad hoc letters have served as laboratory requisitions for follow - up testing when required. in such cases, clinicians simply included the specific test requested and an associated diagnostic code to facilitate billing. example of an emr - generated letter by a physician to his / her patient including laboratory results and interpretation comments for the patient as we discovered, laboratory data in our emr were being utilized by clinicians in a number of ways. the positive and negative features of utilizing the laboratory data in different emr tools are summarized in table 1. it should be noted, however, that our findings are based upon the evaluation of a single institution 's proprietary emr (cerner millennium), with certain tools specific to this emr. while it is acceptable to have laboratory results within an emr be displayed in several ways, these data should not be permitted to be edited by users. allowing laboratory results viewed within an emr to be customized, as was evident in our emr with different flowsheets, meets the needs of many users and specialties. rather than force all users to view data in the same manner, such customized screens (dynamic displays, dashboards, or flowsheets) can be created. moreover, countless test results on a computer screen can be distracting and is a waste of time for busy users to search through. honing into these flowsheets hides distracting test results that are not relevant to the clinical scenario or decision - making process on the screen for users. by collating only relevant data and focusing attention on important data, customized displays because printing laboratory results directly from the emr in draft format may not incorporate pertinent data, for medicolegal purposes users are required to use an alternate medical record publishing (mrp) format. laboratory tests that are ordered and resulted during a hospital encounter post to the results flowsheet. this fits well into the traditional workflow of the hospital - based clinician. in this practice setting, the flowsheet is routinely viewed in the midst of hospital rounds and is accessible when required. for laboratory orders that are ordered in the ambulatory setting, while results still post to the flowsheet, they have been found to be most helpful to users when they are also sent to the clinician 's emr inbox. new or outstanding lab results for review are displayed in the inbox. in this way, the emr provides added value by decreasing the risk of a lost or overlooked result. furthermore, the clinician is afforded the opportunity to both review the relevant data and electronically immediately document any necessary action taken. utilizing the emr messaging system thereby facilitates a paperless environment, eliminating the need to also deliver printed pathology reports to patients ' providers. the emr messaging system has several other advantages such as displaying new or updated chart information for sign off and facilitating communication (e.g. send reminders, request information, forward instructions) among clinical colleagues. however, poorly formatted inboxes may cause important results to be missed. given the potential for medicolegal liability of missing results in one 's inbox, there is some concern about relying solely on an emr messaging system. clinical decision support systems allow the emr to more actively contribute to the clinical care process. available data suggest that decision support tools not only improve the efficiency of patient care (e.g. lower the misuse of tests), but also may enhance outcomes (e.g. reduce the incidence of adverse events). these tools can also support mundane administrative duties (e.g. automated coding) and promote best - practices (e.g. practice guidelines and protocols). they can include alerts (e.g. pop - up screens), reminders, calculators, order sets, and embedded educational content in the emr. alerts may be interruptive (i.e. an action is required by the user) or non - interruptive (i.e. no user action is needed). alerts may certainly be a hindrance when they interrupt user workflow and with overuse they may result in " alert fatigue " causing users to ignore, delete, or override them. modified, abnormal and/or critical laboratory results within the lis can even be used to trigger a timely message to automatically alert clinicians via a cell phone, pager, e - mail, or the emr inbox. at our institution, we also invested in building more complex emr tools that link laboratory data and information about medications. they may generate at the time of online order entry or trigger when laboratory data are resulted. linking laboratory results and pharmacy data has been shown to help with drug choices (e.g. laboratory - based indications and contraindications), drug dosing (e.g. renal or hepatic values, blood level - guided adjustments), drug monitoring (e.g. laboratory signals of toxicity), and broader quality improvement (e.g. surveillance for unrecognized toxicity). computer - based reminders to prompt physicians to implement preventive and other services have been available since the late 1970s. studies have reported improvements in the delivery of preventive services (e.g. vaccination) using automated reminders. by virtue of the automated delivery of results to a health maintenance tool, the clinician is afforded a highlighted alert that particular testing is either imminent or past due. laboratory data such as pap test results posted to the health maintenance tool in our emr were used to generate reminders, prompting physicians to perform a pap test on their patient if indicated. unfortunately, researchers have shown that many clinicians neither pay attention to these reminders nor do they even regularly review the health maintenance needs of their patient before the clinical encounter. perhaps what is required is not just a tool to remind providers of needed preventive services, but a system to ensure optimal delivery of preventive services. as a result of this study, it was apparent that clinicians frequently cut and paste or " pull " laboratory data from the " source of truth " into their clinical notes and letters. this cut and paste mechanism can be utilized by clinicians to quickly summarize other clinical data (e.g. radiology interpretations) in addition to laboratory data. the ability to pull laboratory data into clinical documentation certainly enhances communication, not only between referring and consulting physicians, but also between the clinician and the patient. this feature, however, is not without risk, as it has the potential for users to misreport data if incomplete or incorrect data are pasted or moved. guidelines (e.g. clia 493.1109 standard) exist regarding the specific data elements that should be incorporated in laboratory reports. when appropriate, laboratory reports (i.e. paper and potentially electronic) are required to include the following data elements : unique patient identification, name and address of the performing laboratory, report date, test(s) performed, specimen source, result, units of measure, reference range as determined by the laboratory performing the test, and information regarding specimen(s) that do not meet acceptability criteria. all of these additional data elements, although present in the emr, are not included when clinicians cut and paste laboratory data into their notes and letters. users need to be aware of the data they are omitting. according to the college of american pathologists (cap) checklist question gen.41067 (does an individual meeting cap laboratory director qualifications review and approve the content and format of paper and electronic patient reports at least annually ?), the laboratory director must approve the content and format of laboratory patient reports, including computer screen images, to ensure that they effectively communicate patient test results. pathologists should assume responsibility for the data supplied by their laboratory that get cut and pasted into clinical emr notes and letters, and accordingly, should have a role in the design and/or utilization of this feature in their emr. in summary, flexible electronic laboratory data in the emr have many advantages. users can view, sort, and pool lab information to support trend analysis and clinical decision making. laboratory data can also be used to trigger clinical decision support systems such as alerts and reminders. future studies could survey clinicians to better assess their needs and determine which tools they find most valuable. it may also be of interest to compare and contrast similar tools in other emrs. pathologists need to start participating more actively in the creation of many of these emr tools in order to support the appropriate utilization of laboratory information in the emr.	background : laboratory data account for the bulk of data stored in any given electronic medical record (emr). to best serve the user, electronic laboratory data needs to be flexible and customizable. our aim was to determine the various ways in which laboratory data get utilized by clinicians in our health system 's emr.method:all electronic menus, tabs, flowsheets, notes and subsections within the emr (millennium v2007.13, cerner corporation, kansas city, mo, us) were explored to determine how clinicians utilize discrete laboratory data.results:laboratory data in the emr were utilized by clinicians in five distinct ways : within flowsheets, their personal inbox (emr messaging), with decision support tools, in the health maintenance tool, and when incorporating laboratory data into their clinical notes and letters. conclusions : flexible electronic laboratory data in the emr hava many advantages. users can view, sort, pool, and appropriately route laboratory information to better support trend analyses, clinical decision making, and clinical charting. laboratory data in the emr can also be utilized to develop clinical decision support tools. pathologists need to participate in the creation of these emr tools in order to better support the appropriate utilization of laboratory information in the emr.
slow coronary flow (scf) phenomenon is a coronary microvascular disease diagnosed by detection of delayed dyeopacification in coronary arteries during an angiography in the lack of obstructive coronary artery disease. the incidence of scf has been reported to be approximately 1 - 7% in patients who undergo a diagnostic coronary angiography due to suspected coronary artery disease.1) despite being a well - known phenomenon, the pathophysiologic mechanisms of scf remain unknown. potential mechanisms involved in scf are : small vessel and endothelial dysfunctions,2)3) inflammation,4)5) diffuse atherosclerosis,6) and increased platelet aggregability.7) platelets have been shown to have an important role in the pathogenesis of acute coronary syndrome. some mediators involved in inflammatory processes and coagulatory pathways might have contributed to the development of atherosclerotic and atherothrombotic diseases.8)9) mean platelet volume (mpv), an index of platelet activation, has been shown to be associated with adverse cardiovascular outcomes.10) mpv was found to be elevated in scf cases compared to normal ones.11) platelet distribution width (pdw) is found to be increased during platelet activation.12) platelet larger cell ratio (p - lcr), an idicator of larger platelet circulating (> 12 fl), has also been used to monitor platelet activity.13) given the probabale pathophysiologic roles of inflammation and platelet dysfunction in the development of scf, we sought to determine the relationship between scf and platelet activation using platelet volume indices mpv, pdw, and p - lcr. in a case - control study, a total of 1650 consecutive patients who underwent diagnostic coronary angiography due to unstable angina and/or chest discomfort without positive non - invasive tests were evaluated in our two university centers, including seyyed - al - shohada heart center and taleghani hospital from april 2013 to november 2013. the local ethics committee in the urmia university of medical sciences, west - azerbaijan province, iran, approved this investigation. of evaluated participants, 50 diagnosed with scf based on thrombolysis in myocardial infarction frame counting (tfc) method 30 participants had normal coronary flow (ncf) were considered as control group after fulfilling inclusion and exclusion criteria. exclusion criteria were : 1) diagnosed as coronary artery disease ; 2) coronary artery stenosis > 50% of the coronary artery diameter in angiographic evaluation ; 3) peripheral arterial disease ; 4) valvular heart disease ; 5) thrombocytopenia ; 6) hematologic diseases ; 7) hepatic diseases ; 8) chronic inflammatory disease ; 9) malignancies ; 10) infectious disease ; 11) autoimmune disease ; 12) anemia ; 13) renal failure ; and 14) thyroid diseases. all patients underwent coronary angiographic examination through femoral artery using judkins ' method (siemens, forcheim, germany). the contrast media of iodixanol (ge healthcare, cork, ireland) was used in all cases. two cardiologists who were blinded to the patients ' condition and clinical features evaluated all angiograms to identify scf using tfc method.14) the numbers of angiographic frames were recorded at 30 frames per second. the coronary flow was calculated as reaching contrast media to the defined distal part of coronary arteries. the end distal parts in the left anterior descending (lad) and left circumflex (lcx) arteries were distal bifurcation points. the calculated frame counting for lad artery was divided by 1.7 based on the predefined criteria.14) according to gibson.14) the cut - off values for detecting scf were 36.22.6, 20.43, and 22.24.1 frames for lad, rca, and lcx arteries, respectively. automated cell count analyzer (sysmex, kobe, japan) was used to measure complete blood count. continuous variables were analyzed using t - test, one - way analysis of variance or mann whitney u test as appropriate based on the variables ' distribution normality. receiver operating characteristic curve was constructed to detect the accuracy of platelet volume indices to differentiate scf from ncf. multivariate logistic regression analysis and backward stepwise were conducted to determine the predictors of scf among our studied cohort. in this analysis, demographics and laboratories were entered into the model as covariates, and the status of scf was considered as a dependent variable. all analyses were performed using statistical package for the social sciences (spss) version of 18.0 (spss inc., in a case - control study, a total of 1650 consecutive patients who underwent diagnostic coronary angiography due to unstable angina and/or chest discomfort without positive non - invasive tests were evaluated in our two university centers, including seyyed - al - shohada heart center and taleghani hospital from april 2013 to november 2013. the local ethics committee in the urmia university of medical sciences, west - azerbaijan province, iran, approved this investigation. of evaluated participants, 50 diagnosed with scf based on thrombolysis in myocardial infarction frame counting (tfc) method 30 participants had normal coronary flow (ncf) were considered as control group after fulfilling inclusion and exclusion criteria. exclusion criteria were : 1) diagnosed as coronary artery disease ; 2) coronary artery stenosis > 50% of the coronary artery diameter in angiographic evaluation ; 3) peripheral arterial disease ; 4) valvular heart disease ; 5) thrombocytopenia ; 6) hematologic diseases ; 7) hepatic diseases ; 8) chronic inflammatory disease ; 9) malignancies ; 10) infectious disease ; 11) autoimmune disease ; 12) anemia ; 13) renal failure ; and 14) thyroid diseases. all patients underwent coronary angiographic examination through femoral artery using judkins ' method (siemens, forcheim, germany). the contrast media of iodixanol (ge healthcare, cork, ireland) was used in all cases. two cardiologists who were blinded to the patients ' condition and clinical features evaluated all angiograms to identify scf using tfc method.14) the numbers of angiographic frames were recorded at 30 frames per second. the coronary flow was calculated as reaching contrast media to the defined distal part of coronary arteries. the end distal parts in the left anterior descending (lad) and left circumflex (lcx) arteries were distal bifurcation points. the calculated frame counting for lad artery was divided by 1.7 based on the predefined criteria.14) according to gibson.14) the cut - off values for detecting scf were 36.22.6, 20.43, and 22.24.1 frames for lad, rca, and lcx arteries, respectively. automated cell count analyzer (sysmex, kobe, japan) was used to measure complete blood count. continuous variables were analyzed using t - test, one - way analysis of variance or mann whitney u test as appropriate based on the variables ' distribution normality. receiver operating characteristic curve was constructed to detect the accuracy of platelet volume indices to differentiate scf from ncf. multivariate logistic regression analysis and backward stepwise were conducted to determine the predictors of scf among our studied cohort. in this analysis, demographics and laboratories were entered into the model as covariates, and the status of scf was considered as a dependent variable. all analyses were performed using statistical package for the social sciences (spss) version of 18.0 (spss inc., baseline clinical characteristics of patients were summarized in table 1. a total of 50 patients with scf and 30 patients with ncf were studied. based on the baseline characteristics, patients with scf were more likely to have diabetes and hypertension than ncf (24% vs. 6.7%, p=0.048 and 50% vs. 23.3%, p=0.018, respectively). the platelet volume indices mpv (p=0.005), pdw (p=0.007), and p - lcr (p=0.039) were significantly higher in the scf group compared to those in the ncf group (table 1). based on correlation analysis, positive and significant correlations existed between the platelet volume indices and the tfc values in three coronary arteries except between p - lcr and lad and lcx arteries (table 2). when comparing the differences of platelet volume indices and the extent of scf, the amounts of mpv, pdw, and p - lcr were significantly higher in patients with three scf arteries compared to those of patients without scf (p=0.004, p=0.005, and p=0.014, respectively). in addition, pdw was significantly (p=0.026) higher in patients with two scf arteries compared with patients without scf (fig. receiver operating characteristic curve analysis showed that the best cut - off values of mpv, pdw, and p - lcr to distinguish scf from ncf were 10.4 fl, 13.9 fl, and 28%, respectively (fig., mpv had significantly higher amount of area under the curve (auc) (auc=0.669, p=0.012). all variables whose p were significant (p<0.05) based on bivariate analysis were entered into the linear regression analysis. according to this model, mpv { =32.393, 95% confidence interval (ci) 0.678 - 11.048, p=0.027 }, pdw (=0.428, 95% ci 0.060 - 3.924, p=0.043), and p - lcr (=-0.771, 95% ci -1.917 - -0.117, p=0.027) were independent predictors of mean tfc (table 3). results of multivariate analysis were summarized in the table 4. according to this model, mpv { odds ratio (or)=32.393, 95% ci=1.189 - 882.606, p=0.039 } and p - lcr (or=0.566, 95% ci=0.330 - 0.937, p=0.028) were independent predictors of scf among all variables measured in this study. in this study, for the first time, we showed that the platelet volume indices mpv, pdw, and p - lcr were significantly higher in the patients with scf compared to those in the ncf group. there were positive correlations between platelet volume indices and the tfc measured for three coronary arteries. furthermore, mpv, pdw, and p - lcr were found to be the independent predictors of mean tfc. platelets are important blood cells that participate in the processes of atherothrombotic events, coagulation, and inflammation.8) mpv, pdw, and p - lcr are indicators of platelet size, volume, and activation. elevated mpv value has been shown to be involved in the pathogenesis of atherosclerosis and thrombogenesis.15) gke.7) have deomonastrated that platelet aggregability induced by ristocetin, collagen, and adenosine diphosphate was significantly higher in patients with scf compared to that of ncf, indicating the potential of platelet in scf pathogenesis. in a case - control study, celik.11) have demonstrated that increased mpv and plasma sp - selectin values are higher in scf patients compared to those of ncf ones, with positive correlations between mpv values and the tfc of coronary artery. they concluded that the increased platelet activity existed in scf might be a pathogenesis for scf development. elsherbiny.16) have shown that mpv is higher in scf compared to ncf. nurkelam.17) have reported that scf cases with unstable angina has higher mpv compared to scf ones with stable coronary artery disease and ncf cases. other studies have also confirmed that elevated mpv is associated with the presence of scf.18)19) furthermore, it has been shown that mpv correlated with the extent of scf.19) in consistent with those research findings, our results also reveald that mpv was significantly higher in scf compared to ncf cases. our results also revealed that mpv was associated with mean tfc of three coronary arteries. in addition, for the first time, we found that pdw and p - lcr were also higher in scf. li.4) found that c - reactive protein (crp) and interlukin-6 were higher in scf patients and that scf was was correlated with mean tfc. in additon, some adhesion molecules, including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and e - selectin as markers of endothelial activation or inflammation were reported to be significantly higher in patients diagnosed with scf.5) given the probable role of inflammation in the scf pathogenesis, some clinical trials have been conducted. albayrak.20) have demonstrated that nebivolol reduced crp level and controlled the chest pain of patients with scf. the impact of simvastatin on improving myocardial perfusion in scf is another evidence to support the role of inflammatory processes in the scf development.6) in addition, platelets involve in inflammatory pathways through releasing mediators, and larger ones contain more mediators and activity. considering the results of this study and those other studies demonstrating increased platelet indices in scf patients, further clinical trials are needed to illustrate the effects of anti - platelet and anti - inflammatory agents on scf and to prove the relationship between platelet - related disorders and scf pathogenesis. in order for recurrent chest pain to be controlled, some drugs such as beta - blockers, nitrates, and statins have been used. however, no proper impact of these agents have been demonstrated. in this study. however, due to the above mentioned pathophysiologic mechanisms of scf, including inflammation, thrombogeneity, and atherosclerosis, and due to the relationship between increased platelet volume indices and scf, it may be of great benefit to prescribe anti - platelet and anti - inflammatory agents to reduce platelet volume indices. beside the therapeutic use of these markers, they could be used for risk staritification of scf cases. based on our findings, it can be proposed that increased platelet volume indices mpv, pdw, and p - lcr are inexpensive and widely available tests associated with scf and that they might be involved in the pathophisiology of scf. considering the fact that platelet volume indices were indicators of platelet activation and consequent inflammatory processes, we suggest that increased mpv, pdw, and p - lcr could be indicators of platelet dysfunction and inflammation in patients diagnosed with scf. therefore, platelet volume indices mpv, pdw, and p - lcr could be considered for monitoring drug efficacy and risk stratification of such cases. firstly, the small sample size and the lack of some variables, including lipid profile tests and familial history of cardiovascular disease, were the main limitation of this study. secondly, we did not measure other inflammation and platelet function markers such as crp, which could provide more information regrding the pathophysilogic mechanisms involved in scf. finally, because we chose the participants consecutively based on the inclusion and exclusion criteria, selection bias may have occurred during patients enrollment. platelet volume indices mpv, pdw, and p - lcr were significantly higher in scf patients compared to those in ncf. the increases of platelet volume indices mpv, pdw, and p - lcr were associated with the extent of scf. moreover, mpv, pdw, and p - lcr were found to be indipendent predictors of mean tfc. firstly, the small sample size and the lack of some variables, including lipid profile tests and familial history of cardiovascular disease, were the main limitation of this study. secondly, we did not measure other inflammation and platelet function markers such as crp, which could provide more information regrding the pathophysilogic mechanisms involved in scf. finally, because we chose the participants consecutively based on the inclusion and exclusion criteria, selection bias may have occurred during patients enrollment. platelet volume indices mpv, pdw, and p - lcr were significantly higher in scf patients compared to those in ncf. the increases of platelet volume indices mpv, pdw, and p - lcr were associated with the extent of scf. moreover, mpv, pdw, and p - lcr were found to be indipendent predictors of mean tfc.	background and objectiveswe sought to determine the relationship between mean platelet volume (mpv), platelet distribution width (pdw), and platelet larger cell ratio (p - lcr) with slow coronary flow (scf).subjects and methodseighty participants who underwent coronary angiography were divided into two groups, 50 participants with scf as case group, and 30 with normal coronary flow (ncf) as control group. baseline characteristics and laboratory data were collected before angiography.resultsplatelet volume indices mpv (10.81.2 fl), pdw (14.52.2 fl), and p - lcr (30.58.1%) in the scf group were significantly (p<0.05) higher than those (10.10.9 fl, 13.21.8 fl, and 26.86.8%, respectively) in the ncf group. the patients with three scf arteries had significantly higher platelet volume indices compared to those with ncf arteries ; however, the patients with one scf artery did not. based on linear regression model, mpv, pdw, and p - lcr were independent predictors of mean infarction frame counting (tfc). in multivariate analysis, mpv { odds ratio (or)=32.393, 95% confidence interval (ci)=1.189 - 882.606, p=0.039 } and p - lcr (or=0.566, 95% ci=0.330 - 0.937, p=0.028) were independent predictors of scf.conclusionplatelet volume indices mpv, pdw, and p - lcr were associated with both the presence and extent of scf.
, many countries have made efforts to prevent and reduce tobacco use in this vulnerable age group (1). to achieve this goal, factors that influence smoking in adolescents in different communities need to be understood. a study in turkey showed that household size, higher birth rank, school type, low academic performance, exposure to second - hand smoke, and stress were important factors influencing tobacco use in high - school students (2). in a brazilian study, the period of late adolescence, attending a private school, and work activities were associated with the initiation of hookah use (3). parental tobacco use was also reported to increase the risk of smoking initiation in adolescents (4). a study of canadian students showed that younger age, single - parent family status, stress, impulsivity, low self - esteem, experimentation with smoking, poor school performance, susceptibility to tobacco advertisements, alcohol use, consumption of other tobacco products, and attending a smoking - tolerant school, as well as smoking by parents, siblings, friends, and school staff, were the main determinants of smoking initiation (5). a community - based study in iran showed that older age, poor parental control of children, insufficient parental supervision of adolescents in friend selection, and having a smoker friend or family member were associated with lifetime cigarette use among male adolescents (6). a study in nepal demonstrated that late adolescence, male gender, attending a public school, and a substantial amount of pocket money were associated with tobacco use in secondary - school students (7). another study in nepal showed that substance use and parental smoking were associated with smoking among medical and dental students (8). a study of middle - school students in the u.s. underscored the independent role of seeing tobacco use in films in the initiation of smoking by adolescents (9). in a study in iran, age, grade, the mother s job, and education had statistically significant differences between tobacco smokers and nonsmokers (10). although many common factors seem to influence tobacco use at a young age in different countries, understanding the determinants of using different kinds of tobacco in various populations is necessary to plan international and national preventive programs. the current study aimed to assess the determinants of smoking tobacco and hookah use in a nationally representative sample of iranian students. the data of this study were collected as a part of a national survey of school student high - risk behaviors (2011 - 2012), as part of the fourth survey of a school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non - communicable disease (caspian - iv) study. a total sample size of 480 subjects in each province was calculated as the maximum sample size needed to provide an optimal estimate of all the risk factors of interest. from the 30 provinces, 48 clusters of 10 subjects were selected in each province, in addition to 14,880 students and an equal number of their parents. using the multistage, cluster - sampling method, the students were selected from urban and rural areas of different cities in the 30 provinces of the country (48 clusters of 10 students in each province). stratification was done in each province according to the area of residence (urban / rural) and school grade (elementary / intermediate / high school). the sampling was proportional to the size, with an equal sex ratio (i.e., equal numbers of boys and girls were selected from each province, and the ratios in urban and rural areas were balanced to the population of urban and rural students). in this way, the number of samples in the rural / urban areas and in each school grade was divided equally between the populations of students in each grade. cluster sampling with equal clusters was used in each province to scope the required sample size. the clusters were concluded the level of schools, including 10 sample units (the students and their parents) in each cluster. the maximum sample size in each province required to provide a good estimate of all the risk factors of interest was calculated as 480 students. finally, 48 clusters of 10 subjects in each of the provinces (n = 14,880 students) were selected. with the participation of trained teams of expert health care providers, all processes of examinations and inquiry follow under standard protocols of the world health organization - global school - based student health survey (10). the validity and reliability of the questionnaire were confirmed in a previous study (12). the cronbach s alpha and pearson s correlation coefficient for the reliability and stability of the questionnaire was 0.97 and 0.94, respectively. in the cvi assessment, the questions were about demographic characteristics, parents education levels and occupations, students school grades, birth orders, family sizes, smoking habits, smoking habits of the families, school types (public / private), living with parents, and exposure to tobacco smoke, as well as the time spent with friends, physical activity level, sleep duration, and screen time. individuals who reported having tried smoking any tobacco product were considered ever smokers, and those who reported that they continued smoking at the study time were considered current smokers. a hookah smoker was based on self - reports of hookah use at the time of the study. approval for the study was obtained from the ethical committees of isfahan university of medical sciences, tehran university of medical sciences (tums), and other relevant regulatory organizations at national and provincial levels. after explaining the study objectives and protocols, written consent and verbal assent were obtained from the parents and students, respectively. the students were reassured about the confidentiality of their answers, and the questionnaires were completed anonymously. continuous variables are reported as the mean and 95% confidence interval (ci), and categorical variables are presented as a percentage (95% ci). the association of age with smoking was assessed using an independent sample t - test. chi - square tests were used to compare the prevalence rates in the study groups. multiple logistic regression (mlr) models using the enter method were fitted to assess the factors that increased or decreased the risk of smoking. all variables having a p value of 0.05). according to the self - reports of the students, 2.6% (3.5% of boys and 1.7% of girls) were current tobacco smokers, 5.9% (7.5% of boys and 4.2% of girls) were ever tobacco smokers, and 1.8% (2.49% of boys and table 1 shows the sociodemographic characteristic of the participants, according to their current or ever - smoking habits in the univariate analysis. the mean (95% ci) age of the current smokers (15.55 [15.28, 15.82 ] vs. 12.39 [12.33, 12.45 ]) and ever smokers (15.14 [14.94, 15.34 ] vs. 12.30 [12.25, 12.36 ]) was significantly higher than that of the never smokers. the frequency of current tobacco use was significantly higher in boys than in girls (3.49% [95% ci : 2.91, 4.18 ] vs. 1.66% [95% ci : 1.32, 2.08 ]). likewise, the frequency of ever tobacco use was significantly higher in boys (7.48% [95% ci : 6.54, 8.54 ] vs. 4.19% [95% ci : 3.6, 4.87 ]). overall, 3.01% of the urban students and 1.28% of the rural students were current tobacco smokers. the corresponding figures for ever tobacco use were 6.54% and 3.8%, respectively, with a significantly higher frequency in urban than rural residents (p 3 days (or:1.96 [95% ci : 1.38, 2.79 ]), hookah smoking by the father (or : 3.02 [2.15, 4.25 ]), hookah smoking by sister(s) or brother(s) (or : 4.01 [95% ci : 2.76, 5.8 ]), hookah smoking by other members of the family (or : 4.00 [95% ci : 2.82, 5.67 ]), cigarette smoking by the father (or : 1.63 [95% ci : 1.26, 2.1 ]), and more than 2 hours / day screen time compared to less than 2 hours / day screen time (or : 1.64 [95% ci : 1.26, 2.14 ]). female gender (or : 0.51 [95% ci : 0.38, 0.70 ]) and living in a rural area (or : 0.62 [95% ci : 0.40, 0.97 ]) decreased the risk of current smoking. the association between the independent variables in the univariate model and hookah use are presented in table 3. as shown, the mean age of the participants who smoked a hookah was significantly higher than those who did not (15.75 [95% ci : 15.47, 16.03 ] vs. 12.39 [95% ci : 12.33, 12.45 ]). the frequency of hookah use was significantly higher among boys than girls (2.49% [95% ci : 2.02, 3.08 ] vs. 1.14% [95% ci : 0.87, 1.50 ]) and among urban than rural students (2.14% [1.79, 2.56 ] vs. 0.85% [0.55, 1.32 ]). the association between all the factors, except the mother s educational level, living with parents, family size, and physical activity, with hookah use was statistically significant in the univariate model. in the mlr model, age, the number of days spent with friends, hookah smoking or cigarette smoking by the father, hookah smoking by sister(s) or brother(s), and screen time increased the risk of hookah smoking, and female gender and living in a rural area decreased the risk of hookah smoking in students (table 4). this nationwide survey, which to the best of our knowledge is the first of its kind in the middle east and north africa region, showed that tobacco use is a health concern in iranian children and adolescents. this finding is in line with previous studies in different countries (6, 13 - 17). likewise, some previous studies in iran reported a higher frequency of current and ever smoking among boys than girls (6, 15, 16, 18 - 20). the current study suggested that the presence of a smoker in the family, especially hookah use by a sister or brother, increased the risk of smoking in adolescents. this finding is consistent with a previous nationwide iranian study, which showed that tobacco use at home, especially by a sister or brother, increased the likelihood of smoking in adolescents (13). another study in iran found that smoking by friends and family members was associated with smoking among medical students (20). many previous studies confirmed that tobacco use by parents and other relatives was a risk factor for intention to smoke in adolescents (21 - 25). in the current study, spending more time with friends increased the risk of smoking in adolescents. given the impact of friends on adolescent smoking, it seems that spending more time with friends increases the risk of smoking. inadequate parental supervision of children may also explain the association between the time spent with friends and the increased risk of smoking. some researchers documented that direct peer pressure was positively and independently associated with smoking and suggested that authoritative parents supervised and prevented their children associating with deviant peers, thereby preventing smoking in adolescence (21, 22). some studies also suggested that peer smoking was associated with positive attitudes toward cigarettes smoking (23) and might increase the friendship with deviant peers and the risk of smoking (24). nonsmoker parents and strong family monitoring and bonding were reported to be associated with a lower risk of smoking initiation among children (25). another study reported statistically significant differences between tobacco smokers and nonsmokers according to age, grade, the mother s occupation, and education (10). a study of high - school boys documented a significant relationship between smoking behavior of best friends and the intention to smoke among peers (26). attachment to friends and having friends who smoked were common factors related to the onset of smoking in both male and female adolescents (27). in the present study, screen time of more than 2 h increased the risk of smoking 1.64 times in adolescents. a study conducted in six european countries suggested a causal relationship between exposure to smoking in movies and smoking initiation in adolescents (28). in addition to this mechanism, we suggest that prolonged screen time and smoking can be considered two health risk behaviors as different aspects of an unhealthy lifestyle. the present study showed that the prevalence of current hookah (or kalian in the persian language) use among adolescents was 2.49 (2.02, 3.08) in boys and 1.14 % (0.87, 1.5) in girls. a report by iran gyts in 2007 found that 16.5% of students aged 13 - 15 years were current hookah users (30). a study of tunisians aged 13 - 17 years reported that 5.2% were current hookah users (31). other studies conducted in new jersey in the u.s. and beirut reported that 9.7% of high - school students (32) and 29.6% of secondary - school students were current hookah users (33). our findings suggested that the use of a hookah or cigarettes by family members was associated with hookah use in students. this finding is in line with that of amok and colleagues who reported that the presence of a hookah user at home increased current hookah use by students (34) and the study by jamil. who reported that having a father, mother, or sibling who smoked a hookah at home was a significant risk factor for current hookah smoking (35). a study in iran demonstrated that having a hookah smoker in the family was associated with hookah use (36). rice and colleagues reported that having friends and family members who smoked were predictors of cigarette smoking and hookah use (37). in the current study, increased spent time with friends was associated with hookah use in adolescents. a study of high - school student in san diego county showed that half of students first understand about hookah from friends (38). another study reported that 90.7% of students preferred to use a hookah with friends (3). in the current study, hookah use was more common among older students and male students. many studies have demonstrated that hookah use increased with age (3, 34, 39, 40) and that it was more common among males than females (41). however, in studies conducted by jamil., male gender and younger age were associated with current hookah smoking (35, 42). one of the main strengths of the present study is that it was based on a large national representative sample of iranian children and adolescents. moreover, it adhered to the protocol of the world health organization s global school - based student health survey. the main limitation of this study was its cross - sectional design and some limitation of recall bias of participants in some information. considering the harmful effects of smoking, public health practitioners and health care providers should identify high - risk individuals and design educational programs to prevent the use of all kinds of tobacco products. the preparation of strategies on the promotion of a healthy lifestyle through vast advocacy on the health impacts of tobacco smoking and access to appropriate health services as needed should be considered as the most priorities of youth health.	backgroundthe consumption of tobacco through a hookah is growing in popularity, especially among children and adolescents, but little is known about the determinants of hookah smoking.objectivesthe current study aimed to assess the determinants of tobacco smoking and hookah smoking in a nationally representative sample of iranian children and adolescents.patients and methodsthis study was conducted as part of the fourth cross - sectional survey of a national school - based program. using a cluster random sampling method, a validated questionnaire was completed anonymously by 14,880 students who were aged 6 - 18 years and living in urban and rural areas of 30 provinces in iran.resultsthe final study group consisted of 13,486 children and adolescents (participation rate of 90.6%), of whom 49.2% were girls and 75.6% were urban residents. the mean age was 12.47 3.36 years. according to the self - reports of the students, 2.6% (3.5% of boys and 1.7% of girls) were current tobacco smokers, 5.9% (7.5% of boys and 4.2% of girls) were ever tobacco smokers, and 1.8% (2.49% of boys and 1.14% of girls) were current hookah smokers. based on a multiple logistic regression (mlr) model, the following factors increased the risk of current smoking : age, number of days spent with friends per week, hookah smoking or cigarette smoking by the father, hookah smoking by siblings, hookah smoking by other members of the family, and screen time. the age, number of days spent with friends, hookah or cigarette smoking by the father, hookah smoking by siblings, and screen time increased the risk of hookah smoking. female gender and living in rural areas decreased the risk of current tobacco and hookah smoking.conclusionspreventive measures against tobacco use should be underscored for iranian families. the preparation of strategies on the promotion of a healthy lifestyle should be considered a health priority.
the epic - interact is a large prospective type 2 diabetes case - cohort study (16) nested within the epic study (17) with more than half a million adult participants recruited in the 1990s from the following 10 european countries : denmark, france, germany, greece, italy, the netherlands, norway, spain, sweden, and the united kingdom. with the exception of greece and norway, all epic countries participated in the epic - interact study (n = 455,680). after the exclusion of individuals without stored blood (n = 109,680) or with prevalent diabetes at baseline (5,821), 340,234 participants with 3.99 million person - years of follow - up were included in this study. all participants gave written informed consent, and the study was approved by the local ethics committee in the participating countries and the internal review board of the international agency for research on cancer. a pragmatic, high - sensitivity approach for case ascertainment was used in order to identify all potential incident type 2 diabetes cases and to exclude all individuals with prevalent diabetes (16), using at least two multiple sources of evidence including self - report and linkage to primary care registers, secondary care registers, medication registers, and hospital admissions and mortality data. cases in denmark and sweden were not ascertained by self - report, but were identified via local and national diabetes and pharmaceutical registers, and hence were considered as verified. follow - up was censored either on 31 december 2007, the date of type 2 diabetes diagnosis, or the date of death, whichever occurred first. in total, 12,403 verified incident type 2 diabetic cases were identified. a random subcohort of 16,835 individuals was selected from the 340,234 participants with available stored blood samples, stratified by center. after the exclusion of 681 individuals without information on diabetes status, 16,154 subcohort individuals were included, of whom 778 individuals developed incident type 2 diabetes during follow - up. of the 27,779 participants (12,403 case subjects, of whom 778 were within the subcohort of 16,154 participants) in the epic - interact study, we excluded 619 participants within the lowest and the highest 1% of the distribution of the ratio of reported energy intake (determined from the questionnaire) to estimate energy requirements (calculated from age, sex, body weight, and height). in addition, we excluded 1,072 participants with missing information on nutritional intake or other covariates used in the statistical analysis. this resulted in a final sample of 26,088 participants for inclusion in the current analysis with 11,559 case subjects and a subcohort of 15,258 participants, including 729 case subjects in the subcohort. habitual diet during the 12 months prior to recruitment was recorded using country - specific validated food frequency questionnaires or diet histories (17,18). most centers adopted a self - administered questionnaire of 98 to 266 food items. in spain and ragusa (italy) questionnaires in france, italy, spain, the netherlands, and germany were quantitative, estimating individual average portion size systematically. those in denmark, naples (italy), and ume (sweden) were semiquantitative, with the same standard portion assigned to all subjects. in malm (sweden) and total energy and nutrient intakes were estimated using the standardized epic nutrient database (19). estimated flavonoid and lignan intake was derived from foods included in the dietary questionnaires through a comprehensive food composition database on flavonoids and lignans, as we have previously described (20,21). department of agriculture databases (22), phenol - explorer (23) and the u.k. this database compiles composition data on lignans and the six flavonoid subclasses (supplementary table 1). furthermore, our flavonoid food composition database was expanded by using retention factors when no analytical data were provided by cooked food. the retention factors applied to all flavonoid classes, except isoflavones, were 0.70, 0.35, and 0.25, respectively, after frying, cooking in a microwave oven, and boiling (25). these retention factors were not applied to isoflavones and lignans because their cooking losses are usually minimal. our database was also expanded by calculating the flavonoid content of recipes, estimating missing values based on similar foods (by botanical family and plant part), obtaining consumption data for food group items, and using botanical data for logical zeros (when negligible amounts of flavonoids or lignans would be present in a food type, e.g., anthocyanidins in plant foods without red, blue or purple color). in nature, flavonoids and lignans are usually found as glycosides, mainly with glucose or rhamnose moieties, but other sugars may also be involved. therefore, data on flavonoids and lignans are expressed as aglycone equivalents, after conversion of the flavonoid glycosides into aglycone contents using their respective molecular weights. the final database contains 1,877 food items, including raw foods, cooked foods, and recipes, and 10% of values for these food items are missing. a lifestyle questionnaire was used to collect information about sociodemographic characteristics, smoking status, and medical history (17). occupational and leisure - time physical activity was assessed by questionnaire and classified according to the cambridge physical activity index (26). a history of previous illness included hypertension, hyperlipidemia, previous cancers, and/or cardiovascular diseases (angina, stroke, and myocardial infarction). information on family history of type 2 diabetes in a first - degree relative was collected for all participants except for individuals in italy, spain, germany, and oxford (u.k.). height, weight, and waist circumference were measured by trained health professionals using standardized protocols, except in oxford (u.k.) and france, where self - reported measurements were obtained, and ume (sweden), where waist circumference was not recorded (16). blood samples were collected at baseline, and hemoglobin a1c (hba1c) was measured using high - performance liquid chromatography (diamat automated glycated hemoglobin analyzer ; bio - rad laboratories ltd., hemel hempstead, u.k.). dietary questionnaire - derived means, sds, medians, and 5th and 95th percentiles of total intake and intakes of subclasses of flavonoids and lignans were calculated. total flavonoid intake by country was also visualized in a box - and - whisker plot. baseline characteristics and dietary intakes in the subcohort were summarized by quintiles of total flavonoid intake using means and sds or frequencies. prentice - weighted cox regression models accounting for the case - cohort design (27) were used to estimate the associations between flavonoid and lignan intakes and type 2 diabetes of each epic country. total intake and intakes of subclasses of flavonoids and lignans were categorized using sub - cohort - wide quintiles. tests for linear trend were performed by assigning the medians of each quintile as scores. intakes were also analyzed continuously, after a log2 transformation that indicates a doubling in flavonoid and lignan intakes. age was used as the underlying time scale, with entry time defined as the participant s age at baseline, and exit time as the participant s age at diagnosis of diabetes, censoring, or death (whichever came first). all analyses were stratified by center to control for center effects such as follow - up procedures and questionnaire design. model 1 included age (as underlying time scale), sex, and total energy intake (kilocalories per day). model 2 was additionally adjusted for the following potential lifestyle confounders : educational level (none, primary school, technical / professional, secondary school, higher education) ; physical activity (inactive, moderately inactive, moderately active, and active) ; smoking status (never, former, and current) ; bmi (kilograms per square meter) ; and alcohol intake (grams per day). model 3 was additionally adjusted for the following potential dietary confounders : intakes of red meat, processed meat, sugar - sweetened soft drinks, and coffee (grams per day). model 4 was additionally adjusted for the following potential mediators : intakes of fiber (grams per day), vitamin c (milligrams per day), and magnesium (milligrams per day). hrs and 95% cis were estimated within each country and then combined by using random - effects meta - analysis. between - country heterogeneity was assessed using the i statistic. effect modification by sex, baseline bmi category (bmi 0.8), whereas other flavonoid subclasses (such as anthocyanidins, flavanones, flavones, and isoflavones) had low to moderate correlation (r = between 0.1 and 0.4). the main food sources of total flavonoid intake were fruits (36.4%), tea (33.1%), wine (8.6%), chocolate products (4.2%), fruit juices (3.9%), beer (2.5%), vegetables (2.3%), and legumes (2.3%) (table 1). dietary intake of flavonoids and lignans in the epic - interact subcohort (n = 15,258) baseline characteristics of the subcohort according to quintiles of total flavonoid intake are shown in table 2. participants in the highest quintile of total flavonoid intakes were likely to be older and to have the lowest bmi and waist circumference compared with those participants in the lowest quintile. with increasing total intake of flavonoids, participants tended to have a more health - conscious lifestyle pattern with greater educational level and physical activity ; lower tobacco consumption ; a higher intake of fruits, vegetables, fiber, vitamin c, and magnesium ; and a lower consumption of processed meat. however, participants in the top quintile reported greater alcohol and red meat intake and lower coffee intake. baseline characteristics and dietary intakes of the epic - interact subcohort according to quintiles of total flavonoid intake the pooled hrs (95% cis) for type 2 diabetes by quintiles of total intake and intakes of subclasses of flavonoids and lignans are shown in table 3. significant inverse associations were observed in model 1 (stratified by center and adjusted for age [as underlying time - scale ], sex, and total energy) for total intakes of flavonoids, flavanols (including flavan-3-ol monomers, proanthocyanidins, and theaflavins), anthocyanidins, flavonols, flavones, and lignans. after further adjustment for potential confounders (models 2 and 3), all associations were attenuated but were still statistically significant for flavan-3-ol monomers and flavonols. when fiber, vitamin c, and magnesium intakes were additionally included in the multivariable models (model 4), similar risk estimates were observed between the intake of all flavonoid subclasses and lignans, and the incidence of type 2 diabetes as in model 3, showing significant inverse associations with intakes of flavanols (hr for highest vs. lowest quintile 0.82 [95% ci 0.680.99 ] ; p for trend 0.012) ; flavan-3-ol monomers (hr 0.73 [95% ci 0.570.93 ] ; p for trend 0.029) ; and flavonols (hr 0.81 [95% ci 0.690.95 ] ; p for trend 0.020). a significant trend was also detected for total flavonoids (hr 0.90 [95% ci 0.771.04 ] ; p for trend 0.040). a borderline significant trend was seen for theaflavins (hr 0.83 [95% ci 0.691.01 ] ; p for trend 0.084). no significant association was observed with lignans (hr 0.88 [95% ci 0.721.07 ] ; p for trend 0.119). association between flavonoid and lignan intakes and type 2 diabetes : epic - interact study in multivariable analyses (model 4), similar associations of type 2 diabetes were observed when dietary flavonoid and lignan exposures were assessed as continuous variables after a log2 transformation (fig. 1 and supplementary fig. 2). no statistically significant heterogeneity between countries was detected for the associations of total intake and intakes of subclasses of flavonoids and lignans with type 2 diabetes, except for flavanones (i = 52.8%, p = 0.038) and flavones (i = 53.3%, p = 0.036) (supplementary fig. no interactions were found with sex (p for interaction = 0.609), bmi (p = 0.680), or smoking status (p = 0.526) for total flavonoid intake. hrs (and 95% cis) for incident type 2 diabetes for a doubling of total flavonoid (a) and lignan (b) intakes across countries in the interact study. the pooled hr is based on a random - effects meta - analysis using prentice - weighted cox regression analysis with age as the underlying time scale (model 4 ; see statistical analysis section) ; stratified by center ; and adjusted for sex, educational level, smoking status, physical activity levels, bmi, total energy, and intakes of alcohol, red meat, processed meat, sugar - sweetened soft drinks, coffee, fiber, vitamin c, and magnesium. in sensitivity analyses (supplementary table 2), similar results were observed after the exclusion of diabetes case subjects in whom type 2 diabetes had been diagnosed within the first 2 years of follow - up or participants with prevalent cardiovascular diseases. when family history of diabetes was added in model 4, after the exclusion of 84 non - case subjects from the subcohort with an hba1c level 6.5% (48 mmol / mol) at baseline, the results were almost identical. in this large european case - cohort study, an inverse trend between dietary total flavonoid intake and incidence of type 2 diabetes was observed. flavanols, including flavan-3-ol monomers and flavonols, were the flavonoid subclasses significantly related to a lower hazard of type 2 diabetes. to date, there are only two large u.s. cohort studies that have evaluated the association between the total flavonoid intake and incident type 2 diabetes, each using a different update of the u.s. department of agriculture database on flavonoids (22). only the study using the database release 2.1 (year 2007) observed a consistent inverse association between intake of anthocyanidins and type 2 diabetes risk (8,11). this is in line with the crude, but not the multivariable adjusted, findings in our study, based on the database version from 2007. this inconsistency could be due to the different dietary intakes between studies ; in our study, the median anthocyanidin intake in the first quintile (7.1 mg / day) was similar to that in the third quintile (8.1 mg / day) in the u.s. study, the hrs were almost identical for the third (hr 0.87 [95% ci 0.800.94 ]), fourth (hr 0.88 [95% ci 0.830.94 ]), and fifth quintiles (hr 0.85 [95% ci 0.800.91 ]) compared with the first quintile (8). this suggests that the lower risk of type 2 diabetes due to intake of anthocyanidins might reach a plateau at a certain intake level. two other prospective studies have assessed the relationships between the intake of some flavonoid subclasses and the risk of the development of type 2 diabetes (9,10). the u.s. study reported no association with intake of either flavonols or flavones (9) ; however, the finnish study reported inverse significant trends for two individual flavonols (10), as in our study. these differences in the results for intakes of flavonols and flavanols between european and u.s. studies could be a result of european countries having approximately twice the intake compared with the u.s. (8,21). in both asian studies, inverse associations with isoflavone intakes were reported (12,13), but not in western studies (8,11). asian countries still have the highest isoflavone intakes worldwide (10-fold higher than in european countries) (20,30), which may explain the differences observed in association with type 2 diabetes between asian and western countries. in our study, there was no association between lignan intake and risk of type 2 diabetes, although in a u.s. study, lignan levels were significantly associated with a lower fasting insulin level (31). indeed, in two recent experimental studies lignans have been associated with an improvement of glucose homeostasis by increasing glucose disposal rates and enhancing hepatic insulin sensitivity (14) and an inhibition of -amylase activity (15). the main food sources of flavonoids were fruits and vegetables, tea, and wine. these foods (2,32,33), as well as the mediterranean diet, a dietary pattern based on flavonoid - rich foods (e.g., fruits and vegetables, olive oil, and moderate wine consumption) (3) were associated with a reduced risk of type 2 diabetes in the epic - interact study. studies, where anthocyanidin - rich foods (blueberries and apples / pears) (8) and wine consumption (11), a rich source of anthocyanidins and flavanols, were inversely associated with type 2 diabetes risk. notably, after adjustment for potential compounds co - occurring in flavonoid - rich foods, such as fiber, vitamin c, magnesium, and alcohol, associations between flavonoids and the risk of type 2 diabetes were still statistically significant in the current study, suggesting that it is unlikely that these compounds confound or mediate the association between intake of flavonoids and type 2 diabetes risk. the potential mechanisms underlying these inverse associations between flavonoids and type 2 diabetes risk may include the modulation of the postprandial glucose levels by reducing the activity of digestive enzymes (e.g., -amylase and -glucosidase) (34) and decreasing the active transport of glucose across intestinal brush border membrane, inhibiting sodium glut2 (35). furthermore, some flavonoid - rich extracts improved hyperglycemia and insulin sensitivity in type 2 diabetic mice via activation of amp - activated protein kinase and accompanied by an upregulation of glut4 (36). in vitro, flavonoids also had a protective effect on pancreatic -cells by reducing the inducible form of nitric oxide synthase gene expression mediated through the suppression of nuclear factor-b and c - jun nh2-terminal kinase signaling pathways (37,38). other antioxidant, anti - inflammatory, and antiangiogenic activities of flavonoids may also contribute to their potential protective effect against type 2 diabetes (5). strengths of the current study include the multicenter design and the large sample size at recruitment, from which a large number of verified incident cases of type 2 diabetes accrued during 3.99 million person - years of follow - up. this study also includes a wide variation in flavonoid and lignan intakes among participants in eight european countries. furthermore, we were able to control for a number of plausible confounders and factors that may mask the etiological pathway of the association between flavonoid and lignan intake and type 2 diabetes. in all sensitivity analyses, the associations were almost identical, denoting the robustness of our results. limitations of the current study included the use of a single baseline assessment of diet and other lifestyle variables. in addition, our results may be influenced by measurement errors of the dietary questionnaires that may have attenuated our findings, although country - specific validated questionnaires for some flavonoid - rich foods, such as fruits, vegetables, tea, and wine (17,18), were used. furthermore, flavonoid and lignan intakes are likely to be underestimated since the flavonoid database was incomplete (although an extensive common database was used) (20,21) and herb / plant supplement intakes were omitted in these analyses (up to 5% in denmark, the highest consumer country) (39). nutritional biomarkers offer an alternative and objective method for estimating dietary intake and provide more accurate measures than self - reported questionnaires. to date, there are only a few validated biomarkers of flavonoid and lignan intakes, so further research in this field is warranted (40). however, we were unable able to evaluate the association between the intakes of other polyphenols, such as phenolic acids and stilbenes, and type 2 diabetes because data on these are not yet available in the epic cohort. moreover, the association of dietary intakes of flavonoids and lignans with type 2 diabetes risk might be susceptible to confounding since high flavonoid and lignan intake reflects a healthier lifestyle. in our models, we have adjusted for other determinants of healthy lifestyle ; however, possible residual confounding can not be excluded. in conclusion, this large case - cohort study conducted in eight european countries supports a role for dietary intake of flavonoids in the prevention of type 2 diabetes in men and women. high total intakes of flavonoids, flavanols, flavan-3-ol monomers, and flavonols were associated with a 10, 18, 27, and 19% lower risk, respectively, of type 2 diabetes. these results highlight the potential protective effect of eating a diet rich in flavonoids (a dietary pattern based on plant - based foods) on type 2 diabetes risk.	objectiveto study the association between dietary flavonoid and lignan intakes, and the risk of development of type 2 diabetes among european populations.research design and methodsthe european prospective investigation into cancer and nutrition - interact case - cohort study included 12,403 incident type 2 diabetes cases and a stratified subcohort of 16,154 participants from among 340,234 participants with 3.99 million person - years of follow - up in eight european countries. at baseline, country - specific validated dietary questionnaires were used. a flavonoid and lignan food composition database was developed from the phenol - explorer, the u.k. food standards agency, and the u.s. department of agriculture databases. hazard ratios (hrs) from country - specific prentice - weighted cox regression models were pooled using random - effects meta-analysis.resultsin multivariable models, a trend for an inverse association between total flavonoid intake and type 2 diabetes was observed (hr for the highest vs. the lowest quintile, 0.90 [95% ci 0.771.04 ] ; p valuetrend = 0.040), but not with lignans (hr 0.88 [95% ci 0.721.07 ] ; p valuetrend = 0.119). among flavonoid subclasses, flavonols (hr 0.81 [95% ci 0.690.95 ] ; p valuetrend = 0.020) and flavanols (hr 0.82 [95% ci 0.680.99 ] ; p valuetrend = 0.012), including flavan-3-ol monomers (hr 0.73 [95% ci 0.570.93 ] ; p valuetrend = 0.029), were associated with a significantly reduced hazard of diabetes.conclusionsprospective findings in this large european cohort demonstrate inverse associations between flavonoids, particularly flavanols and flavonols, and incident type 2 diabetes. this suggests a potential protective role of eating a diet rich in flavonoids, a dietary pattern based on plant - based foods, in the prevention of type 2 diabetes.
nonmelanocytic tumors are the most frequent.1 the national cancer institute estimates that approximately 2 million cases of nonmelanocytic skin tumors occurred in 2012 in the usa.2 these tumors cost more than us $ 400 million per year in the united states alone.3 basal cell carcinoma (bcc) is a nonmelanocytic skin cancer that arises from basal cells. eighty percent of nonmelanoma skin cancers are bcc.4 the real incidence of bcc is difficult to determine since there is no cancer registry that collects such specific data.5 published studies suggest that the incidence of bcc is increasing worldwide, and there is significant geographic variability.6,7 in caucasian individuals, the lifetime risk of developing bcc is 30%.4,8 australia has the highest rate of bcc in the world.9 the prognosis for patients with bcc is excellent ; it is generally considered as a curable disease.3 bcc occurs most frequently in caucasian persons with environmental exposure ; predisposing genetics may also play a role, such as in the case of albinism, xeroderma pigmentosum, bazex dupre christol syndrome (follicular atrophoderma and bcc), gorlin syndrome, and rombo syndrome. this rare, autosomal, heritable, basal - cell nevus syndrome was found to be responsible for the development of simultaneous bcc in the same patient.10 the clinical presentation of bcc is variable. the nodular form is the most common (70%).11 it appears as pink pearly papule with prominent telangiectatic surface vessels. superficial bcc occurs on 20% of cases ; it appears as reddish patches on the skin that resemble eczema. the morpheaform type (10%) resembles to a yellowish ill - defined mass ; it looks like localized scleroderma.11 bcc lesions generally grow slowly ; however, in some cases, they can become highly aggressive and invade local tissue. in the head and neck region, which is the most common location, the tumor penetrates into underlying tissues (the eyelid or internal canthus) and causes local destruction and disfigurement.4 these tumors are considered as locally advanced. the metastatic dissemination of bcc is exceptional (range : 0.0029%0.55%).12 common sites for metastases are the lymph nodes, lung, bone, and liver.13 the prognosis of metastatic bcc is poor, and the mean survival rates range from 8 months to 3.6 years.14 the majority of patients are successfully treated with cryotherapy, curettage, electrodessication, mohs micrographic surgery, topical treatments (5-fluorouracil [5-fu ], imiquimod), surgical excision, radiation therapy, or photodynamic therapy. in an advanced setting that comprises both locally advanced and metastatic disease, none of these treatments are effective, and systemic therapy remains the unique option. before 2012, physicians had very limited effective drugs for these conditions. a better understanding of the pathogenesis of bcc has led to a revolution in the treatment of this disease. vismodegib was approved on january 30, 2012 by the us food and drug administration (fda). it is the first available oral - targeted therapy for advanced bcc.15 here, we will review bcc s molecular biology, and examine the studies that have led to this major development. bcc is the most common cancer worldwide ; however, metastatic disease remains extremely rare. limited data are available on the treatment of this condition, and there are no prospective phase iii studies. most of the available data come from published case reports or small case series that include bcc and squamous cell carcinomas. chemotherapeutic agents commonly used are doxorubicin, 5-fu, cisplatin, cyclophosphamide, vincristine, etoposide, bleomycin, and methotrexate. a polychemotherapy with cisplatin seems to be the most active.16 a significant response rate was reported with a combination of paclitaxel, 5-fu or blemomycin.17,18 in one report, a complete regression of the tumor was observed after three cycles of combined cisplatin and 5-fu chemotherapy.19 in another case report, the authors observed a complete response with a combination of paclitaxel and carboplatin in lung metastatic bcc.17 the authors also reviewed the literature and found a very high response to platinum - based chemotherapy in 12 other patients ; five with a complete response and four with a partial response. although cisplatin - based regimens are effective, their use is limited by renal and hematological toxicity in a population with advanced age and multiple comorbidities. the epidermal growth factor receptor (egfr) is a member of the tyrosine kinase growth factor receptor family. activation of this tyrosine kinase receptor plays an important role in cell cycle progression, angiogenesis, metastasis, and reduced apoptosis.20 a total of 57% of bccs express egfr.21 cetuximab is a monoclonal antibody that competitively inhibits egfr.22 it has been approved for ras wild - type metastatic colorectal cancer and in recurrent or metastatic head and neck cancer.23 it is a well - tolerated drug. in a phase ii study, cetuximab achieved a 69% disease control rate in patients with unresectable skin squamous cell carcinoma.24 concerning bcc, no prospective trials have been performed, and only isolated cases are available.25 in a retrospective study, eight patients had received cetuximab for cutaneous carcinoma ; three of the four patients who had bcc maintained remission while on treatment. this study suggested that this treatment had a beneficial role for patients that are refractory to standard therapy.26 the hedgehog (hh) signaling pathway was identified in 1980 through an analysis of the fruit fly, drosophila. the discovery was awarded the nobel prize in 1995.27 the hh signaling pathway is a key cascade that plays a major role in cellular growth and differentiation during embryonic periods. the first demonstration of the role of the hh signaling pathway in carcinogenesis has been done through the discovery of the genetic human mutation (ptch1) in gorlin syndrome. this mutation is responsible for the dysregulation of the hh signaling pathway and the development of bcc. a dysregulated hh signaling pathway was also been reported in sporadic bcc, medulloblastoma, and many other cancers, such as those of the gastrointestinal tract, brain, lung, breast, and prostate.28 the hh ligand and two receptor proteins (patched 1 [ptch1 ] and smoothened [smo ]) are involved in the cascade. in the physiologic condition, ptch1, a suppressor protein, forms a complex with smo, and the hh signaling pathway is inactive. when the hh ligand binds with ptch1, smo becomes free and promotes the transcription of the different genes responsible for cellular proliferation and tumor growth. in the stratified epithelium, they cause epidermal hyperplasia with uncontrolled proliferation of the basal cells, leading to bcc.29 ptch mutations were found in 90% of sporadic bcc, while smo mutations were found in 10%. these mutations were also found in other solid tumors. in bcc, studies have suggested that the inactivation of ptch1 or the oncogenic activation of smo are responsible for hh signaling pathway activation;29 their targeting is relevant. vismodegib (gdc-0449, erivedge ; genentech usa, inc., san francisco, ca, usa) is a synthetic, first - in - class, oral, small - molecule agent that binds selectively to smo and inhibits its activity. inhibiting the activation of smo is responsible for the downstream hh target genes, and it stops proliferation of the tumor cell. the discovery of vismodegib was made by a high throughput screening of a small molecule compound library.30 vismodegib was very active in preclinical studies ; it was found to inhibit the growth of tumor cells without inhibiting the proliferation of the normal tissue cells.30 in a phase i trial testing vismodegib in refractory solid tumors, 33 of 68 patients had advanced bcc. a total of 58% of the 33 patients obtained a response (complete or partial), while 6% had a complete response and 48.5% had a partial response. grade 3 adverse events included fatigue, hyponatremia, muscle spasm, and atrial fibrillation. the choice of the dose of 150 mg / day was based on pharmacological data (maximal plasma concentration and pharmacodynamics).31 this study confirms the role played by the hh signaling pathway in the pathogenesis of basal cell tumors, and the importance of inhibiting this cascade. vismodegib has a unique pharmacokinetic (pk) profile ; there is nonlinearity between dose and time. a phase ib, randomized, open - label, multicenter study compared the effect of different dosing schedules on the safety and steady - state plasma pk profiles of vismodegib. a total of 67 patients with advanced solid tumors were included, and they received 150 mg per day for 11 days, and they were then randomized between a 150 mg maintenance dose administered every day, or one time (qw), or three time a week (tiw). the qw or tiw groups failed to maintain unbound plasma concentrations, such as daily maintenance.32 a pivotal multicenter, nonrandomized study was realized. this two - cohort phase ii study (erivance bcc) included 104 patients : 33 patients in the metastatic setting, and 71 with unresectable bcc that were not amenable to radiation therapy. the most relevant adverse events were muscle spasms, dysgeusia, weight loss, alopecia, and fatigue, which affected 30% of patients. moreover, 25% of patients experienced serious adverse events that caused seven deaths.33 based on the positive results of this study, vismodegib was reviewed under the fda s priority review program, and it was approved for use on january 30, 2012.15 vismodegib was also tested in patients with gorlin syndrome.34 a randomized, double - blind, phase ii study compared vismodegib to placebo in basal cell nevus syndrome. no residual disease was present in 83% of the biopsy of the clinically regressed bcc. chang.35 performed an expanded access study testing vismodegib in 119 patients with advanced bcc. the objective responses were 46.4% and 30.8% in locally advanced and metastatic bcc, respectively. the most common adverse events were muscle spasms (70.6%), dysgeusia (70.6%), alopecia (58.0%), and diarrhea (25.2%). these studies (table 1) confirm the clinical relevance of targeting hh pathway treatment guided by biology will change our daily practice and our way of conceiving treatment trials. actually, several ongoing studies are continuing to investigate vismodegib in the treatment of several tumors. active phase ii studies on sarcoma, medulloblastoma, pancreatic adenocarcinoma, glioma, and advanced head and neck bcc are currently in the recruiting stage.36 bcc is the most common cancer worldwide ; however, metastatic disease remains extremely rare. limited data are available on the treatment of this condition, and there are no prospective phase iii studies. most of the available data come from published case reports or small case series that include bcc and squamous cell carcinomas. chemotherapeutic agents commonly used are doxorubicin, 5-fu, cisplatin, cyclophosphamide, vincristine, etoposide, bleomycin, and methotrexate. a polychemotherapy with cisplatin seems to be the most active.16 a significant response rate was reported with a combination of paclitaxel, 5-fu or blemomycin.17,18 in one report, a complete regression of the tumor was observed after three cycles of combined cisplatin and 5-fu chemotherapy.19 in another case report, the authors observed a complete response with a combination of paclitaxel and carboplatin in lung metastatic bcc.17 the authors also reviewed the literature and found a very high response to platinum - based chemotherapy in 12 other patients ; five with a complete response and four with a partial response. although cisplatin - based regimens are effective, their use is limited by renal and hematological toxicity in a population with advanced age and multiple comorbidities. the epidermal growth factor receptor (egfr) is a member of the tyrosine kinase growth factor receptor family. activation of this tyrosine kinase receptor plays an important role in cell cycle progression, angiogenesis, metastasis, and reduced apoptosis.20 a total of 57% of bccs express egfr.21 cetuximab is a monoclonal antibody that competitively inhibits egfr.22 it has been approved for ras wild - type metastatic colorectal cancer and in recurrent or metastatic head and neck cancer.23 it is a well - tolerated drug. in a phase ii study, cetuximab achieved a 69% disease control rate in patients with unresectable skin squamous cell carcinoma.24 concerning bcc, no prospective trials have been performed, and only isolated cases are available.25 in a retrospective study, eight patients had received cetuximab for cutaneous carcinoma ; three of the four patients who had bcc maintained remission while on treatment. this study suggested that this treatment had a beneficial role for patients that are refractory to standard therapy.26 the hedgehog (hh) signaling pathway was identified in 1980 through an analysis of the fruit fly, drosophila. the discovery was awarded the nobel prize in 1995.27 the hh signaling pathway is a key cascade that plays a major role in cellular growth and differentiation during embryonic periods. the first demonstration of the role of the hh signaling pathway in carcinogenesis has been done through the discovery of the genetic human mutation (ptch1) in gorlin syndrome. this mutation is responsible for the dysregulation of the hh signaling pathway and the development of bcc. a dysregulated hh signaling pathway was also been reported in sporadic bcc, medulloblastoma, and many other cancers, such as those of the gastrointestinal tract, brain, lung, breast, and prostate.28 the hh ligand and two receptor proteins (patched 1 [ptch1 ] and smoothened [smo ]) are involved in the cascade. in the physiologic condition, ptch1, a suppressor protein, forms a complex with smo, and the hh signaling pathway is inactive. when the hh ligand binds with ptch1, smo becomes free and promotes the transcription of the different genes responsible for cellular proliferation and tumor growth. in the stratified epithelium, they cause epidermal hyperplasia with uncontrolled proliferation of the basal cells, leading to bcc.29 ptch mutations were found in 90% of sporadic bcc, while smo mutations were found in 10%. these mutations were also found in other solid tumors. in bcc, studies have suggested that the inactivation of ptch1 or the oncogenic activation of smo are responsible for hh signaling pathway activation;29 their targeting is relevant. vismodegib (gdc-0449, erivedge ; genentech usa, inc., san francisco, ca, usa) is a synthetic, first - in - class, oral, small - molecule agent that binds selectively to smo and inhibits its activity. inhibiting the activation of smo is responsible for the downstream hh target genes, and it stops proliferation of the tumor cell. the discovery of vismodegib was made by a high throughput screening of a small molecule compound library.30 vismodegib was very active in preclinical studies ; it was found to inhibit the growth of tumor cells without inhibiting the proliferation of the normal tissue cells.30 in a phase i trial testing vismodegib in refractory solid tumors, 33 of 68 patients had advanced bcc. a total of 58% of the 33 patients obtained a response (complete or partial), while 6% had a complete response and 48.5% had a partial response. grade 3 adverse events included fatigue, hyponatremia, muscle spasm, and atrial fibrillation. the choice of the dose of 150 mg / day was based on pharmacological data (maximal plasma concentration and pharmacodynamics).31 this study confirms the role played by the hh signaling pathway in the pathogenesis of basal cell tumors, and the importance of inhibiting this cascade. vismodegib has a unique pharmacokinetic (pk) profile ; there is nonlinearity between dose and time. a phase ib, randomized, open - label, multicenter study compared the effect of different dosing schedules on the safety and steady - state plasma pk profiles of vismodegib. a total of 67 patients with advanced solid tumors were included, and they received 150 mg per day for 11 days, and they were then randomized between a 150 mg maintenance dose administered every day, or one time (qw), or three time a week (tiw). the qw or tiw groups failed to maintain unbound plasma concentrations, such as daily maintenance.32 a pivotal multicenter, nonrandomized study was realized. this two - cohort phase ii study (erivance bcc) included 104 patients : 33 patients in the metastatic setting, and 71 with unresectable bcc that were not amenable to radiation therapy. the most relevant adverse events were muscle spasms, dysgeusia, weight loss, alopecia, and fatigue, which affected 30% of patients. moreover, 25% of patients experienced serious adverse events that caused seven deaths.33 based on the positive results of this study, vismodegib was reviewed under the fda s priority review program, and it was approved for use on january 30, 2012.15 vismodegib was also tested in patients with gorlin syndrome.34 a randomized, double - blind, phase ii study compared vismodegib to placebo in basal cell nevus syndrome. significant responses and few complete responses were obtained. no progress was reported during treatment. no residual disease was present in 83% of the biopsy of the clinically regressed bcc. chang.35 performed an expanded access study testing vismodegib in 119 patients with advanced bcc. the objective responses were 46.4% and 30.8% in locally advanced and metastatic bcc, respectively. the most common adverse events were muscle spasms (70.6%), dysgeusia (70.6%), alopecia (58.0%), and diarrhea (25.2%). these studies (table 1) confirm the clinical relevance of targeting hh pathway treatment guided by biology will change our daily practice and our way of conceiving treatment trials. actually, several ongoing studies are continuing to investigate vismodegib in the treatment of several tumors. active phase ii studies on sarcoma, medulloblastoma, pancreatic adenocarcinoma, glioma, and advanced head and neck bcc are currently in the recruiting stage.36 before 2012, we had few therapeutic options in metastatic or locally advanced cases of bcc. vismodegib confirms the important role of the hh cascade and its direct implication on bcc pathogenesis. it has a distinct a mechanism of action, a unique pk profile, and a good tolerance profile. it has become the standard treatment of bcc, and has subsequently expanded our therapeutic arsenal.	although basal cell carcinoma (bcc) is the most common cancer worldwide, its metastatic dissemination is exceptional. before 2012, we had a few treatment options available for metastatic or locally advanced cases. management of these patients was complicated due to the lack of scientific data, the deterioration of a patient s general status, the patient s advanced age, and the presence of multiple comorbidities. the hedgehog signaling pathway is dysregulated in bcc. the exploration of this signaling pathway yielded to a major milestone in the treatment of advanced bcc. vismodegib (gdc-0449), an oral small - molecule agent that targets the hedgehog signaling pathway, demonstrates high levels of activity in clinical trials. it was approved in january 2012 for the treatment of locally advanced or metastatic bcc. vismodegib confirms, once again, the interest in exploring the signal transduction pathways in cancers.
periodontal plastic surgery is defined as a surgical procedure performed to correct or eliminate anatomic, developmental, or traumatic deformities of gingival or alveolar mucosa. the presence of adequate zone of gingiva was considered critical for the maintenance of marginal tissue health and for the prevention of continuous loss of connective tissue attachment (naber 's 1954). the prevailing concept is thus that a narrow zone of gingiva was insufficient (a) to protect the periodontium from injury caused by friction forces encountered during mastication and (b) to dissipate the pull on the gingival margin created by the muscles of the adjacent alveolar mucosa. gingival recession displaces the gingival margin apically, reducing the vestibular depth, which is measured from the gingival margin to the bottom of the vestibule. multiple techniques have been developed to obtain predictable root coverage ; however, need for a graft that has its own blood supply, which can be harvested adjacent to the recession defect in sufficient amounts without requiring any second surgical site and has the potential for promoting the regeneration of lost periodontal tissue, is a long - felt need. the present case report describes a technique where the vestibular deepening was carried out with the fenestration technique and the layer of periosteum that was scraped to create the fenestration was used as a pedicle flap for the treatment of a single tooth gingival recession. a 17-year - old girl reported to the department of periodontics with the chief complaint of unesthetic appearance of her front lower teeth [figure 1 ]. on examination, it was found that 6 mm deep and 4 mm wide class ii gingival recession (miller, 1985) was there on the lower left central incisor [figures 2 and 3 ]. the tooth was slightly labially placed and patient also gave the history of tooth brush trauma. the vestibular depth and there was no mobility associated with the tooth. for the root coverage, increase in width of attached gingiva and vestibular deepening the periodontal plastic surgery was planned with a single stage fenestration technique and root coverage using the periosteal pedicle graft. she was explained about the surgery and signed informed consent was taken by the patient. a general assessment of the patient was made through her history, clinical examination and routine laboratory investigations. before surgery, the patient received phase - i therapy, which included oral hygiene instructions and scaling and root planning with ultrasonic and hand instruments. two weeks after phase i therapy, the patient was planned for surgical procedures. on the day of surgery 15 surgical blade at the mucogingival junction retaining all of the attached gingiva [figures 4 and 5 ]. a split thickness flap was reflected sharply, dissecting muscle fibers and tissue from the periosteum. this was then sutured in the depth of the vestibule using resorbable 5 - 0 sutures [figure 6 ]. a strip of periosteum was then removed at the level of the mucogingival junction, causing a periosteal fenestration exposing the bone. the care was taken not to remove the periosteal strip completely and to leave it pedicled to the bone and the rest of the surrounding periosteum at the lateral end [figures 7 and 8 ]. first, intracrevicular incision and a second incision made parallel and apical to the first incision [figure 9 ]. the incisions were followed by split - thickness dissection of the facially located tissue up to the level of the vestibular incision so as to create a tunnel [figures 10 and 11 ]. the exposed root surface was root planed with curettes to remove bacterial contamination and was biomodified using the tetracycline powder mixed with saline. the pedicled periosteal donor tissue was then moved vertically towards the recession area, passing through the tunnel [figures 12 - 14 ]. at repositioning, the osteoperiosteal portion was closely adapted to the recipient site by pressing for 3 min and then sutured along with the overlying gingival tissue, to the recipient bed, using 5 - 0 resorbable sutures [figures 15 and 16 ]. class ii miller 's recession in lower left central incisor 6 mm length of recession 4 mm width of recession vestibular incision placed extending from canine to canine exposing the periosteum diagrammatic representation (a) incision for vestibular deepening and (b) periosteum exposed labial mucosa sutured to the periosteum at the apical level diagrammatic representation : (a) elevated periosteum and (b) bone fenestration after elevating periosteal pedicle flap periosteum scraped to create the fenestration, periosteum remains attached to bone at one end diagrammatic representation : (a) crevicular incision and (b) 2 horizontal incision tunnel created at the recipient site diagrammatic representation of the preparation of the tunnel at the reciepient site after raising a partial thickness flap pedicled periosteal graft placed over the area of recession through the prepared tunnel diagrammatic representation : (a) periosteal pedicle flap attached at lateral end to bone which acts as a pedicle, (b) periosteum, and (c) bone fenestration after elevating periosteal pedicle flap periosteal graft closely adapted to exposed root diagrammatic representation : (a) periosteal pedicled flap sutured on the recession after passing through the tunnel and (b) periosteal pedicle flap elevated and rotated toward the recession defect graft sutured with 5 - 0 resorbable suture periodontal dressing (coe - pak ; gc america inc.) was applied over the operated area covering the exposed bone [figure 17 ]. antibiotic therapy (amoxicillin 500 mg, thrice daily and analgesic (ibuprofen 400 mg twice daily) was prescribed for 5 days. tooth - brushing was discontinued for the first 2 weeks at the surgical site and 0.2% chlorhexidine mouth rinse was instructed till 4 weeks after surgery. coepak was removed 10 days after the surgical procedure and the patient was asked to maintain meticulous oral hygiene. healing had proceeded uneventfully, with secondary wound closure [figure 18 ]. in 3 weeks, healing was nearly complete, with minimal post - operative discomfort to the patient [figure 19 ]. at 6 months post - operative, root coverage was nearly 100% of the recipient site, with minimal probing depths, no inflammation, and a favorable esthetic result [figure 20 ]. post - operative view after 10 days 3 weeks post - operative view 6 months post - operative view showing complete root coverage the major therapeutic goals of mucogingival surgery are esthetics, treatment of hypersensitivity and prevention of root surface caries. it was believed that an inadequate zone of gingiva would (a) facilitate subgingival plaque formation because of the improper pocket closure resulting from the movability of the marginal tissue and (b) favor attachment loss and soft - tissue recession because of less tissue resistance to apical spread of plaque - associated gingival lesion. it was also considered that a narrow gingiva in combination with a shallow vestibular fornix might (a) favor the accumulation of food particles during mastication, and (b) impede proper oral hygiene measurement. hence, vestibular deepening should be considered where patients experience discomfort during brushing and chewing. the indications for surgical treatment of gingival recession include reducing root sensitivity, minimizing cervical root caries, increasing the zone of attached gingiva, and improving esthetics. miller defined complete root coverage as the location of soft - tissue margin at the cemento - enamel junction presence of clinical attachment to the root, sulcus depth of 2 mm or less and absence of bleeding on probing. the purposes of developing new techniques are to increase predictability and to reduce patient discomfort, number of surgeries and the number of surgical sites, together with the need to satisfy the patient 's esthetic demands, which include the final color and a tissue blend of the grafted area. the ideal requirement of graft is that it should have its own blood supply and the potential for promoting the regeneration of lost periodontal structures. the adult human periosteum is highly vascular and is known to contain fibroblasts and their progenitor cells (i.e., osteoblasts) and stem cells. in all age groups, the cells of the periosteum retain the ability to differentiate into fibroblasts, osteoblasts, chondrocytes, adipocytes, and skeletal myocytes. the tissues produced by these cells include cementum with the periodontal ligament fibers and bone ; in addition, the presence of the periosteum adjacent to gingival recession defects occurs in sufficient amounts, making it suitable for a graft. the adult human periosteum is highly vascular and comprises of at least two layers, an inner cellular layer or cambium layer and outer fibrous layer. because of the osteogenic potentiality of periosteum it has been considered as a grafting material for the repair of bone and joint defects. there are very limited studies, which have mentioned the use of periosteum for the treatment of gingival recession defects successfully. mahajan reported the successful treatment outcome by using the periosteal pedicle graft for treating gingival recession defects. used periosteum as a barrier membrane for the treatment of periodontal defects in their studies. the present case report successfully demonstrated a single stage technique for vestibular deepening and recession coverage utilizing the periosteum as autograft for the treatment of gingival recession defect. the success rates of root coverage procedures vary because coverage depends on several factors, including location and classification of the gingival recession and the technique used. an increase in gingival height independent of the number of millimeters is considered a successful outcome of gingival augmentation procedures. the present case demonstrates that an adequate attachment occurred after the use of the pedicled periosteal flap in the treatment of denuded root. an increase in the width of keratinized gingiva occurred after the combined vestibular deepening procedure. according to lomelcher such periosteal activation may result in the differentiation of cells portraying the ability to produce cementum and connective tissue and may lead to enhanced cementogenesis and fiber reattachment to tooth structure, demineralized in situ. histologic studies done by wilderman and wentz have shown connective tissue attachment of the replaced tissues to previously denuded root surfaces. thus, it seems obvious that some kind of connective tissue reattachment is a possibility with the osteostimulated repositioned periosteal flap. the success of the technique may be due to the high vascularity of the graft, the single surgical site, patient comfort, reduced intraoperative time and minimum post - operative complications and the low cost of treatment. throughout the follow - up period, the patient maintained good plaque control and hence the plaque did not have any influence on the final stable attachment that was achieved. the limitation of the technique remains that it is technique sensitive and it can be used only for single tooth recessions with an inadequate width of attached gingiva. however, the lack of a second surgical site and good post - operative results achievable makes it a viable procedure for miller 's class ii recessions with an inadequate width of attached gingiva. the technique used here is proposed to give better results than the previous techniques described in literature owing to the dual blood supply, i.e., from the pedicled periosteum and secondly from the periosteum present below the gingival mucosa used to create the tunnel. the present case reported an excellent post - operative outcome showing great coverage of exposed root surface and increase in width of attached gingiva and keratinized gingiva. the advantages of periosteal pedicled flap over the gold standard technique i.e., subepithelial connective tissue graft for treating the gingival recession is that : the periosteal pedicle flap does not require a second operation to obtain a donor tissuesufficient amount of tissue can be obtained from adjacent to the defectless surgical traumavestibular deepening and root coverage in a single stagedual blood supply to the periosteumless post - operative complicationsbetter patient satisfaction. the periosteal pedicle flap does not require a second operation to obtain a donor tissue sufficient amount of tissue can be obtained from adjacent to the defect vestibular deepening and root coverage in a single stage dual blood supply to the periosteum less post - operative complications better patient satisfaction. thus, it can be concluded that a periosteal pedicled flap, when combined with a fenestration technique for vestibular deepening, offers a successful and viable alternative for the coverage of localized gingival recessions with an inadequate width of attached gingiva.	gingival recession along with reduced width of attached gingiva and inadequate vestibular depth is a very common finding. multiple techniques have been developed to obtain predictable root coverage and to increase the width of attached gingiva. usually, the width of gingiva is first increased and then the second surgery is caried out for root coverage. the newer methods of root coverage are needed, not only to reconstruct the lost periodontal tissues but also to increase predictability, reduce the number of surgical sites, reduce the number of surgeries and improve patient comfort. hence, this paper describes a single stage technique for increasing the width of attached gingiva and root coverage by using the periosteal pedicle flap.
in contrast to bladder tumors (bt), ureteral urothelial tumors (uut) are uncommon ; the bt : uut ratio is 33:1. the incidence of uut is higher in areas affected by balkan endemic nephropathy (ben), with low grade lesions prevalent. all urothelial tumors have a propensity for multicentric growth in space and time and a high rate of recurrence ; almost every second bt will reoccur. patients with uut face a bt development risk of 2050%, while the risk is 10 times lower inversely. however, it has been proved that the efficacy of conservative surgery is comparable to that of nu in patients from areas affected by ben. endoscopic treatment is indicated for single, small (< 1.5 cm) low grade tumors in older patients at risk from open radical surgery, a solitary kidney, or bilateral disease. if there is a recurrence, it can be treated with an additional endoscopic procedure. the lady was born in 1929 in a small village in south eastern serbia ; she has never smoked. in march 1994 a pathological examination revealed transitional cell cancer (tcc) stage ta, grade g1. over the next 20 years the woman had 24 recurrent low grade bts and five ureteroscopic fulgurations for about 30 urothelial tumors in the left ureter (figure 1). the positions and size of the tumors in the urinary bladder and left ureter from march 1994 march 2012. the first ureteral tumor was seen during a routine cystoscopy, emerging from the left orifice. the tumor was biopsied and fulgurated : pathological examination revealed ureteral tcc, minimal tagi. all further recurrent ureteral tumors were tag1 ; they were discovered by urs after two and after five months and fulgurated immediately. ten months after the appearance of the first ureteral tumor, intravenous urography (ivu) revealed the presence of multiple defects in the left ureter suggestive of tumors (figure 2). intravenous urography presenting multiple tumors in the lower part of the left ureter (april 2009). subsequently, urs confirmed the presence of at least five tumors in the upper ureter and at least 10 tumors in the lower ureter. the tumors in the lower ureter were biopsied, while all other tumors were fulgurated without biopsy. the next evidence of recurrence was in the left ureter after 10 months (one tumor) and after 20 months (10 tumors) (figure 3). the three dimensional (3d) reconstruction based on ct urography showing at least 10 recurrent tumors in the left ureter (february 2012). ten months later, three 35 mm large tumors had occurred in the bladder, around the left orifice. today, the patient is 84 years old and shows no signs of the disease. the author finds this case interesting for a number of reasons : primarily due to the long history of bladder tumors without progression. furthermore, the history of the disease can be divided into two parts : in the first 10 years malignant transformation took place in various parts of the bladder ; in later years the left ureter was the source of malignant cells. the appearance of multiple small bts around the left orifice was most probably the result of downstream seeding of malignant cells from the ureter. in addition, the ureteral tumors were small and low grade, without progression, which is not typical. fulguration, despite multiple recurrences and repeated procedures, the patient is quite well and disease free. intravesical bcg therapy was administered twice, in 1994 and 1998 ; the duration of the therapy was 36 months. after 2001, there was a recurrence free interval, followed by a period of small low grade intraurothelial neoplasias, so bcg therapy was not continued. intraureteral bcg therapy is generally recommended after conservative treatment of uut, especially where carcinoma is present in situ by percutaneous nephrostomy (pcn), or through a ureteric stent. however, the long term results have not been confirmed. in this case, the patient did not receive intraureteral bcg : the tumors were low nevertheless, the fact that massive recurrence appeared nine months later is convincing evidence that topical bcg was actually indicated. however, indications did not quite accord with eau guidelines (unifocal tumor), rather the tumors were small, non infiltrative and low grade. in addition, the patient was elderly and came from an area affected by ben. given the lack of laser equipment, which is in all likelihood superior, urs with fulguration was performed. in the period 20012004, the follow up consisted of control cystoscopy and urine cytology every six months, with a three month interval between cystoscopy and urine cytology., urs has been repeated every 35 months, in combination with 3d reconstruction ct urography. although it is recommended, especially in muscle invasive disease, ureteral cytology has not been performed, due to frequent urs. it is interesting that despite the patient having 24 bts and over 30 uuts this does not reflect the extent of the disease. specifically, if tumor volume is calculated using the sphere formula, the total volume of all bts is around 60 ml, yet the total volume of all uuts is only 1.1 ml. this fact also justifies the ureter sparing procedure, despite the large number of tumors. the only complications were three stenoses in the left ureter, which were mild and did not progress. it can be successfully performed in a highly select group of elderly patients with multiple small superficial low grade uut, especially if they originate from areas known for a low uut malignancy potential.	ureteral urothelial tumors (uut) are uncommon ; their incidence is higher in areas affected by balkan endemic nephropathy (ben), with low grade lesions prevalent. in these patients, the efficacy of conservative surgery is comparable to that of nephroureterectomy. endoscopic treatment is indicated for single, small and low grade uut, in older patients with significant comorbidity.the case of an 84year old lady from an area affected by ben is presented. over 20 years, the patient underwent five endoscopic interventions for multiple uut, and numerous endoscopic interventions for recurrent bladder tumors.among a highly select group of patients, endoscopic treatment of multiple small superficial low grade ureteral tumors may prove successful.
a 55-year - old female presented with 6-month history of progressively worsening right posterior calf pain. on physical exam, the right posterior calf appeared erythematous and warm with decreased range of motion. core needle biopsy of the right calf mass demonstrated diffuse - type giant cell tumor [also known as diffuse - type tenosynovial giant cell tumor (tgct) ] and the patient underwent surgical resection. approximately 7 weeks after surgery, the patient noticed swelling and warmth of the right lower extremity with associated slow growth of friable red soft tissue, approximately 1 cm lateral to the incision. the patient returned to the operating room for resection of suspected recurrent tgct and debridement of devitalized tissue, ultimately followed by amputation. the slowing of disease was short - lived and, despite multiple subsequent systemic therapies first with imatinib, followed by adriamycin ifosfamide, the patient developed extensive metastatic disease with significant tumor burden in the chest and right inguinal region. radiographs taken on presentation revealed mild osteoarthritis in bilateral knees, with fullness posterior to the right knee [figure 1 ]. post - operative ultrasound performed 7 weeks after initial resection showed a complex cystic and solid mass with hypervascular solid components [figure 2 ]. mri demonstrated a large, well - circumscribed, mixed - density soft tissue 20-cm mass, with both solid and cystic components extending from the popliteal fossa to the mid - calf [figure 3 ]. ct of the chest, abdomen, and pelvis showed no evidence of metastatic disease. 55-year - old female with right posterior calf pain diagnosed with tenosynovial giant cell tumor. lateral radiograph of the knee reveals soft tissue fullness in the popliteal fossa (arrow). 55-year - old female with right posterior calf pain diagnosed with tenosynovial giant cell tumor. doppler ultrasound shows prominent vascularity within the solid, echogenic components of the lesion (arrow). 55-year - old female with right posterior calf pain diagnosed with tenosynovial giant cell tumor. sagittal t1-weighted mr post - contrast imaging reveals enhancement of solid areas (arrow). mri done 7 weeks after initial resection showed a heterogeneous lobulated lesion with enhancement of the solid components, centered within the gastrocnemius and extending 7 cm superior to the tibiofemoral joint [figure 4 ]. low - volume lymph nodes in the right inguinal region were unchanged. given the aggressive appearance of the lesion, the patient underwent a below the knee amputation. 55-year - old female 7 weeks post - resection of tenosynovial giant cell tumor with complaints of pain and friable tissue growth at the incision site. (a) coronal t1-weighted post - contrast mri of the right knee shows a heterogeneous lobulated lesion extending to the mid - calf. margins of soft tissue components of the lesion are ill - defined and somewhat infiltrative in some areas (arrowhead). (b) sagittal t2-weighted mr of the right knee shows both cystic (thick arrow) and solid (thin arrow) areas within the heterogeneous lesion. core needle biopsy of the right calf mass demonstrated diffuse - type giant cell tumor (also known as diffuse - type tgct) with a heterogeneous cell population consisting of sheets of larger, epithelioid cells with round nuclei and discrete cell borders in a background of histocytes, lymphocytes, and scattered hemosiderin [figure 5a ], the characteristic appearance of diffuse - type tgct. numerous giant cells and focal necrosis were also present. although the cytology of the tumor was atypical with a high mitotic rate of 27 per 10 high - power fields (hpfs), a diagnosis of malignant diffuse - type tgct was not warranted in this limited sample as the sample did not meet five out of the eight criteria needed for a diagnosis of malignancy (see discussion). immunohistochemical stains were negative for smooth muscle actin (sma), desmin, cd34 (cluster of differentiation protein), pan - k, and s100 protein in the small amount of tissue present. 55-year - old female with right posterior calf pain initially diagnosed with tenosynovial giant cell tumor with subsequent resection demonstrating malignant tenosynovial giant cell tumor. (a) core biopsy sample of the mass stained with hematoxylin and eosin, 400, shows epithelioid cells with round nuclei and a background of lymphocytes, histiocytes, several multinucleated giant cells (thick arrows), and scattered mitoses (thin arrow). (b) although the majority of the resection specimen appears similar to the core biopsy, select areas of the resection stained with hematoxylin and eosin (400), display pleomorphic cytology with large, highly atypical cells (arrow) and atypical mitoses (arrowhead), consistent with the diagnosis of malignant diffuse - type tgct. the right lower extremity amputation specimen, which was received 7 weeks after the initial resection, revealed a large, cystic and solid mass extending from the popliteal fossa to the mid - calf [figure 6 ]. microscopic examination showed areas of large, bizarre, and anaplastic epithelioid cells, warranting a diagnosis of malignant tgct. atypical mitoses were also present, and the tumor showed an infiltrative pattern, with invasion of the skin, associated ulceration and necrosis, and a mitotic rate of 23 per 10 hpfs [figure 5b ]. hemosiderin deposition and characteristic cleft - like spaces were readily apparent, along with widespread areas with relatively bland cytology, the characteristic appearance of benign diffuse - type tgct. 55-year - old female who initially presented with progressive right posterior calf pain now status post right leg amputation for malignant tenosynovial giant cell tumor. gross pathology of the amputated right lower extremity reveals a heterogeneous tumor with cystic (thick arrow) and solid (thin arrow) components extending from the popliteal fossa to the mid - calf. immunohistochemical stains for sma, d2 - 40 (focal), and desmin (rare) were positive in lesional cells, with cd163-positive histiocytes, while s100, pan - k, cd34, and leukocyte common antigen (lca or cd45) immunostains were negative. these differences from the original immunoprofile were minor and also likely due to tumor heterogeneity. core needle biopsy of the right calf mass demonstrated diffuse - type giant cell tumor (also known as diffuse - type tgct) with a heterogeneous cell population consisting of sheets of larger, epithelioid cells with round nuclei and discrete cell borders in a background of histocytes, lymphocytes, and scattered hemosiderin [figure 5a ], the characteristic appearance of diffuse - type tgct. numerous giant cells and focal necrosis were also present. although the cytology of the tumor was atypical with a high mitotic rate of 27 per 10 high - power fields (hpfs), a diagnosis of malignant diffuse - type tgct was not warranted in this limited sample as the sample did not meet five out of the eight criteria needed for a diagnosis of malignancy (see discussion). immunohistochemical stains were negative for smooth muscle actin (sma), desmin, cd34 (cluster of differentiation protein), pan - k, and s100 protein in the small amount of tissue present. 55-year - old female with right posterior calf pain initially diagnosed with tenosynovial giant cell tumor with subsequent resection demonstrating malignant tenosynovial giant cell tumor. (a) core biopsy sample of the mass stained with hematoxylin and eosin, 400, shows epithelioid cells with round nuclei and a background of lymphocytes, histiocytes, several multinucleated giant cells (thick arrows), and scattered mitoses (thin arrow). (b) although the majority of the resection specimen appears similar to the core biopsy, select areas of the resection stained with hematoxylin and eosin (400), display pleomorphic cytology with large, highly atypical cells (arrow) and atypical mitoses (arrowhead), consistent with the diagnosis of malignant diffuse - type tgct. the right lower extremity amputation specimen, which was received 7 weeks after the initial resection, revealed a large, cystic and solid mass extending from the popliteal fossa to the mid - calf [figure 6 ]. microscopic examination showed areas of large, bizarre, and anaplastic epithelioid cells, warranting a diagnosis of malignant tgct. atypical mitoses were also present, and the tumor showed an infiltrative pattern, with invasion of the skin, associated ulceration and necrosis, and a mitotic rate of 23 per 10 hpfs [figure 5b ]. hemosiderin deposition and characteristic cleft - like spaces were readily apparent, along with widespread areas with relatively bland cytology, the characteristic appearance of benign diffuse - type tgct. 55-year - old female who initially presented with progressive right posterior calf pain now status post right leg amputation for malignant tenosynovial giant cell tumor. gross pathology of the amputated right lower extremity reveals a heterogeneous tumor with cystic (thick arrow) and solid (thin arrow) components extending from the popliteal fossa to the mid - calf. immunohistochemical stains for sma, d2 - 40 (focal), and desmin (rare) were positive in lesional cells, with cd163-positive histiocytes, while s100, pan - k, cd34, and leukocyte common antigen (lca or cd45) immunostains were negative. these differences from the original immunoprofile were minor and also likely due to tumor heterogeneity. tgct was first described in 1852 by chassaignac as synovial membrane proliferation involving the flexor tendons of the fingers and was later redefined by jafee., to encompass lesions involving the synovium, tendon sheath, bursa, and joint. the most common type of localized tgct is also referred to as giant cell tumor of the tendon sheath, which typically occurs as a localized nodule on the tendon sheaths of the fingers or toes. on the contrary, the diffuse type tends to infiltrate the joint, and more frequently involves large joints, most commonly the knee, followed by the hip. diffuse - type tgct tends to be more aggressive, often recurring locally (856%), and is capable of malignant transformation. enzinger., first described malignant tgct as uncontrolled growth within a benign lesion or as the recurrence of a previously benign lesion. despite aggressive management, including surgical resection, chemotherapy, and radiation, prognosis in malignant tgct is poor. lesions most commonly metastasize to regional lymph nodes and lungs, with a median survival of 22.5 months after diagnosis. time to metastatic disease ranges from 4 months to 5 years, with a median of 11 months. li., and bertoni., described the following histologic characteristics as suggestive of malignancy : nodular and/or solid infiltrative growth pattern, large round or oval cells, cells with large nuclei and nucleoli, areas of necrosis, and atypical mitoses. criteria for malignant tgct were also developed at the armed forces institute of pathology (afip) to include five out of eight characteristics including diffuse pleomorphism, prominent nucleoli, high cytoplasmic to nuclear ratios, mitotic ratio greater than 10 per 10 hpfs, necrosis, discohesion of tumor cells, paucity of giant cells, and a diffuse growth pattern. our case demonstrated features described by both bertoni., and the afip to be classified as malignant [figure 5b ] and the initial biopsy of the lesion was atypical including a mitotic rate greater than 10 per 10 hpfs as well as necrosis ; however, it did not fulfill enough of the criteria to be classified as malignant tgct. radiographic appearance of tgct is non - specific, ranging from soft tissue swelling to soft tissue mass and bony involvement. radiographs of tgct lesions most commonly demonstrate joint effusion, soft tissue swelling, bony erosion, and lack of calcification in the context of normal bone mineralization and preservation of the joint space. similarly, ultrasound is non - specific ; however, it is useful for characterizing its solid and cystic components. mri is superior due to its better delineation of soft tissues and its ability to more accurately determine the extent of synovial proliferation, joint effusion, bone erosion, and deposits of hemosiderin. tgct lesions generally have hemosiderin deposits, which will appear as dark, low - signal regions on t1-weighted and t2-weighted images, with associated susceptibility artifact on gradient echo sequences. the presence of hemosiderin is characteristic of tgct lesions ; however, it can also be seen in other lesions such as synovial hemangioma or hemophiliac arthropathy and does not correlate with malignant potential. regions of enhancement can be seen in tgct in the regions of synovial proliferation, but do not predict the aggressiveness of the lesion. imaging of malignant tgct is rarely reported, and the only description of the mri characteristics suggestive of malignant tgct is reported in the pathology and rheumatology literature and includes frequent lobulation, infiltrative lesions with poorly defined margins, and close association with tenosynovial structures and/or joint spaces. murphey., suggest that findings most consistent with a malignant lesion are bony invasion and presence of metastases. our patient 's first post - resection mri included multiple areas of lobulation and close association with the joint ; however, these findings are also often seen in benign disease. the most concerning feature of the post - resection mri was diffuse involvement of the medial and lateral gastrocnemius muscles with an infiltrative appearance and loss of clear tissue planes, a finding that was not seen on the initial mri. in all reported cases, the diagnosis of malignant tgct was not made until after histopathologic examination of the biopsied lesion, most likely because of the low incidence of the lesion and the paucity of imaging of malignant tgct in the literature. this case highlights the appearance of malignant tgct on multiple imaging modalities. while no single clinical, histopathologic, or radiographic feature separates benign from malignant lesions, a compilation of findings including rapid recurrence, and infiltrative appearance on imaging with invasion of adjacent soft tissues suggest malignancy. therefore, the current standard of practice remains local wide resection of tgct lesions with close follow - up and tissue sampling when clinically indicated.	malignant tenosynovial giant cell tumor (tgct) is a rare clinical entity that can arise as a recurrent lesion or can co - exist with a benign tgct lesion. malignant tgct most commonly arises in the lower extremity and tends to be clinically aggressive, with most patients developing recurrent lesions or dying. much of the literature describes the histopathologic features and classifies this broad group of tumors, with little description of the imaging characteristics of this disease. we present the multimodality appearance of a case of malignant diffuse - type tgct that recurred 2 months after resection with subsequent rapid clinical progression.
chronic infection with hepatitis b virus (hbv) affects more than 350 million people worldwide and continues to be an important cause of morbidity and mortality. the current therapy for chronic hepatitis b (chb) is based on the use of immunomodulators like pegylated interferon or nucleos(t)ide analogues (nucs) that inhibit both the priming and the elongation steps of viral dna replication [210 ]. the high cost, side effects, and the fact that potent antiviral response can be only achieved in a minor population of patients limit the clinical use of peg - ifn. in addition, as the off - treatment durability of response to nucs is generally low, it is required to maintain a long - term continuous therapy when patients treated with nucs. however, long - term continuous therapy with nucs carries significant risks of occurrence of viral resistance, drug toxicity as well as unsustainable cost for many of the most heavily affected countries. all of these guidelines support both nucs and peg - ifn as first - line treatment options, but the optimal choice for individual patients remains controversial. the choice of therapy is determined by many factors including the stage of the disease, serum alanine transaminase (alt), hbv dna levels, and eag status of the patient. both tenofovir (tenofovir disoproxil fumarate) and adefovir dipivoxil are very potent and effective nucleotide analog against hbv [9, 11, 12 ]. immunologic mechanisms involved in the control of hbv replication in vivo are not yet completely understood [1317 ]. understanding the mechanisms of therapy - induced antiviral immune responses could further direct novel therapeutic strategies. most studies examined the functionality of hbv - specific cd4 t cells and regulatory t cells in blood of patients and found that their functionality is improved in nuc - treated chb patients [1922 ]. however, this effect was only transient. besides, hbv - specific t cells also natural killer (nk) are important effector cells during antiviral immune responses. an early rise in circulating nk cells has been documented in the incubation phase of hbv infection, suggesting that they may contribute to the initial viral containment in this setting. nk cells are innate immune cells and play important roles in the defense against viral infections. they can kill virus - infected cells directly as well as indirectly via antibody - dependent, cell - mediated cytotoxicity. nk cells are usually defined as cd3cd56 lymphocytes and are comprised of about 5%20% of peripheral blood lymphocytes. however, the frequency of nk cells in intrahepatic lymphocytes can increase to about 30%50%. nk cells are a diverse population and nk cells do not possess a single well - defined receptor to recognize antigens on target cells like t cells. instead, their function depends on the expression of activating and inhibitory receptors that recognize various classes of cell surface ligands. these ligands include classical and nonclassical mhc class i antigens, mhc - like proteins, and a variety of other self- and virus - derived molecules. they may be expressed constitutively and/or de novo on the surface of virus - infected cells. only limited information has been published on the role of nk cells in hbv infection. in acute hbv, an early rise in circulating nk cells has been documented, suggesting their contribution to the initial viral containment [25, 28, 29 ]. in the context of persistent hbv infection, nk cell studies mainly focused on nk cell induced tissue injury [30, 31 ]. however, very little is known about the quality of the antiviral functions of nk cells during chronic hbv. it has recently shown that inhibition of chronic hepatitis b virus replication by antiviral therapeutic medicine such as entecavir, lamivudine, and adefovir helps to partially restore function of nk cells in peripheral blood [32, 33 ]. this restoration of nk cell activity was accompanied by an enhanced frequency of ifn--producing cd56 nk cells in blood as well as normalization of the expression of the activating receptor nkg2a on circulating nk cells. controlling of viral replication by antiviral treatment can partially correct this defect but little is known about the impact of antiviral therapy on the frequency of different subsets of nk cells. to this aim, we performed this longitudinal study to examine the dynamic changes of the frequency of different subsets of nk cells in chb patients after initiation of tenofovir or adefovir therapy. we found that nk cell numbers and subset distribution differ between chb patients and normal subjects, furthermore the association was found between alt level and cd158b nk cell in hbv patients. in chb patients treatment with tenofovir, the frequency of nk cells was increased during the treatment, accompanied downregulated expression of nkg2a and kir2dl3, which correlated with serum hbv - dna load. in chb patients treatment with adefovir, nk cell numbers did not differ during the treatment, but also accompanied downregulated expression of nkg2a and kir2dl3. a total of 24 chb patients and 12 gender- and age - matched healthy subjects were recruited at the outpatient clinic of the department of hepatology of the first hospital of jilin university, china from may 2011 to october 2011. individual patients with chb were diagnosed, according to the criteria of detectable hbv virions and hbeag positive or negative (hbvdna 105 copies / ml, an interval of 14 days or more than twice elevated alt of hbeag - positive patients 2 uln(upper limit of normal) ; hbeag - negative patients > 1 uln and 10 uln). there are seven hbeag - positive patients in tdf - group and eight in adv - group. individuals with a history of hepatitis c and hepatitis d, positive antibodies against hcv and hdv, human immunodeficiency virus (hiv), or other inflammatory diseases, such as rheumatoid arthritis, diabetes, autoimmune hepatitis, hypertension, kidney disease, or recent infectious diseases were excluded. written informed consent was obtained from all patients, and the experimental protocol was approved by the ethics committee of the first hospital of jilin university. individual patients with chb were randomized and treated with tenofovir disoproxil fumarae (gsk pharma uk) 300 mg once daily. another group of patients with chb were treated with adefovir disoproxil (gsk pharma uk) 10 mg once daily. all of the patients met the most recent european guideline criteria for treatment of chb and were followed up for 24 weeks. peripheral edta anticoagulant blood samples were obtained from individual participants before and after the treatment longitudinally. hbvserology (hbsag / anti - hbs, hbeag / anti - hbe, quantitative hbsag, quantitative hbeag) were determined by microparticle enzyme immunoassay (meia). the virus loads in individual plasma samples were measured by quantitative pcr using roche cobas taqman hbv test (roche, uk), and the limitation of detecting hbv was 20 iu / ml.the levels of serum aspartate aminotransferase (ast) and alanine transaminase (alt) were detected by biochemistry automatic analyzer (roche diagnostics, branchburg, usa). to determine the percentages of different subsets of nk cells, anticoagulated blood was incubated for 30 min with a cocktail of allophycocyanin (apc)-conjugated cd56 (clone b159), fluorescence isothiocyanate (fitc)-conjugated cd3 (clone ucht1), and percp - cy 5.5-conjugated cd16 (clone 3g8), and stained with pe - conjugated antibodies against cd158a (clone hp-3e4), cd158b (clone ch - l), nkp30 (clone p30 - 15), nkp44 (clone p44 - 8.1), nkp46 (clone 9e2/nkp46, bd pharminge, san diego, usa), nkb1 (clone dx9, bd biosciences, belgium), kir2dl3 (clone 180701), nkg2d - pe (clone 149810), nkg2a (clone 131411), nkg2c (clone 134591, r&d systems, usa), or isotope controls, respectively. the frequency of different subsets of nk cells was characterized by flow cytometry analysis. the difference between two independent groups was analyzed by the mann - whitney u test, and the difference between paired variables by the wilcoxon matched - pairs test using the prism 5.0 (graphpad software, usa). the potential correlation between two variables was analyzed by the spearman 's rank correlation coefficient test. to determine the frequency of nk cells, 24 chb patients and 12 healthy age and gender matched subjects were recruited into this study. characterization of peripheral blood nk cells by flow cytometry analysis indicated that the frequency of cd3cd56 nk cells in chb patients was lower than that in healthy controls (figures 1 and 2). furthermore, analysis of different subsets of nk cells revealed that the frequency of activating receptor nkp30 nk cells in chb patients was significantly higher than that in healthy controls while the frequency of inhibitory receptor cd158b nk cells in chb patients was found to be significantly lower than that in healthy controls(figures 1 and 3). we observed no significant difference in the expression of nkp44, nkp46, nkg2a, nkg2c, nkg2d, nkb1, and cd158a between the health controls and chb patients. interestingly, the concentrations of plasma alt in chb patients were negatively associated with the frequency of cd158b nk cells in chb patients (figure 4). these data indicated that alteration in the frequency of nk cells was associated with liver damage in chb patients. these patients were treated with tenofovir or adefovir. in patients treated with tenofovir or adefovir, alt, ast, and hbv dna there were 7 patients (rate = 58%) whose hbv dna reaches < 3log10 iu / ml in the tdf treated group while only one patient 's hbv dna reaches this level in adv treated group (rate = 8.3%) (table 2). < 3log10 iu / ml) was significantly higher than adv - group at 8 weeks (p = 0.028, fisher test). in seven hbeag - positive patients at 24 weeks after treatment of tenofovir disoproxil, three cases achieved hbeag seroconversion. however, no hbeag seroconversion occurred in 8 patients with adefovir dipivoxil treatment. therefore, different treatments may yield different responses, tdf at a daily dose of 300 mg had superior antiviral efficacy compared with adv at a daily dose of 10 mg through week 24. we found that the frequency of nk cells transiently increased in tenofovir treated group at 8, 12, and 24 weeks after initial treatment (figure 5). these data clearly indicated that treatment with tenofovir modulated the frequency of nk cells. given that different subsets of nk cells have different functions, we characterized the frequency of different subsets of nk cells longitudinally after treatment. treatment with tenofovir or adefovir decreased the frequency of nkg2a and kir2dl3 nk cells at 12 and 24 or 24 weeks after initial treatment in both groups of chb patients (figure 6), respectively. in contrast, treatment with adefovir significantly increased the frequency of cd158b nk cells at 24 weeks after initial treatment in chb patients (figure 7). therefore, treatment with different kinds of nucleos(t)ide analogues (nucs) had differential effects on modulating activating and inhibitory receptor nk cells in chb patients. in this study, we examined the frequency of different subsets of nk cells in patients with chb following nucleos(t)ide analogues (nucs) antiviral therapy. we found that, there was significant difference in the frequency of nk cells between chb patients and healthy controls, the frequency of nkp30 nk cells in chb patients was significantly higher than those in healthy controls. in contrast, the frequency of cd158b nk cells in chb patients was significantly lower than that in healthy controls. more interestingly, the concentrations of serum alt were negatively correlated with the percentages of cd158b nk cells in chb patients. these data indicated that a lower frequency of inhibitory receptor nk cells contributed to liver damages. apparently, a lower frequency of cd158b nk cells may be valuable for the evaluation of liver damage in chb patients. previous studies have shown controversial results on the frequency of nk cells in chb patients following standard therapies [33, 35 ]. we treated chb patients with tenofovir or adefovir and characterized the frequency of different subsets of nk cells by flow cytometry analysis. we found that treatment with tenofovir transiently increased the frequency of nk cells in chb patients at 4, 12, and 24 weeks after initiation of treatment. in contract, the frequency of nk cells does not change significantly over the course of treatment with adefovir. these data suggest that treatment with tenofovir may promote nk cell proliferation during the early periods after treatment. maybe because of tdf had superior antiviral efficacy compared with adv. characterization of different subsets of nk cells indicated that treatment with the antiviral therapy transiently decreased the frequency of nkg2a and kir2dl3 nk cells after treatment. more importantly, the decreased percentages of nkg2a nk cells were negatively correlated with the reduced levels of hbv loads in chb patients early after treatment. these data were similar to that of previous reports in chronic hepatitis b virus infected and chronic hepatitis c virus infected patients [32, 36 ]. although we could not establish significant correlations between the expression of nk cell receptors, such as nkg2a, kir2dl3, and activation status of function, it does not exclude a role for these receptors in the regulation of nk cell activation and/or function. since nkg2a triggering have been shown to regulate ifn- production [37, 38 ], it is tempting to speculate that in chronic hbv infection, increased expression of nkg2a prohibits nk cells from being activated and from functioning. in that respect, the downregulation of nkg2a upon viral load reduction as demonstrated herein may be in part responsible for the improved nk cell function. these data suggest that nkg2a nk cells are crucial for the clearance of hbv in chb patients and that the frequency of peripheral blood nkg2a nk cells may be a valuable biomarker for the evaluation of hbv clearance in chb patients. the balance of activating signals via ncr and inhibitory signals via kir is crucial for systemic nk cell responses, and hbv infection can modulate the interplay between inhibitory and activation signals [39, 40 ]. to further understand the mechanisms of therapy - induced antiviral immune responses, we characterized the frequency of different subsets of nk cells in chb patients with different nucs treatments. we found that treatment with either protocol decreased the frequency of nkg2a, kir2dl3nk cells later after treatment. apparently, treatment with nucs down - regulates inhibitory receptor expression in nk cells in chb patients. these data further indicate that downregulates inhibitory receptor expression in nk cells which can increase nk activity is associated with the clearance of hbv in chb patients. therefore, given that treatment with antiviral therapy significantly reduced the levels of plasma hbv loads and serum alt, the higher frequency of nk cells and decreased inhibitory receptors suggested that activated nk cells participated in the clearance of hbv in chb patients. we compared the different subsets between tenofovir group and adefovir group, adefovir promoted the percentage of cd158b nk cells. the frequency of cd158b nk cells significantly correlates with the alt level in chb patients before initiation of antiviral treatment. the increased percentage of cd158b nk cells may be associated with the decreased concentrations of plasma alt. several groups have studied nk cells during the antiviral treatment of chb patients, but the results regarding nk cell frequency and receptor expression are conflicting [32, 40, 41 ]. one important question is how the responses of individual patients to the antiviral therapy are associated with the change in the frequency of different subsets of nk cells. we noted a rapid decline in expression of nkg2a and kir2dl3, but not activating receptors, following the administration of nucs. this implies that these cells have this balance altered in favour of activation. viral load reduction may be an indirect effect on nk cells due to changes in the cytokine milieu, or due to interactions with other cells of the immune system that can subsequently effect nk cell receptor expression. during the treatment of chc patients, the frequency of nkg2a and kir2dl3 nk cells in the evr group was significantly higher than those in the non - evr group. interestingly, a recent study has shown that kir2dl3 nk cells have higher degranulation activity in patients with self - limited hcv infection. different from hepatitis c treatment, besides the decrease of viral load, hbeag seroconversion was also an important standard to judge the antiviral effect. in this study, subjects of hbeag seroconversion were limited to analysing the difference frequency of nkg2a and kir2dl3 nk cells bewteen hbeag seroconversion and none - hbeag seroconversion at 24 weeks after initial treatment. therefore, the frequency of nkg2a and kir2dl3 nk cells may be a prognostic marker for the evaluation of individual responses to the standard therapy in the clinic. in summary, our data indicate that the frequency of cd3cd56 nk cells in chb patients was lower than that in healthy controls, the frequency of nkp30nk cells in chb patients was significantly higher than those in healthy controls. in contrast, the frequency of cd158b nk cells in chb patients was significantly lower than that in healthy controls. the percentages of inhibitory cd158b nk cells were negatively associated with liver damage in chb patients. treatment with the tenofovir therapy transiently increased the frequency of nk and decreased frequency of inhibitory receptor nk cells in chb patients. therefore, our findings provide a unique insight in the nk cell compartment in chb patients during tenofovir induced viral load reduction. our study demonstrates modest changes in the frequency of inhibitory receptor nk cells after successful antiviral therapy. we recognized that our study had limitations of small sample size and the lack of analysis of nk cells function and nk cells in the target tissue. thus, further studies with a large population, combined with liver tissue analysis, nk cells function, to validate the findings are warranted.	natural killer (nk) cells play a critical role in innate antiviral immunity, but little is known about the impact of antiviral therapy on the frequency of nk cell subsets. to this aim, we performed this longitudinal study to examine the dynamic changes of the frequency of different subsets of nk cells in chb patients after initiation of tenofovir or adefovir therapy. we found that nk cell numbers and subset distribution differ between chb patients and normal subjects ; furthermore, the association was found between alt level and cd158b+ nk cell in hbv patients. in tenofovir group, the frequency of nk cells increased during the treatment accompanied by downregulated expression of nkg2a and kir2dl3. in adefovir group, nk cell numbers did not differ during the treatment, but also accompanied by downregulated expression of nkg2a and kir2dl3. our results demonstrate that treatment with tenofovir leads to viral load reduction, and correlated with nk cell frequencies in peripheral blood of chronic hepatitis b virus infection. in addition, treatments with both tenofovir and adefovir in chronic hbv infected patients induce a decrease of the frequency of inhibitory receptor+ nk cells, which may account for the partial restoration of the function of nk cells in peripheral blood following treatment.
damage control (dc) procedures for patients with major truncal or extremity trauma have evolved over the past 20 years and are recognized as major factors in decreasing morbidity and mortality in severely injured patients. early physiologic criteria, such as hypothermia, coagulopathy, and acidosis are used to select patients that would benefit from dc techniques. injury severity scoring or the estimated time required to accomplish definitive repair of injuries are further useful parameters in determining the need for dc. numerous studies have reported indications to perform dc operations in severely injured trauma patients.[26 ] however, investigations evaluating predictors of poor outcome among the subset of trauma patients undergoing dc procedures are scarce. thus, the objectives of this study were to review our institutional experience with dc management and to investigate risk factors for early mortality in patients undergoing dc procedures. after approval by the institutional review board, the trauma registry of the division of trauma surgery, university hospital of zurich, a verified level i trauma centre, was reviewed to identify all severely injured patients [injury severity score (iss) 17 ] from january 1, 1996, through december 31, 2005 who underwent initial dc procedures. a dc management was defined by limited operations for control of hemorrhage and contamination, or temporary fixation of extremity injuries. this included management of solid organ injuries by packing, resection of gastrointestinal tract injuries without re - anastomosis, use of temporary closure techniques at a site of surgical exploration, or temporary stabilization of extremity and pelvic fractures. demographic and clinical information collected included age, sex, mechanism of injury (blunt vs. penetrating), systolic blood pressure, glasgow coma score (gcs) upon admission, iss, and abbreviated injury scale (ais) for each body area (head, chest, abdomen, and extremity). for the analysis, continuous variables were dichotomized using clinically relevant cut - points : gcs on admission (8 vs. > 8), systolic blood pressure (sbp) on admission (90 mmhg vs. > 90 mmhg) and iss (25 vs. 6 mmol / l and hemoglobin 4 mmol / l on admission to icu and > 10 units prbc transfused until icu admission were found to be independent predictors of early mortality (r=0.373) [table 5 ]. independent risk factors at icu admission for early mortality in patients undergoing damage control management intensive care unit length of stay for early survivors was 15.70.8 days and the mean hospital length of stay was 36.51.5 days. overall, 59.6% (159 of 267) patients developed major in - hospital complications with the most frequent being sepsis (n=95, 35.6%), pneumonia (n=82, 30.7%), wound infection (n=64, 24.0%), intra - abdominal infection (n=17, 6.4%), and acute respiratory distress syndrome (ards) (n=12, 4.5%). twenty - two early survivors (8.2%) died 22.55.3 days later due to multiple organ failure (n=16) or severe head injury (n=6). various factors, potentially associated with early mortality in patients undergoing dc procedures, were identified using bivariate analysis. mmol / l, ais head 3, body core temperature 6 mmol / l and hemoglobin 4 mmol / l on admission to icu and > 10 units prbc transfused until icu admission were found to be independent predictors of early mortality (r=0.373) [table 5 ]. intensive care unit length of stay for early survivors was 15.70.8 days and the mean hospital length of stay was 36.51.5 days. overall, 59.6% (159 of 267) patients developed major in - hospital complications with the most frequent being sepsis (n=95, 35.6%), pneumonia (n=82, 30.7%), wound infection (n=64, 24.0%), intra - abdominal infection (n=17, 6.4%), and acute respiratory distress syndrome (ards) (n=12, 4.5%). twenty - two early survivors (8.2%) died 22.55.3 days later due to multiple organ failure (n=16) or severe head injury (n=6). managing severely injured patients is a diagnostic and therapeutic challenge for every trauma team. during the last decade, several studies have shown that the dc concept for abdominal, thoracic, and orthopedic injuries improves outcome in blunt and penetrating trauma.[711 ] johnson. recently demonstrated that the continued application of dc principles has led to improved survival in patients with penetrating abdominal injury by comparing a historical cohort with a current study population. in their study, overall survival they concluded that the early treatment of hypothermia and coagulopathy and the increasing experience with the open abdomen significantly contributed to improved survival. for multiple injured patients with concomitant orthopedic trauma, it has been demonstrated that dc procedures significantly reduce the incidence of general systemic complications such as ards and multiple organ failure. likewise in our study cohort, although 84% of the 267 patients surviving the first 3 days presented with critical injuries (iss 25), only 22 patients (8.2%) died in the later in - hospital course due to multiple organ failure or severe traumatic brain injury. analysis of our data has identified several hospital and icu admission parameters that predict early mortality in severely injured patients despite being managed with dc procedures. among those variables, early deaths presented with significantly deranged coagulation parameters including an elevated inr and lower platelet counts on hospital and icu admission. an inr > 1.2 on hospital admission represented the most important independent risk factor (adjusted odds ratio, 10.64) associated with early mortality. likewise, in numerous previous studies, the development of coagulopathy in trauma patients has been associated with increased morbidity and a several - fold increased mortality.[1219 ] in the study by macleod and colleagues, coagulation abnormalities upon admission, defined by a prothrombin time > 14 seconds and a partial thromboplastin time > 36 seconds, were independent predictors of in - hospital mortality with an adjusted odds ratio of 1.35 and 4.26, respectively. in the analysis by mitra., the presence of acute coagulopathy was independently associated with death within 24 hours of hospital admission (odds ratio, 8.77). additionally, coagulopathy is common following severe traumatic brain injury, mediated by the systemic release of tissue factor from the injured brain parenchyma, resulting in an unregulated activation of the extrinsic coagulation pathway.[172024 ] talving. demonstrated that the development of traumatic brain injury - associated coagulopathy, defined by thrombocytopenia and/or elevated inr and/or activated thromboplastin time, is associated with longer icu lengths of stay and an almost ten - fold increased risk of death. also, in a prospective study by olson and colleagues, elevated fibrin degradation products and an increased consumptive coagulopathy at admission were identified as independent predictors of mortality after isolated head injury. in our study, a significantly higher proportion of patients dying within the first 72 hours suffered severe traumatic brain injury compared to early survivors (71.2% vs. 43.1%, p 3 mmol / l, lactate level > 6 mmol / l on hospital admission, and the inability to achieve a lactate level 35c, the relative risk of death for patients with a temperature between 35c and 33c was 4.0, and that for patients with a temperature 33c was 7.1. we identified several risk factors present upon hospital and icu admission for early mortality such as severe head injury and the lethal triad (coagulopathy, acidosis and hypothermia). however, when surviving the first 72 hours after injury, survival in severely injured trauma patients managed with damage control protocol exceeded 90%.	background : this study reviews our 10-year institutional experience with damage control management and investigates risk factors for early mortality.materials and methods : the trauma registry of our level i trauma centre was utilized to identify all patients from 01/96 through 12/05 who underwent initial damage control procedures. demographics, clinical and physiological parameters, and outcomes were abstracted. patients were categorized as either early survivors (surviving the first 72 hours after admission) or early deaths.results:during the study period, 319 patients underwent damage control management. overall, 52 patients (16.3%) died (early deaths) and 267 patients (83.7%) survived the first 72 hours (early survivors). early deaths showed significantly deranged serum lactate (5.810.55 vs. 3.460.13 mmol / l ; p 1.2, base deficit > 3 mmol / l, head abbreviated injury scale 3, body temperature 6 mmol / l, and hemoglobin < 7 g / dl proved to be independent risk factors for early mortality on hospital admission.conclusions:several risk factors for early mortality such as severe head injury and the lethal triad (coagulopathy, acidosis and hypothermia) in patients undergoing damage control procedures were identified and should trigger the trauma surgeon to maintain aggressive resuscitation in the intensive care unit.
type 2 diabetes (t2d) is a metabolic disorder characterized by high blood glucose due to insulin resistance and insulin deficiency. t2d accounts for more than 90% of all diabetes, and the prevalence of t2d and prediabetes is rapidly rising worldwide. better treatment and prevention of t2d and its complications are needed to overcome the disease. to date, many animal models of t2d have been established such as the goto - kakizaki (gk) rat, otsuka long - evans tokushima fatty (oletf) rat, spontaneously diabetic torii (sdt) rat, sdt fatty rat, wistar fatty rat, zucker diabetic fatty (zdf) rat, db / db mouse, kk - a mouse, nagoya shibata yasuda (nsy) mouse, new zealand obese (nzo) mouse, tallyho (th) mouse, and the tsumura suzuki obese diabetic (tsod) mouse. among rat models of the disease, it is well recognized that a mutation in the leptin receptor gene (lepr) causes infertility in the homozygous state. thus, rats homozygous for a missense mutation (fatty, fa) in lepr are infertile ; these include the sdt fatty rat, wbn / kob fatty rat, wistar fatty rat, zdf rat, and the zucker fatty (zf) rat, all of which harbor the fa mutation. the infertility of fa / fa homozygous animals requires maintenance and production of the strains by mating between fa/+ heterozygous animals. only one male of eight pups obtained by this type of mating is expected to be fa / fa homozygous and serve as research material. we establish here a novel diabetic rat strain, zfdm, in which the fa / fa male rats have reproductive ability. we performed initial characterization of the strain to compare their phenotypic characteristics with those of the original zf rats. zfdm rats (hos : zfdm - lepr, fa / fa and fa/+) were provided by hoshino laboratory animals, inc. normoglycemic zf rats (slc : zucker, fa / fa and + /+) were purchased from japan slc, inc. all animals were maintained under specific pathogen free conditions at 23 2c and 55 10% relative humidity with a 12 h light - dark cycle and were provided with water and a commercial diet ce-2 (clea japan, inc., tokyo, japan) at the animal facility of kobe biotechnology research and human resource development center of kobe university. all animal experiments were approved by the committee on animal experimentation of kobe university and carried out in accordance with the guidelines for animal experimentation at kobe university. the zfdm and zf rats were checked for body weight and nonfasting blood glucose level by a portable glucose meter (antsense iii, horiba, ltd., diabetes was defined as a blood glucose level equal to or higher than 300 mg / dl under ad libitum dietary conditions. at 6, 8, 12, 16, and 20 weeks of age, nonfasting whole blood samples were collected from the tarsal vein, and plasma samples were separated by centrifugation and stored at 80c for later insulin measurement. in different batches, zfdm rats at 8, 12, 16, 20, and 24 weeks of age were checked for body weight, body length (head to anus), and nonfasting blood glucose level and were sacrificed for histological analysis. fasting whole blood samples were collected from the heart, and serum samples were separated by centrifugation and stored at 80c for later leptin, adiponectin, and lipid parameter measurements. body mass index (bmi) was calculated by dividing body weight (in grams) by body length (in centimeters) squared. plasma insulin levels were measured by insulin elisa kit (shibayagi co., ltd., serum leptin and adiponectin levels were measured by leptin elisa kit (morinaga institute of biological science, inc., serum lipid parameters were measured by hitachi automatic analyzer 7070 (hitachi high - technologies corporation, tokyo, japan). the fixed specimens were embedded in paraffin, sectioned at 4 m, and stained with hematoxylin and eosin for histopathological examination. differences in blood glucose level, body weight, body length, bmi, plasma insulin level, serum leptin level, serum adiponectin level, and serum lipid parameters were assessed using welch 's t - tests. to improve the reproductive efficiency of our zf rat colony, we have been trying to obtain fa / fa animals having reproductive ability since 1992 (figure 1). in 1997, we fortunately identified fa / fa males having reproductive ability and tried to maintain the colony by using these fa / fa males. in 2005, we successfully established an outbred zf rat colony in which fa / fa males having reproductive ability were frequently and continuously obtained. we have been maintaining this colony by mating between fa/+ females and 10- to 15-week - old fa / fa males. in 2008, during maintenance of the above - mentioned zf rat colony, we incidentally identified one 25-week - old fa / fa male rat exhibiting diabetes (figure 1). afterward, we measured blood glucose levels of this fa / fa male 's descendants at 10 weeks of age and selected the animals showing relatively high blood glucose levels to maintain the colony. consecutive maintenance by this type of selective breeding for more than 10 generations resulted in establishment of an outbred diabetic rat colony, which we designated hos : zfdm - lepr (zfdm). it is most noteworthy that the fa / fa males in this zfdm rat colony are fertile and diabetic. we then compared body weight and nonfasting blood glucose level of fa / fa and fa/+ male rats in the zfdm strain from 5 to 21 weeks of age (figures 2(a) and 2(b)). differences in body weight between fa / fa and fa/+ rats were evident as early as 6 weeks of age (fa / fa 158.4 4.5 g versus fa/+130.6 4.8 g, p = 0.0004) and were increased gradually until 21 weeks of age (fa / fa 448.5 15.3 g versus fa/+ 375.3 5.8 g, p = 0.0004). differences in nonfasting blood glucose level between fa / fa and fa/+ rats were evident as early as 8 weeks of age (fa / fa 128.2 4.7 mg / dl versus fa/+ 104.7 2.1 mg / dl, p = 0.0003) and were increased markedly until 21 weeks of age (fa / fa 464.2 30.0 mg / dl versus fa/+ 118.6 2.1 mg / dl, p < 0.0001). nonfasting plasma insulin level was significantly higher in fa / fa rats than that in fa/+ rats at 6 weeks of age (fa / fa 0.89 0.12 ng / ml versus fa/+ 0.56 0.04 ng / ml, p = 0.026), and that in fa / fa rats was increased until 16 weeks of age and then decreased but remained significantly higher than that of fa/+ rats at 20 weeks of age (fa / fa 1.23 0.18 ng / ml versus fa/+ 0.62 0.03 ng / ml, p = 0.006) (figure 2(c)). the fa / fa rats developed diabetes (nonfasting blood glucose levels 300 mg / dl) as early as 10 weeks of age and the cumulative incidence of diabetes reached 100% by 21 weeks of age (figure 2(d)). in contrast, none of the fa/+ rats developed diabetes. in different batches of the experiment, we compared body weight, nonfasting blood glucose level, body length, and bmi of fa / fa and fa/+ male rats in the zfdm strain at 8, 12, 16, 20, and 24 weeks of age (figure 3). body weight and nonfasting blood glucose level of both animals showed similar tendencies to those described above (figures 3(a) and 3(b)). body length was significantly larger in fa/+ rats than that in fa / fa rats at 16 weeks of age and later (16 weeks of age : fa / fa 22.9 0.2 cm versus fa/+ 23.9 0.2 cm, p = 0.007) (figure 3(c)). differences in bmi between fa / fa and fa/+ rats were evident at 8 weeks of age (fa / fa 0.59 0.01 g / cm versus fa/+ 0.54 0.01 g / cm, p = 0.028), were increased markedly by 12 weeks of age, and were maintained until 24 weeks of age (fa / fa 0.84 0.02 g / cm versus fa/+ 0.65 0.01 g / cm, p < 0.0001) (figure 3(d)). most of the lipid parameters and leptin levels were higher in fa / fa rats than those in fa/+ rats. in contrast, the adiponectin level in fa / fa rats was decreased with age and was lower than that in fa/+ rats at 24 weeks of age (figure 4(h)). to compare the phenotype of the zfdm strain and that of the normoglycemic zf strain, we measured body weight and nonfasting blood glucose level of fa / fa and + /+ rats in the zf strain from 5 to 21 weeks of age (figures 5(a) and 5(b)). differences in body weight between fa / fa and + /+ rats in the zf strain were evident as early as 5 weeks of age (data at 6 weeks of age for comparison with the zfdm strain : fa / fa 161.8 3.0 g versus + /+ 129.2 2.3 g, p < 0.0001) and were gradually increased until 21 weeks of age (fa / fa 558.8 7.4 g versus + /+ 367.0 6.9 g, p < 0.0001) (figure 5(a)). while the body weights of fa / fa rats in both zfdm and zf strains were similar at earlier ages, those of fa / fa rats in the zfdm strain were significantly lower at 11 weeks of age and later, most likely due to the onset of diabetes (supplementary figure 1(a) available online at http://dx.doi.org/10.1155/2013/103731). there was no difference in nonfasting blood glucose level between fa / fa and + /+ rats in the zf strain during the experimental period, and none of the fa / fa and + /+ rats in the zf strain exhibited diabetes by 21 weeks of age (figure 5(b)) ; the character is different from that of the zfdm strain (supplementary figure 1(b)). nonfasting plasma insulin level was significantly higher in fa / fa rats than that in + /+ rats at 6 weeks of age (fa / fa 1.49 0.27 ng / ml versus + /+ 0.83 0.09 ng / ml, p = 0.045) and that in fa / fa rats was increased until 20 weeks of age (fa / fa 2.00 0.26 ng / ml versus + /+ 0.62 0.06 ng / ml, p = 0.0007) (figure 5(c)). while there were no obvious pathological changes in endocrine and exocrine pancreas of fa/+ rats in the zfdm strain (figures 6(a), 6(b), 6(e), 6(f), 6(i), and 6(j)), various degrees of pathological change were observed in the pancreas of the fa / fa rats at 16 and 24 weeks of age, including loss of islet architecture and fibrosis (figures 6(g), 6(h), 6(k), and 6(l)). to determine whether these pathological changes are specific to the fa / fa rats in the zfdm strain, we performed histological characterization of the pancreas in the normoglycemic zf strain. although the pancreatic islets of fa / fa rats in the zf strain were enlarged, islet architecture was maintained even at 24 weeks of age (figures 6(o) and 6(p)). similarly to the fa/+ rats in the zfdm strain, + /+ rats in the zf strain exhibited no obvious pathological changes in the pancreas (figures 6(m) and 6(n)). in this study, we describe the establishment and initial characterization of a novel type 2 diabetes model derived from normoglycemic zf rats. we first established an outbred zf rat colony in which fa / fa males have reproductive ability. we then incidentally identified an fa / fa male rat exhibiting diabetes and performed selective breeding using fa / fa male rats showing relatively high blood glucose levels at 10 weeks of age. we successfully maintained this colony by mating between fa / fa male and fa/+ female rats, which resulted in establishment of an outbred diabetic rat colony that we designated hos : zfdm - lepr. the male fa / fa rats in the zfdm strain are fertile, exhibit obesity, and develop diabetes as early as 10 weeks of age, reaching 100% incidence at around 20 weeks of age. the zdf strain was established in 1990 and has been widely used to study t2d associated with obesity. the zdf strain is an inbred rat model of early - onset diabetes in which all of the fa / fa male rats develop diabetes at 10 to 12 weeks of age when fed a special diet of purina 5008 (charles river laboratories international, inc., wilmington, ma, usa). the phenotype is homogeneous, mainly due to the facts that the strain is genetically inbred and the special diet is provided. in contrast, the zfdm strain is an outbred rat model of young- to middle - aged adult - onset diabetes in which the fa / fa male rats develop diabetes as early as 10 weeks of age and most develop diabetes at 12 to 20 weeks of age (table 1). after the onset of diabetes, high blood glucose levels are maintained, a feature is similar to that of the zdf strain. since the prediabetic stage of the zfdm strain is much longer than that of the zdf strain, the zfdm strain is more suitable for pathophysiological studies of diabetes and therapeutic intervention studies. in the zdf strain, the fa / fa male rats exhibit diabetic complications including nephropathy and neuropathy. given that the zfdm strain harbors a similar genetic background to that of the zdf strain and exhibits severe diabetes, diabetic complications found in the zdf strain may well occur in the zfdm strain. usually, humans, mice, and rats lacking normal lepr are infertile due to hypogonadotropic hypogonadism. however, there is some evidence for spontaneous pubertal development in humans homozygous for a splicing mutation resulting in lepr lacking both the transmembrane and intracellular domains, among whom one woman was able to give birth to a healthy son [18, 19 ]. in addition, human subjects with lepr missense mutations have been reported to show less severe clinical features than those with the splicing mutation described above. in rat species, l. m. zucker and t. f. zucker reported that fa / fa female rats are always infertile, while chelich and edmonds found that a small portion of young fa / fa female rats are fertile. furthermore, young fa / fa male rats are more likely to be fertile ; about 70% of fa / fa male rats were reported to be fertile in a colony at vassar college, but their current status is unknown. in our zf rat colony, more than 10 generations of selective breeding for fertility of fa / fa male rats enabled us to obtain fertile fa / fa male rats stably. we have established a novel diabetic rat strain, zfdm, in which fa / fa male rats are fertile. in this strain, fa / fa male rats develop diabetes as early as 10 weeks of age, which reaches 100% incidence at around 20 weeks of age, while no fa/+ rats develop diabetes. the zfdm rat strain possesses high reproductive efficiency and therefore should serve as a useful model of young- to middle - aged adult - onset t2d in studies of the pathophysiology, therapeutic interventions, and complications of the disease.	the zucker fatty (zf) rat harboring a missense mutation (fatty, fa) in the leptin receptor gene (lepr) develops obesity without diabetes ; zucker diabetic fatty (zdf) rats derived from the zf strain exhibit obesity with diabetes and are widely used for research on type 2 diabetes (t2d). here we establish a novel diabetic strain derived from normoglycemic zf rats. in our zf rat colony, we incidentally found fa / fa homozygous male rats having reproductive ability, which is generally absent in these animals. during maintenance of this strain by mating fa / fa males and fa/+ heterozygous females, we further identified fa / fa male rats exhibiting diabetes. we then performed selective breeding using the fa / fa male rats that exhibited relatively high blood glucose levels at 10 weeks of age, resulting in establishment of a diabetic strain that we designated hos : zfdm - leprfa (zfdm). these fa / fa male rats developed diabetes as early as 10 weeks of age, reaching 100% incidence by 21 weeks of age, while none of the fa/+ male rats developed diabetes. the phenotypic characteristics of this diabetic strain are distinct from those of normoglycemic zf rats. zfdm rat strain having high reproductive efficiency should serve as a more useful animal model of t2d.
the lac insects (coccoidea : tachardiidae (= kerriidae)) have been commercially harnessed to yield three useful products, viz., resin, wax and dye, which have found a remarkably wide range of applications in food, pharmaceuticals, cosmetics, perfumes, varnishes, paints, polishes, adhesives, jewellery and textile dyes, since ancient times (dave, 1950 ; sarkar, 2002 ; ramani., 2007). the importance of this commodity lies in its safety for human use, and as a renewable and eco - friendly resource. lac production is confined to a few south, southeast and east asian countries in the tropical forest region (ramani., 2007) with india is the leading lac - producer, with an annual production of about twenty thousand tons (pal., 2011). they are phytosuccivorous and sessile ; only the crawlers and adult males are free moving. they thrive well only on certain plant species known as lac hosts (kapur, 1962 ; varshney, 1985). information on the taxonomy of lac insects is based on a monograph and its supplement (chamberlin 1923, 1925), as well as subsequent works by kapur (1958), varshney (1977) and kondo and gullan (2007). ninety species, under nine genera, have so far been reported worldwide (varshney, 2009). distribution is mainly restricted to tropical and subtropical regions between the latitudes 40 n and 40 s (kapur, 1962). nearly all the production comes from the indian lac insect kerria lacca, represented by two infrasubspecific forms, viz., kusmi and rangeeni, which differ by host preference, life - cycle pattern, the quality and amount of lac produced, etc. other minor species are k. sharda (mishra and sushil, 2000) and k. chinensis. palas (butea monosperma), ber (ziziphus mauritiana) and kusum (schleichera oleosa) are the most common hosts used for lac production in india (roonwal 1962), which is mainly restricted to the states of jharkhand, chhattisgarh, madhya pradesh, west bengal, maharashtra, besides a few others (pal., 2011). notwithstanding, wild populations of kerria are distributed throughout the length and breadth of the country, except in the colder regions (varshney 1977). the taxonomy of coccoids is based on adult - female morphology (varshney, 1977 ; kondo and gullan, 2007). even so, they are highly degenerate, and undergo tremendous changes in size and shape during the post - metamorphic stage. differentiated populations, due to geographic separation and host - choice, and which have not diverged morphologically, pose an additional challenge to identification. molecular approaches would therefore serve as useful complementary tools for characterizing such lac - insect taxa with greater reliability. a wide range of markers are employed for understanding insect - population genetics (behura, 2006). rapd - pcr is widely used for identifying cultivars, clones, natural populations, etc. despite the limitation posed by reproducibility, unless reaction conditions are stringent (baruffi., 1995 and bertin., 2007), this technique offers the advantages of simplicity, independence from prior dna sequence information, and the evaluation of a large number of loci across the genome (hadrys., 1992 ; lynch and milligan, 1994 ; weising., 2005), besides providing the basis for developing more reliable scar (sequence characterized amplified regions) markers (kethidi., 2003). the technique has already been widely employed for assessing the genetic diversity of other insect populations (reyes and ochando, 1998 ; castiglioni and bicudo, 2005 ; dvorak., 2006 ; lopes - da - silva and vieira, 2007 ; martins., 2007 ; magaa., 2007 ; karam., 2007 ; sosa - gomez., 2008 ; this constitutes a report on genetic diversity in lac - insect populations belonging to the genus kerria, the true lac - producing insects, from different parts of india, using the rapd technique (williams., 1990 ; welsh and mcclelland, 1990). the study material included lac - insect populations collected from different parts of the country, both natural and cultured populations, besides inbred lines derived from k. lacca. the insects used in the study were obtained from lac - insect cultures maintained at the research farm, iinrg campus, ranchi (231951 n 852218 e ; elevation 2080 ft). a few were also drawn from collections of natural field populations (table 1 ; figure 1). the lac - insect cultures were maintained on a common lac host flemingia macrophylla, under potted conditions. the cultures, enclosed in synthetic mesh sleeves to exclude parasite and predator infestation, were regularly sprayed with fungicide (carbendazim, 0.01%) to maintain cleanness. field - collected insects were carefully screened, in order to select only healthy ones. the lines studied mainly included the most commonly used species for lac production, viz., k. lacca (kusmi and rangeeni infrasubspecific forms), a collection of k. sharda, and two collections of k. chinensis (india and thailand), as well as inbred and crossbred lines, and collections from wild populations collected from all over of india. these lines were divided into four groups, depending on the nature (table 1). mature female insects were kept in 100% ethanol for 48 h at room temperature, so as to dissolve the resinous covering, whence they were individually cleaned with sable - hair brushes under a stereo - zoom microscope, and serially washed with alcohol to eliminate waxy secretions. the cleaned insects were kept in 200 l absolute ethanol in 1.5 ml microcentrifuge tubes and stored in 80 c freezer. dna was extracted from mature females, adopting a phenol - chloroform procedure described by de barro. the extracted dna was individually quantified with a shimadzu uv - vis 1700 spectrophotometer using a dna program pack, and checked by electrophoresis on 1% agrose gel together with 100 bp dna ladder - plus (fermentas, germany). we screened 120 decamer primers (operon bio - technologies gmbh, germany) for satisfactory amplification of products, using three selected lines. rapd - pcr was carried out with 48 samples of pooled genomic dna from three female insects, to shortlist primers exhibiting polymorphism and reproducibility. for each of these primers, annealing temperatures and other parameters were standardized by repeated experiments (table 2). all the rapd reactions were done in 25 l of reaction mixtures containing 20 ng of template dna, 1x taq buffer [750 mm tris - hcl (ph 8.8), 200 mm (nh4)2so4, 0.1% (v / v) tween 20 ; fermentas gmbh, germany ], 2.5 mm of mgcl2 (fermentas gmbh, germany), 0.2 mm of each dntp mix (fermentas gmbh, germany), 20 pmol of each primer, and 1.5 units of taq dna polymerase (fermentas gmbh, germany). all the pcr reactions were carried out in a thermal cycler (biorad icycler, usa) programmed with the following cycling conditions : initial denaturation of template dna was carried out at 95 c for 5 min followed by 35 cycles programmed for denaturation step at 95 c for 1 min, primer annealing step at specific tm for the particular primer (table 2) for 45 s, and extension step at 72 c for 2 min. the final extension of the pcr products was carried out at 72 c for 7 min. all pcr amplified products were resolved on 2% agarose gel containing 0.5 g / ml, ethidium bromide, prepared with 0.5 x tbe buffer [45 mm tris - borate, 1.0 mm edta (ph 8.0) ] and electrophoresed in 0.5 x tbe at 4 v cm for 2 h in an amersham submarine electrophoresis unit. either fermentas 100 bp ladder (1001000) or 100 bp plus ladder (1003000) were used as reference, depending on the band - size range. sufficiently resolved dna bands were documented using the bioimaging system (gene genius, syngene, u k), through genesnap. clear and unambiguous bands present across the dna samples from 48 lac - insect lines at a particular locus (based on size) were scored as 1, whereas their absence or only a very faint outline were scored as 0, to so generate a binary matrix, to be used for analysis. for line diagnosis and the analysis of marker discrimination power, the average band frequency obtained for each primer, marker index (mi) and resolving power (rp), using band informativeness (ib), were calculated (prevost and wilkinson 1999). ib=1(2\|0.5pi\|)where pi is the proportion of lines showing the i band, and i = 1 to n where primer resolving power (rp) was also calculated using the following formula rp=ibwhere ib is band informativeness, as calculated above. marker index (mi) is the parameter for determining the utility of the marker in distinguishing different genotypes. the estimation of mi was by applying the following formula provided by archak. mi=1/n ib x emrwhere emr (effective multiplex ratio) is the product of the number of polymorphic bands (i.e. a band absent in at least one genotype at a particular locus) per primer and the fraction of polymorphic bands. in order to study the genetic relationships among kerria lines, the scored binary data matrix was analyzed using the ntsyspc version 2.02e software program (exeter software, new york, usa) (rohlf, 1998). the dendrogram was generated by applying the neighbor - joining method (saitou and nei, 1987), using midpoint rooting. only wild - type insects were considered for assessing the variation of similarity indices, in the case of mixed populations. the same software was used for principal coordinate (pcoord) analysis (sneath and sokal, 1973) of the data. the confidence level for distinguishing genotypes, using the selected primers, was estimated through the analysis of probability of identical match by chance (pimc), as proposed by wetton. the insects used in the study were obtained from lac - insect cultures maintained at the research farm, iinrg campus, ranchi (231951 n 852218 e ; elevation 2080 ft). a few were also drawn from collections of natural field populations (table 1 ; figure 1). the lac - insect cultures were maintained on a common lac host flemingia macrophylla, under potted conditions. the cultures, enclosed in synthetic mesh sleeves to exclude parasite and predator infestation, were regularly sprayed with fungicide (carbendazim, 0.01%) to maintain cleanness. field - collected insects were carefully screened, in order to select only healthy ones. the lines studied mainly included the most commonly used species for lac production, viz., k. lacca (kusmi and rangeeni infrasubspecific forms), a collection of k. sharda, and two collections of k. chinensis (india and thailand), as well as inbred and crossbred lines, and collections from wild populations collected from all over of india. these lines were divided into four groups, depending on the nature (table 1). mature female insects were kept in 100% ethanol for 48 h at room temperature, so as to dissolve the resinous covering, whence they were individually cleaned with sable - hair brushes under a stereo - zoom microscope, and serially washed with alcohol to eliminate waxy secretions. the cleaned insects were kept in 200 l absolute ethanol in 1.5 ml microcentrifuge tubes and stored in 80 c freezer. dna was extracted from mature females, adopting a phenol - chloroform procedure described by de barro. the extracted dna was individually quantified with a shimadzu uv - vis 1700 spectrophotometer using a dna program pack, and checked by electrophoresis on 1% agrose gel together with 100 bp dna ladder - plus (fermentas, germany). we screened 120 decamer primers (operon bio - technologies gmbh, germany) for satisfactory amplification of products, using three selected lines. rapd - pcr was carried out with 48 samples of pooled genomic dna from three female insects, to shortlist primers exhibiting polymorphism and reproducibility. for each of these primers, annealing temperatures and other parameters were standardized by repeated experiments (table 2). all the rapd reactions were done in 25 l of reaction mixtures containing 20 ng of template dna, 1x taq buffer [750 mm tris - hcl (ph 8.8), 200 mm (nh4)2so4, 0.1% (v / v) tween 20 ; fermentas gmbh, germany ], 2.5 mm of mgcl2 (fermentas gmbh, germany), 0.2 mm of each dntp mix (fermentas gmbh, germany), 20 pmol of each primer, and 1.5 units of taq dna polymerase (fermentas gmbh, germany). all the pcr reactions were carried out in a thermal cycler (biorad icycler, usa) programmed with the following cycling conditions : initial denaturation of template dna was carried out at 95 c for 5 min followed by 35 cycles programmed for denaturation step at 95 c for 1 min, primer annealing step at specific tm for the particular primer (table 2) for 45 s, and extension step at 72 c for 2 min. the final extension of the pcr products was carried out at 72 c for 7 min. all pcr amplified products were resolved on 2% agarose gel containing 0.5 g / ml, ethidium bromide, prepared with 0.5 x tbe buffer [45 mm tris - borate, 1.0 mm edta (ph 8.0) ] and electrophoresed in 0.5 x tbe at 4 v cm for 2 h in an amersham submarine electrophoresis unit. either fermentas 100 bp ladder (1001000) or 100 bp plus ladder (1003000) were used as reference, depending on the band - size range. sufficiently resolved dna bands were documented using the bioimaging system (gene genius, syngene, u k), through genesnap. clear and unambiguous bands present across the dna samples from 48 lac - insect lines at a particular locus (based on size) were scored as 1, whereas their absence or only a very faint outline were scored as 0, to so generate a binary matrix, to be used for analysis. for line diagnosis and the analysis of marker discrimination power, the average band frequency obtained for each primer, marker index (mi) and resolving power (rp), using band informativeness (ib), were calculated (prevost and wilkinson 1999). ib=1(2\|0.5pi\|)where pi is the proportion of lines showing the i band, and i = 1 to n where n is the total number of bands. primer resolving power (rp) was also calculated using the following formula rp=ibwhere ib is band informativeness, as calculated above. marker index (mi) is the parameter for determining the utility of the marker in distinguishing different genotypes. the estimation of mi was by applying the following formula provided by archak. mi=1/n ib x emrwhere emr (effective multiplex ratio) is the product of the number of polymorphic bands (i.e. a band absent in at least one genotype at a particular locus) per primer and the fraction of polymorphic bands. in order to study the genetic relationships among kerria lines, the scored binary data matrix was analyzed using the ntsyspc version 2.02e software program (exeter software, new york, usa) (rohlf, 1998). the dendrogram was generated by applying the neighbor - joining method (saitou and nei, 1987), using midpoint rooting. only wild - type insects were considered for assessing the variation of similarity indices, in the case of mixed populations. the same software was used for principal coordinate (pcoord) analysis (sneath and sokal, 1973) of the data. the confidence level for distinguishing genotypes, using the selected primers, was estimated through the analysis of probability of identical match by chance (pimc), as proposed by wetton. out of the 120 rapd primers screened, 26 produced the satisfactory, clear and reproducible banding patterns used herein. these produced 173 loci in the 48 lines studied, of which 169 (97.7%) were found to be polymorphic. the maximum size - range of amplified products for a single primer was obtained with oph5 (4502.0 kbp), whereas the minimum, i.e., one single band (600 bp), was obtained with ops12. the number of bands produced by the primers ranged from 1 (ops 12 and ops 16) to 12 (ops 19), with an average of 6.7 per primer. polymorphism in the bands produced by all the rapd primers, except for ops 9, ops 10, ops 19 and opt 16, was 100%. the primers used, their sequences, tm, number of scored bands, band - size range and the number of polymorphic bands, are given in table 2. the total number of amplified bands in all the forty - eight lac - insect lines generated, when using all the twenty - six rapd primers, was 3,380, with an average of 70.4 bands per line. the number of bands amplified through all the primers in all the lines ranged from 33 (li036) to 95 (li025). the resolving power (rp) of the primers screened ranged from 0.08 (ops9) to 4.08 (opa13), with a mean of 1.97. a total 33 unique bands were generated, with 15 rapd primers (ops9, ops10, ops13, ops15, ops17, ops19, opt7, opt16, oph5, oph12, oph19, opb15, opb18, opa2 and opa9) in eight lac - insect lines (li002, li010rr, li09b, li068, li05, li044, li087 and li069). the highest number of unique bands (21 out of 33), compared to the remainder, was in line li002, kerria chinensis from india. only three (li002, li003 and li015) presented unique null bands with four rapd primers, opt16, ops19, ops10 and ops9. in line, ops19, ops10 and ops9. out of the 48 lac - insect lines analyzed, nine, viz., li009a, li002, li068, li043, li085, li026, li027, li003 and li028, could be identified by the presence of two bands by eight primers. with most of the primers, lines li002 and li068, k. chinensis lines, respectively from india and thailand, were found to share a common pattern, by the presence or absence of two bands. pair - wise jaccard s similarity indices varied from 0.15 (li068, li003) to 0.81 (li012, li015 ; li029, li032 ; li048, li032), with a mean of 0.58 for all the cultivated lines, geographic races of k. lacca and two other species (groups i, ii and iv in table 1). the index varied between 0.27 (li036, li085) and 0.81 among the 33 cultivated lines and geographic races of k. lacca (groups i and ii in table 1), with a mean of 0.61, indicative of significant intra - specific genetic diversity. the six inbred lines of k. lacca (rangeeni), derived from a single mother population, presented divergence with a mean similarity index of 0.77 (range : 0.620.92). analysis of the above data showed that the probability of identical chance matching was 4.74 x 10, thus indicative of a high level of reliability. the dendrogram generated from the jaccard s similarity matrix, by using the neighbor - joining method, appears in figure 2. basically, it resolves the lines into two major clusters (nodes a and b), as well as outgroups comprising eight lines under six branches (1 to 6). the cluster originating from node a, the major one comprising 32 lines, basically includes cultivated lines of the rangeeni form, geographic races and inbred lines of k. lacca. subcluster a1 is a heterogeneous group of seven lines, comprised of two (li003 and li031) from jharkhand state, two (li42 and li36) from chhattisgarh state, two from andhra pradesh state, and a cream recombinant mutant line (li001a). the subcluster from node a2 is comprised of 15 lines, mainly from the eastern region, and consists of six lines derived from cultivated populations of four adjoining lac - growing states (jharkhand, west bengal, chhattisgarh and madhya pradesh), six inbred lines (also developed from a cultivated population from jharkhand), two geographic races from northern and eastern india, and the wild - type color form of a recombinant line (li001b). the subcluster of ten lines originating from a3, is mainly composed of those from western india (five from gujarat and rajasthan), one from the northern state of uttar pradesh (li72b), two crossbred lines (li058 and li061), and the yellow mutant of a kusmi line from orissa (li078), whereas subclusters a2 and a3 mainly consist of lines from eastern and western regions of india, respectively., seven of the kusmi form and one of the rangeeni of k. lacca from the southern state of karnataka. these forms of k. lacca appear to have originated through a common ancestor at o. kusmi insects are naturally distributed mainly in the eastern part of peninsular india, whereas the rangeeni are distributed countrywide. these two forms differ as to host preference -rangeeni insects are characterized by their ability to thrive well on butea monosperma, and kusmi on schleichera oleosa (varshney, 1977). based on crosses between the two, it has been shown that the kusmi form is genetically endowed for survival on s. oleosa (chauhan and mishra, 1970). the remaining eight lines form a series of outgroups (branches 16), which include one of the kusmi form (li005), one collection from the central state of madhya pradesh (li085), and a group of three yellow mutant lines (9b, 19a and 13) of k. lacca, besides two lines of k. chinensis (li068 and li002) and one of k. sharda. the dendrogram differentiated the species of kerria studied, as well as li085, represented as an outgroup, and which requires re - examination as to taxonomic status. three color mutants in k. lacca (white, yellow and cream), which affect body and resin color, have been shown to be recessive (chauhan, 1977 ; chauhan and mishra, 1977 ; ramani, 2002). in the present study, the dendrogram generated always separated colour mutants (yellow and cream) from their wild - type counterparts in the same population, due to their distinctive rapd banding profiles, indicative of their distinct genetic makeup. principal component analysis revealed that 27.2% of the variation could be attributed to the first three components. figure 3 presents the 3-d plot of the principal coordinate analysis of the similarity matrix data of the lines studied. the k. lacca populations are well - spread over the dimensions, thereby indicating their diversity. two lines of k. chinensis derived from different geographic locations (li002 and li068), as well as k. sharda (li066), are well separated from those of k. lacca, thus consistent with the nj dendrogram. according to pcoord analysis, the seven kusmi lines of k. lacca, which formed a group at node b in this dendrogram, appeared to spread out. the lines li075 (kalamati, west bengal) and li077 (hesadih, jharkhand) appeared distinct. spurious larval emergence, a phenomenon whereby some crawler emergence takes place during a non - typical period (nov dec) in kusmi populations, is indicative of interbreeding in kusmi and rangeeni forms. oviposition peaks in the segregating progenies of crosses of these two forms correspond to the parental types. four lines of the rangeeni form of k. lacca, li19a & b, li020, li085 and li087, tended to form a cluster at variance with the dendrogram. lac insects, by depending on perennial trees for survival, are specialists in the preference for host - plants with limited dispersal. they may thus become locally adapted, thereby forming genetically distinct geographic and host races without morphological differentiation. human intervention, with systematic lac - cultivation and the transport of insect populations across various regions, also exerts an influence on dispersal and interpopulation gene flow. thus, divergence in k. lacca, can be expected, through being both the most commonly used species for lac - production and widely distributed in india. intermixing of cultivated populations is also expected in lac - producing areas, due to the transportation of insects within the cultivated areas, discernible in line - clustering in node a2 from the principal lac growing region. the six inbred lines derived from the same mother population also diverged from each other, with only li024 and li020 remaining similar. the two forms of kerria lacca, kusmi and rangeeni, commonly referred to as ` strains in lac - insect literature, are distinct as regards certain commercial and biological traits. based upon morphological characteristics, they were allocated as infrasubspecific forms by varshney (1977). even so, they hybridize freely under laboratory conditions, thereby producing viable progeny. premating barriers, such as differences in host preference and asynchrony in sexual - maturity periods, due to differences in life - cycle patterns, prevent interbreeding under natural conditions. nevertheless, through occasional period - overlaps during the rainy - season generation of both forms, interbreeding is possible. a probable illustration is a subgroup of the a1 node cluster, comprising two collections from geographically close locations, i.e., li042, a line of the rangeeni form, collected from mainpur, chhattisgarh, and li036 a line of the kusmi form from kurubhatta, also chhattisgarh. spurious crawler emergence in li077, indicative of interbreeding in these two forms, has already been discussed. the usefulness of rapd - pcr for assessing genetic diversity in indian lac insects has been demonstrated. considering the general recommendation of fifty polymorphic markers to establish precise genetic distances (nei, 1978), 26 rapd primers having produced 169 polymorphic bands is sufficient for distinguishing species to complement pertinent taxonomic studies. the dendrogram generated from the similarity matrix also throws light on the interrelationships of the otus investigated. the above molecular evidence supports the current status of kusmi and rangeeni, as infraspecific forms of k. lacca. color mutants also need to be examined in greater detail, in order to understand the basis for their differentiation. based on rapd profiles, the lac - insect populations of k. lacca collected from different locales presented considerable variation. further studies using other markers and dna sequence variation, are likely to throw fresh light on these populations and their respective relationships.	the lac insects (homoptera : tachardiidae), belonging to the genus kerria, are commercially exploited for the production of lac. kerria lacca is the most commonly used species in india. rapd markers were used for assessing genetic variation in forty - eight lines of kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of k. lacca. in the 48 lines studied, the 26 rapd primers generated 173 loci, showing 97.7% polymorphism. by using neighbor - joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. the major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of k. lacca. the second cluster consisted of eight lines of k. lacca, seven of the kusmi form and one of the rangeeni from the southern state of karnataka. the remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of k. lacca and other species of the kerria studied, among others. color mutants always showed distinctive banding patterns compared to their wild - type counterparts from the same population. this study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of k. lacca.
2,1-borazaronaphthalenes serve as versatile isosteres of all - carbon naphthalene substructures, which allows facile emplacement of a variety of substituents about the ring in an efficient and selective manner. because of electronic desymmetrization brought about by the incorporation of a b n bond in these aromatic systems, borazines have proven to be far more easily elaborated than naphthalenes themselves. previous studies have focused on decorating these ring systems by utilizing arylation reactions. 2-alkenylnaphthalenes can be synthesized through a variety of transition - metal - catalyzed transformations, including heck, mizoroki heck, hiyama, and suzuki miyaura reactions. however, installation of an alkenyl substituent on a functionalized naphthalene is more challenging and often requires four or five steps. one method to afford alkenyl - substituted naphthalenes is to perform free - radical bromination of a methyl - substituted derivative followed by a wittig olefination. aside from the generation of a stoichiometric amount of difficult - to - remove phosphine oxide waste, additional substitution on similarly, titanium - mediated carbonylation with dichloromethyl methyl ether followed by wittig olefination afforded 2-alkyl-3-alkenylnaphthalene in moderate yield over two steps. in addition to the drawbacks described above, the use of a strong lewis acid limits functional group tolerability in this approach. further, there is only one example demonstrating the installation of an alkenyl substituent on a 2-arylnapthalene through the cross - coupling of naphthyl iodide and alkenylboronic acid, which provides 2-aryl-3-alkenylnaphthalene in good yield. the naphthyl iodide in this case was synthesized through gold - catalyzed cyclization / iodination, which required preparation of the requisite starting material (1-arylalka-2,3-dienyl acetate). additionally, to the best of our knowledge, installation of an alkenyl substituent at the c3 position of a 1,2-disubstituted naphthalene has not been reported. accessing 2,1-borazaronaphthalenes, a class of azaborines that are bn - isosteres of naphthalene, with alkenyl substituents at the c3 position under mild reaction conditions would therefore serve as an example of how functionalized azaborines could be prepared more efficiently than their corresponding all - carbon analogues. recently, we reported the bromination of 2,1-borazaronaphthalenes in high yield under mild reaction conditions with complete regiochemical control. because of the inherent reactivity of azaborine, bromination occurs selectively at the c3 position of the azaborine in the presence of alkyl / aryl substituents on boron and nitrogen. miyaura cross - coupling reactions with potassium alkenyltrifluoroborates as a route to c3-alkenyl - substituted 2,1-borazaronaphthalenes, a compound class that has not been previously reported. optimization of the reaction of 3-bromo-2-methyl-2,1-borazaronaphthalene 1 with functionalized alkenyltrifluoroborate 2 was carried out by screening palladium sources, solvents, and bases. side products represent the sum of homocoupling of the azaborine, protodebromination of the azaborine, and unidentifiable side products. conditions for the cross - coupling of potassium aryltrifluoroborates with brominated 2,1-borazaronaphthalenes did not afford the desired product in appreciable yield (table 1, entry 1). therefore, several other ligands were screened in this reaction (entries 24 and 7). of the ligands tested, triphenylphosphine (pph3) and 1,1-bis(diphenylphosphino)ferrocene (dppf) provided the highest conversions to products. several solvents / ratios were tested for these ligands, and the best result was obtained using toluene as the cosolvent and pd(dppf)cl2 as the palladium source (entry 7). a representative set of potassium alkenyltrifluoroborates were subjected to the developed reaction conditions with 3-bromo-2-methyl-2,1-borazaronaphthalene as the electrophilic partner (table 2). cyclic alkenyltrifluoroborates were successful nucleophiles in the reaction by providing the desired product in yields up to 90% (entries 5, 6, 8, and 10). alkenyltrifluoroborates with alkyl substituents afforded the desired product in high yields (entries 2, 3, and 9). vinyltrifluoroborate was a suitable nucleophile for the reaction because the azaborine was obtained in 70% yield (entry 7). most importantly, cis-1-propenyltrifluoroborate was successfully employed in the reaction, affording the product in high yield without isomerization of the alkene (entry 3). the scalable nature of the cross - coupling reaction was demonstrated by performing the coupling on a 4.5 mmol scale (1 g of azaborine) with one - third palladium loading (2 mol %), which provided the desired product in similar yield (entry 8). reaction completed on a 4.5 mmol scale with 2.0 mol % pd(dppf)cl2. reaction conditions (unless otherwise noted) : 1.0 equiv of 3-bromo-2-methyl-2,1-borazaronaphthalene, 1.1 equiv of potassium alkenyltrifluoroborate, 6.0 mol % pd(dppf)cl2, 3.0 equiv of base, 1:1 toluene / h2o, and 60 c for 18 h. the developed reaction conditions were extended to cross - coupling of 3-bromo-2-phenyl-2,1-borazaronaphthalene with an array of potassium alkenyltrifluoroborates to demonstrate that azaborine can also be substituted with an aryl group on the boron. six different alkenyltrifluoroborates were employed in the coupling and provided the desired product in yields up to 90%. the mild reaction conditions of the coupling were demonstrated in the successful coupling of an alkenyltrifluoroborate with an alkyl chloride substituent, affording the desired product in 90% yield (table 3, entry 2). cyclic alkenyltrifluoroborates were successfully engaged in the reaction as the desired products were obtained in high yields (entries 3, 4, and 6). reaction conditions (unless otherwise noted) : 1.0 equiv of 3-bromo-2-phenyl-2,1-borazaronaphthalene, 1.1 equiv of potassium alkenyltrifluoroborate, 6.0 mol % pd(dppf)cl2, 3.0 equiv of base, 1:1 toluene / h2o, and 60 c for 18 h. to demonstrate the versatility of this method, an array of brominated 2,1-borazaronaphthalenes were synthesized and subjected to the developed reaction conditions with 1-decenyltrifluoroborate as the nucleophile in the reaction. the cross - coupled products were obtained with secondary cyclic and acyclic groups on boron in yields up to 90% (table 4, entries 1 and 2). azaborines with various substitution patterns on the arene of boron were suitable electrophiles for coupling as the cross - coupled products were isolated in high yield (entries 35). substitution on the nitrogen of the 2,1-borazaronaphthalenes did not affect the coupling as reactions with both n - allyl and n - benzyl substituents provided the desired product in high yields (entries 6 and 7). reaction conditions (unless otherwise noted) : 1.0 equiv of 3-bromo-2,1-borazaronaphthalene, 1.1 equiv of potassium alkenyltrifluoroborate, 6.0 mol % pd(dppf)cl2, 3.0 equiv of base, 1:1 toluene / h2o, and 60 c for 18 h. to demonstrate that the developed reaction conditions can be directly applied to the functionalization of other azaborines, 6-bromo-2-methyl-3-phenyl-2,1-borazaronaphthalene was subjected to the reaction (eq 1). the desired product was obtained in 83% yield to afford c6-alkenyl - substituted 2,1-borazaronaphthalenes, examples of which are absent in the literature.1 the reaction conditions were then extended to cross - coupling of 3,6-dibrominated 2,1-borazaronaphthalene. the addition of 2.2 equiv of 1-propenyltrifluoroborate provides a doubly cross - coupled product in 76% yield (eq 2).2 in conclusion, a general method for suzuki miyaura cross - coupling of brominated 2,1-borazaronaphthalenes with potassium alkenyltrifluoroborates has been developed. azaborines with alkyl and aryl groups on boron and with or without substitution on nitrogen are suitable reagents for the coupling. through this route, b nmr spectra were obtained on a spectrometer equipped with the appropriate decoupling accessories. all b nmr chemical shifts were referenced to external bf3oet2 (0.0 ppm) with a negative sign indicating an upfield shift. data are presented as follows : chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, br = broad), coupling constant j (hz), and integration. analytical thin - layer chromatography (tlc) was performed on tlc silica gel plates (0.25 mm) precoated with a fluorescent indicator. hrms data were obtained by either esi or ci using a tof mass spectrometer. 2,1-borazaronaphthalenes were synthesized and brominated according to methods previously described in the literature. the title compound was obtained as a white solid in 90% yield (1.0 mmol scale, 316.7 mg) : mp 98103 c ; h nmr (500 mhz, cdcl3) 8.49 (s, 1h), 8.07 (s, 1h), 7.98 (d, j = 7.8 hz, 2h), 7.72 (d, j = 7.8 hz, 2h), 7.64 (d, j = 7.9 hz, 1h), 7.517.49 (m, 1h), 7.34 (d, j = 8.1 hz, 1h), 7.307.27 (m, 1h) ; c nmr (125.8 mhz, cdcl3) 147.3, 139.0, 133.6, 131.2 (d, j = 32.5 hz), 129.7, 129.2, 126.5 (q, j = 275.8 hz), 125.3, 124.6 (q, j = 3.6 hz), 122.5, 118.4 ; b nmr (128.38 mhz, cdcl3) 32.9 ; ir (neat) 3367, 2923, 1556, 1427, 1120, 1070 cm ; hrms (ci) m / z [m + h ] calcd for c15h11bnf3br 352.9825, found 352.9802. to a biotage 10 ml microwave vial equipped with a magnetic stir bar were added 3-bromo-2,1-alkenyltrifluoroborate (1.1 mmol, 1.1 equiv), cs2co3 (3.0 mmol, 977 mg), pd(dppf)cl2 (6.0 mol %, 42 mg), and potassium alkenyltrifluoroborate (1.0 mmol, 1.0 equiv). the vial was sealed with a cap, lined with a teflon septum, and evacuated and purged with argon gas three times. anhydrous degassed toluene (1.0 ml) and deionized h2o (1.0 ml) were added under argon gas. the reaction mixture was heated at 60 c for 18 h. after cooling to rt, the reaction mixture was extracted with etoac (3 10 ml) and dried (mgso4). after being concentrated in vacuo, the product was isolated by flash column chromatography and eluted with a gradient of etoac in hexanes (0 to 10% etoac). the title compound was obtained as a yellow solid in 88% yield (235.8 mg) : mp 8993 c ; h nmr (500 mhz, cdcl3) 7.79 (s, 1h), 7.72 (s, 1h), 7.58 (d, j = 7.5 hz, 1h), 7.347.37 (m, 1h), 7.18 (d, j = 8.1 hz, 1h), 7.157.12 (m, 1h), 5.245.23 (m, 1h), 5.12 (d, j = 2.5 hz, 1h), 3.67 (t, j = 4.6 hz, 4h), 3.31 (s, 2h), 2.48 (br, 4h), 0.79 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 149.0, 140.1, 129.6, 128.1, 125.0, 121.0, 117.4, 114.2, 67.3, 64.8, 53.9 ; b nmr (128.38 mhz, cdcl3) 36.7 ; ir (neat) 3319, 2928, 1566, 1434, 1109, 891 cm ; hrms (esi+) m / z [m + h ] calcd for c16h22bn2o 269.1825, found 269.1820. the title compound was obtained as a yellow solid in 98% yield (179.3 mg) : mp 6975 c;h nmr (500 mhz, cdcl3) 7.76 (s, 1h), 7.65 (s, 1h), 7.56 (d, j = 7.8 hz, 1h), 7.367.29 (m, 1h), 7.16 (d, j = 8.0 hz, 1h), 7.137.10 (m, 1h), 6.59 (d, j = 15.6 hz, 1h), 6.196.12 (m, 1h), 1.91 (dd, j = 6.6, 1.5 hz, 3h), 0.85 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 139.5, 138.7, 134.8, 129.3, 127.6, 126.5, 125.6, 121.0, 117.4, 19.4 ; b nmr (128.38 mhz, cdcl3) 37.2 ; ir (neat) 3364, 2924, 1614, 1457, 1343 cm ; hrms (ci) m / z [m ] calcd for c12h14bn 183.1219, found 183.1216. the title compound was obtained as a brown solid in 87% yield (159.2 mg) : mp 98103 c ; h nmr (500 mhz, cdcl3) 7.42 (s, 1h), 7.68 (s, 1h), 7.59 (d, j = 7.8 hz, 1h), 7.377.34 (m, 1h), 7.19 (d, j = 8.0 hz, 1h), 7.187.13 (m, 1h), 6.576.54 (m, 1h), 5.725.79 (m, 1h), 1.85 (dd, j = 7.0, 1.8 hz, 3h), 0.74 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 140.8, 139.6, 132.1, 129.4, 127.8, 125.3, 125.0, 121.0, 117.6, 14.8 ; b nmr (128.38 mhz, cdcl3) 37.3 ; ir (neat) 3364, 2925, 1611, 1560, 1454 cm ; hrms (ci) m / z [m ] calcd for c12h14bn 183.1219, found 183.1213. the title compound was obtained as a yellow oil in 69% yield (126.2 mg). h nmr (500 mhz, cdcl3) 7.80 (s, 1h), 7.68 (s, 1h), 7.62 (d, j = 7.7 hz, 1h), 7.397.36 (m, 1h), 7.197.15 (m, 2h), 5.215.11 (m, 1h), 5.07 (d, j = 1.9 hz, 1h), 2.16 (s, 3h), 0.87 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 148.4, 139.6, 139.2, 129.6, 128.0, 125.1, 121.0, 117.4, 113.0, 23.9 ; b nmr (128.38 mhz, cdcl3) 36.9 ; ir (neat) 3368, 2933, 1567, 1462, 1453 cm ; hrms (ci) m / z [m ] calcd for c12h14bn 183.1219, found 183.1220. the title compound was obtained as a yellow oil in 82% yield (184.5 mg). h nmr (500 mhz, cdcl3) 7.75 (s, 1h), 7.73 (s, 1h), 7.59 (d, j = 7.8 hz, 1h), 7.347.38 (m, 1h), 7.18 (d, j = 8.1 hz, 1h), 7.167.13 (m, 1h), 5.835.82 (m, 1h), 4.364.35 (m, 2h), 3.97 (t, j = 5.4 hz, 2h), 2.522.49 (m, 2h), 0.84 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 138.6, 137.6, 137.5, 128.5, 127.0, 124.1, 121.9, 120.1, 116.4, 65.2, 63.9, 28.1 ; b nmr (128.38 mhz, cdcl3) 36.7 ; ir (neat) 3325, 2923, 1572, 1458, 1032 cm ; hrms (ci) m / z [m ] calcd for c14h16bno 225.1325, found 225.1336. the title compound was obtained as a white solid in 81% yield (227.6 mg) : mp 8388 c ; h nmr (500 mhz, cdcl3) 7.73 (s, 1h), 7.69 (s, 1h), 7.547.56 (m, 1h), 7.327.35 (m, 1h), 7.16 (d, j = 8 hz, 1h), 7.137.10 (m, 1h), 5.665.65 (m, 1h), 4.054.02 (m, 4h), 2.632.59 (m, 2h), 2.502.49 (m, 2h), 1.93 (t, j = 6.5 hz, 2h), 0.81 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 140.9, 139.5, 138.7, 129.4, 127.8, 125.2, 121.1, 120.9, 117.3, 108.3, 64.7, 36.5, 31.8, 28.7 ; b nmr (128.38 mhz, cdcl3) 37.0 ; ir (neat) 3308, 2927, 1609, 1460, 1047 cm ; hrms (ci) m / z [m ] calcd for c17h20bno2 281.1587, found 281.1593. the title compound was obtained as a light yellow solid in 70% yield (118.3 mg) : mp 5256 c ; h nmr (500 mhz, cdcl3) 7.70 (s, 1h), 7.64 (s, 1h), 7.55 (d, j = 7.8 hz, 1h), 7.337.30 (m, 1h), 7.15 (d, j = 8.0 hz, 1h), 7.107.08 (m, 1h), 6.736.66 (m, 1h), 5.875.85 (m, 1h), 5.745.71, (m, 1h), 0.70 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 140.7, 140.3, 139.9, 129.6, 128.2, 125.3, 121.2, 117.5, 114.8 ; b nmr (128.38 mhz, cdcl3) 37.2 ; ir (neat) 3365, 2927, 1610, 1457 cm ; hrms (ci) the title compound was obtained as a white solid in 90% yield (233.1 mg) : mp 7276 c ; h nmr (500 mhz, cdcl3) 7.73 (s, 1h), 7.69 (s, 1h), 7.60 (d, j = 8.0 hz, 1h), 7.417.37 (m, 1h), 7.207.15 (m, 2h), 5.595.56 (m, 1h), 2.782.72 (m, 2h), 2.672.64 (m, 2h), 2.252.17 (m, 2h), 0.82 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 140.8, 139.5, 139.1, 129.4, 128.0, 124.9, 123.3 (t, j = 239.6 hz), 121.0, 118.2 (t, j = 4.9 hz), 117.3, 35.3 (t, j = 26.4 hz), 31.0 (t, j = 24.4 hz), 27.8 (t, j = 5.2 hz) ; b nmr (128.38 mhz, cdcl3) 36.2 ; ir (neat) 3397, 2936, 1613, 1560, 1425, 1058, cm ; hrms (ci) the title compound was obtained as a yellow solid in 75% yield (210.7 mg) : mp 5862 c ; h nmr (500 mhz, cdcl3) 7.79 (s, 1h), 7.65 (s, 1h), 7.57 (d, j = 7.75 hz, 1h), 7.347.31 (m, 1h), 7.167.11 (m, 2h), 6.58 (d, j = 15.5 hz, 1h), 6.186.12 (m, 1h), 2.262.22 (m, 2h), 1.531.47 (m, 2h), 1.401.28 (m, 10h), 0.92 (t, j = 6.9 hz, 3h), 0.86 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 139.5, 138.8, 133.4, 132.2, 129.3, 127.6, 125.6, 121.0, 117.4, 34.0, 32.2, 30.0, 29.9, 29.7, 29.6, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 37.1 ; ir (neat) 3366, 2921, 1556, 1463, 977 cm ; hrms (ci) m / z [m ] calcd for c19h28bn 281.2315, found 281.2328. the title compound was obtained as a white solid in 65% yield (210.6 mg) : mp 8387 c ; h nmr (500 mhz, cdcl3) 7.75 (s, 1h), 7.69 (s, 1h), 7.57 (d, j = 7.7 hz, 1h), 7.367.33 (m, 1h), 7.17 (d, j = 8.0 hz, 1h), 7.147.11 (m, 1h), 5.71 (br, 1h), 4.07 (br, 2h), 3.64 (t, j = 5.5 hz, 2h), 4.47 (br, 2h), 1.52 (s, 9h), 0.80 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 155.3, 139.9, 139.6, 138.7, 129.5, 128.0, 125.1, 121.1, 120.7, 117.4, 79.7, 44.3, 40.1, 34.9, 29.4, 28.8 ; b nmr (128.38 mhz, cdcl3) 32.0 ; ir (neat) 3327, 2975, 1677, 1563, 1423, 1163 cm ; hrms (ci) m / z [m + h ] calcd for c19h26bn2o2 325.2087, found 325.2080. the title compound was obtained as a yellow solid in 83% yield (226.5 mg) : mp 7477 c ; h nmr (500 mhz, cdcl3) 7.66 (s, 1h), 7.59 (s, 1h), 7.55 (d, j = 7.7 hz, 1h), 7.387.36 (m, 1h), 7.357.31 (m, 2h), 7.227.13 (m, 5h), 6.58 (dd, j = 11.5, 1.4 hz, 1h), 5.69 (dt, j = 11.5, 7.2 hz, 1h), 2.79 (t, j = 7.7 hz, 2h), 2.702.45 (m, 2h), 0.73 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 142.3, 140.6, 139.7, 132.1, 129.9, 129.5, 128.9, 128.6, 127.9, 126.1, 125.3, 121.0, 117.5, 36.7, 30.6 ; b nmr (128.38 mhz, cdcl3) 37.4 ; ir (neat) 3373, 2925, 1556, 1452, 746, 703 cm ; hrms (ci) m / z [m ] calcd for c19h20bn 273.1689, found 273.1683. the title compound was obtained as a white oil in 73% yield (250.3 mg). h nmr (500 mhz, cdcl3) 8.05 (s, 1h), 7.91 (s, 1h), 7.777.75 (m, 2h), 7.69 (d, j = 8.0 hz, 1h), 7.517.47 (m, 3h), 7.427.39 (s, 1h), 7.26 (d, j = 8.0 hz, 1h), 7.22 (t, j = 7.5 hz, 1h), 6.70 (d, j = 15.5 hz, 1h), 6.07 (dt, j = 15.4, 7.0 hz, 1h), 2.21 (q, j = 7.0 hz, 2h), 1.501.45 (m, 2h), 1.401.35 (m, 10h), 0.96 (t, j = 7.0 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 139.8, 139.6, 133.2, 132.7, 132.3, 129.5, 128.8, 128.2, 128.0, 125.9, 121.0, 118.0, 33.7, 32.2, 29.9, 29.8, 29.7, 29.6, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 34.4 ; ir (neat) 3370, 2923, 2852, 1421, 749 cm ; hrms (ci) m / z [m + na ] calcd for c24h30bnna 366.2369, found 366.2375. the title compound was obtained as a brown oil in 90% yield (288.9 mg). h nmr (500 mhz, cdcl3) 8.03 (s, 1h), 7.92 (s, 1h), 7.72 (dd, j = 7.25, 1.5 hz, 2h), 7.68 (d, j = 7.5 hz, 1h), 7.507.46 (m, 3h), 7.427.39 (m, 1h), 7.26 (d, j = 8 hz, 1h), 7.237.20 (m, 1h), 6.68 (d, j = 15.5 hz, 1h), 6.00 (dt, j = 15.4, 7.0 hz, 1h), 3.57 (t, j = 6.5 hz, 2h), 2.232.19 (m, 2h), 1.861.83 (m, 2h), 1.621.58 (m, 2h) ; c nmr (125.8 mhz, cdcl3) 140.1, 139.6, 133.4, 133.1, 131.1, 129.5, 128.8, 128.2, 128.1, 125.7, 121.7, 118.0, 45.3, 32.7, 32.3, 27.0 ; b nmr (128.38 mhz, cdcl3) 35.0 ; ir (neat) 3373, 2933, 1558, 1421, 969, 752 cm ; hrms (ci) m / z [m ] calcd for c20h21bncl 321.1456, found 321.1463. the title compound was obtained as a yellow solid in 79% yield (225.1 mg) : mp 8892 c ; h nmr (500 mhz, cdcl3) 7.95 (s, 1h), 7.85 (s, 1h), 7.797.7.78 (m, 2h), 7.66 (d, j = 7.5 hz, 1h), 7.457.39 (m, 4h), 7.28 (d, j = 8.0 hz, 1h), 7.227.19 (m, 1h), 5.735.71 (m, 1h), 2.192.13 (m, 4h), 1.661.65 (m, 4h) ; c nmr (125.8 mhz, cdcl3) 142.3, 140.5, 139.6, 132.8, 129.5, 128.9, 128.1, 128.0, 125.6, 124.2, 121.5, 117.9, 29.9, 26.1, 23.5, 22.6 ; b nmr (128.38 mhz, cdcl3) 34.0 ; ir (neat) 3375, 2920, 1558, 1451, 751 cm ; hrms (ci) m / z [m ] calcd for c20h20bn 285.1689, found 285.1705. the title compound was obtained as a white solid in 86% yield (246.8 mg) : mp 112115 c ; h nmr (500 mhz, cdcl3) 8.02 (s, 1h), 7.91 (s, 1h), 7.767.68 (m, 2h), 7.69 (d, j = 7.5 hz, 1h), 7.477.42 (m, 4h), 7.29 (d, j = 8.1 hz, 1h), 7.247.21 (m, 1h), 5.755.73 (m, 1h), 4.314.29 (m, 2h), 3.84 (t, j = 5.5 hz, 2h), 2.30 (td, j = 5.3, 2.7 hz, 2h) ; c nmr (125.8 mhz, cdcl3) 140.9, 139.7, 139.4, 132.7, 129.6, 128.9, 128.4, 128.2, 125.3, 122.8, 121.7, 118.0, 66.2, 64.9, 29.5 ; b nmr (128.38 mhz, cdcl3) 34.0 ; ir (neat) 3287, 2930, 1564, 1455, 1229, 758 cm ; hrms (ci) m / z [m ] calcd for c19h18bno 287.1481, found 287.1487. the title compound was obtained as a brown oil in 76% yield (186.2 mg). h nmr (500 mhz, cdcl3) 7.96 (br, 2h), 7.927.81 (m, 2h), 7.70 (d, j = 7.5 hz, 1h), 7.477.43 (m, 4h), 7.29 (d, j = 8.0 hz, 1h), 7.257.22 (m, 1h), 5.065.02 (m, 2h), 1.99 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 149.2, 140.9, 139.5, 132.6, 129.5, 128.7, 128.2, 128.0, 125.2, 121.5, 117.8, 113.0, 24.1 ; b nmr (128.38 mhz, cdcl3) 33.6 ; ir (neat) 3367, 2925, 1573, 1463, 757 cm ; hrms (ci) m / z [m ] calcd for c17h16bn 245.1376, found 245.1377. the title compound was obtained as a white solid in 81% yield (277.8 mg) : mp 108112 c ; h nmr (500 mhz, cdcl3) 7.98 (s, 1h), 7.90 (s, 1h), 7.807.78 (m, 2h), 7.65 (d, j = 7.5 hz, 1h), 7.457.39 (m, 4h), 7.28 (d, j = 8.0 hz, 1h), 7.217.18 (m, 1h), 6.625.60 (m, 1h), 4.053.98 (m, 4h), 2.472.46 (m, 2h), 2.38 (td, j = 6.3, 1.6 hz, 2h), 1.79 (t, j = 6.5 hz, 2h) ; c nmr (125.8 mhz, cdcl3) 141.9, 141.1, 139.6, 132.7, 129.4, 128.9, 128.1, 128.1, 125.3, 121.5, 121.1, 117.8, 108.2, 64.5, 36.4, 31.6, 29.0 ; b nmr (128.38 mhz, cdcl3) 34.4 ; ir (neat) 3326, 1560, 1421, 1112, 755 cm ; hrms (ci) m / z [m ] calcd for c22h22bno2 343.1744, found 343.1756. the title compound was obtained as a colorless oil in 81% yield (247.8 mg). h nmr (500 mhz, cdcl3) 7.92 (s, 1h), 7.67 (s, 1h), 7.62 (d, j = 7.8 hz, 1h), 7.397.35 (m, 1h), 7.24 (d, j = 8.0 hz, 1h), 7.187.15 (m, 1h), 6.69 (dd, j = 15.6, 0.4 hz, 1h), 6.136.18 (m, 1h), 2.312.26 (m, 2h), 1.961.92 (m, 1h), 1.571.54 (m, 2h), 1.431.35 (m, 10h), 1.23 (d, j = 7.3 hz, 6h), 0.97 (t, j = 6.9 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 139.6, 138.4, 132.2, 131.1, 129.3, 127.6, 125.7, 121.2, 117.7, 33.8, 32.2, 30.0, 29.9, 29.7, 29.6, 23.0, 19.6, 14.4 ; b nmr (128.38 mhz, cdcl3) 38.8 ; ir (neat) 3424, 2924, 1560, 1427, 969 cm ; hrms (ci) m / z [m ] calcd for c21h32bn 309.2628, found 309.2633. the title compound was obtained as a yellow solid in 90% yield (276.3 mg) : mp 5155 c ; h nmr (500 mhz, cdcl3) 7.92 (s, 1h), 7.56 (d, j = 7.7 hz, 1h), 7.327.29 (m, 1h), 7.18 (s, 1h), 7.11 (t, j = 7.9 hz, 2h), 6.69 (d, j = 15.6 hz, 1h), 6.386.24 (m, 1h), 2.25 (q, j = 7.2 hz, 2h), 1.521.48 (m, 2h), 1.391.28 (m, 10h), 0.920.89 (m, 5h), 0.660.55 (m, 3h) ; c nmr (125.8 mhz, cdcl3) 139.5, 138.5, 132.8, 132.4, 129.3, 127.6, 125.5, 121.0, 117.4, 34.0, 32.2, 30.0, 29.9, 29.7, 29.6, 23.0, 14.4, 6.0 ; b nmr (128.38 mhz, cdcl3) 37.1 ; ir (neat) 3374, 3010, 2920, 1557, 1428, 1105 cm ; hrms (ci) m / z [m ] calcd for c21h30bn 307.2471, found 307.2484. the title compound was obtained as a yellow oil in 88% yield (317.6 mg). h nmr (500 mhz, cdcl3) 8.01 (s, 1h), 7.85 (s, 1h), 7.717.65 (m, 3h), 7.417.37 (m, 1h), 7.25 (d, j = 8.5 hz, 1h), 7.227.19 (m, 1h), 7.177.14 (m, 2h), 6.62 (dd, j = 15.6, 0.7 hz, 1h), 6.01 (dt, j = 15.5, 7.0 hz, 1h), 2.17 (qd, j = 7.3, 1.3 hz, 2h), 1.481.49 (m, 2h), 1.361.29 (m, 10h), 0.92 (t, j = 7.0 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 163.6 (d, j = 252 hz), 139.8, 139.3, 134.9 (d, j = 7.4 hz), 132.4, 132.3, 129.3, 127.9, 125.7, 121.6, 117.8, 115.1 (d, j = 19.6 hz), 33.5, 32.0, 29.7, 29.6, 29.5, 29.4, 22.8, 14.2 ; b nmr (128.38 mhz, cdcl3) 33.9 ; ir (neat) 3375, 2923, 1595, 1224, 832 cm ; hrms (ci) m / z [m ] calcd for c24h29bnf 361.2377, found 361.2368. the title compound was obtained as a colorless oil in 81% yield (302.1 mg). h nmr (500 mhz, cdcl3) 8.04 (s, 1h), 7.94 (s, 1h), 7.68 (d, j = 7.7 hz, 1h), 7.437.40 (m, 2h), 7.32 (d, j = 7.2 hz, 1h), 7.297.27 (m, 2h), 7.22 (t, j = 7.5 hz, 1h), 7.01 (dd, j = 8.2, 2.7 hz, 1h), 6.68 (d, j = 15.6 hz, 1h), 6.106.04 (m, 1h), 3.89 (s, 3h), 2.212.16 (m, 2h), 1.471.43 (m, 2h), 1.361.28 (m, 10h), 0.93 (t, j = 6.9 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 159.4, 139.8, 139.5, 132.5, 132.3, 129.5, 129.4, 128.0, 125.9, 125.5, 121.7, 118.3, 118.0, 114.4, 55.4, 33.7, 32.2, 29.9, 29.8, 29.6, 29.6, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 34.8 ; ir (neat) 3366, 2924, 1558, 1451, 1236, 971, 761 cm ; hrms (ci) m / z [m + h ] calcd for c25h33bno 374.2655, found 374.2661. the title compound was obtained as a light brown oil in 83% yield (341.3 mg). h nmr (500 mhz, cdcl3) 8.03 (s, 1h), 7.91 (s, 1h), 7.80 (d, j = 7.7 hz, 2h), 7.707.68 (m, 3h), 7.427.39 (m, 1h), 7.27 (d, j = 8.1 hz, 1h), 7.227.21 (m, 1h), 6.56 (dd, j = 15.6, 1.0 hz, 1h), 5.605.94 (m, 1h), 2.14 (m, 2h), 1.421.39 (m, 2h), 1.271.32 (m, 10h), 0.90 (t, j = 5.1 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 140.1, 139.1, 133.1, 132.8, 131.9, 130.4 (d, j = 32.3 hz), 129.4, 128.1, 126.3 (q, j = 272.2 hz), 125.8, 124.6 (q, j = 3.7 hz), 121.9, 117.9, 33.5, 32.0, 29.6, 29.5, 29.3, 22.8, 14.2 ; b nmr (128.38 mhz, cdcl3) 34.5 ; ir (neat) 3395, 2924, 1559, 1323, 969, 836 cm ; hrms (ci) m / z [m ] calcd for c25h29bnf3 411.2345, found 411.2353. the title compound was obtained as a light yellow oil in 62% yield (199.0 mg). h nmr (500 mhz, cdcl3) 7.84 (s, 1h), 7.61 (d, j = 7.0 hz, 1h), 7.39 (dd, j = 4.7, 1.2 hz, 2h), 7.177.13 (m, 1h), 6.66 (dd, j = 15.5, 1.0 hz, 1h), 6.106.01 (m, 2h), 5.15 (dd, j = 10.5, 1.4 hz, 1h), 4.99 (dd, j = 17.2, 1.3 hz, 1h), 4.72 (dd, j = 4.1, 2.0 hz, 2h), 2.272.22 (m, 2h), 1.541.49 (m, 2h), 1.411.27 (m, 10h), 0.930.89 (m, 6h) ; c nmr (125.8 mhz, cdcl3) 141.2, 137.7, 134.7, 132.7, 131.3, 130.2, 127.8, 126.7, 120.8, 115.9, 115.5, 49.8, 33.8, 32.2, 30.1, 29.9, 29.7, 29.6, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 38.7 ; ir (neat) 2923, 1556, 1405, 1212, 968 cm ; hrms (ci) m / z [m ] calcd for c22h32bn 321.2628, found 321.2633. the title compound was obtained as a light brown oil in 90% yield (333.9 mg). h nmr (500 mhz, cdcl3) 7.97 (s, 1h), 7.68 (d, j = 7.0 hz, 1h), 7.357.27 (m, 5h), 7.197.16 (m, 3h), 6.78 (dd, j = 15.6, 0.6 hz, 1h), 6.19 (dt, j = 15.5, 6.9 hz, 1h), 5.38 (s, 2h), 2.362.37 (m, 2h), 1.631.57 (m, 2h), 1.501.39 (m, 10h), 1.000.98 (m, 6h) ; c nmr (125.8 mhz, cdcl3) 141.3, 138.8, 138.02, 132.7, 131.4, 130.2, 129.0, 127.9, 127.1, 126.9, 126.1, 120.9, 115.9, 51.4, 33.8, 32.2, 30.1, 29.9, 29.8, 29.7, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 39.0 ; ir (neat) 2924, 2853, 1610, 1492, 1402, 1360, 967 cm ; hrms (ci) m / z [m ] calcd for c26h34bn 371.2784, found 371.2791. the title compound was obtained as a light yellow oil in 80% yield (285.6 mg). h nmr (500 mhz, cdcl3) 8.04 (s, 1h), 7.91 (s, 1h), 7.68 (d, j = 7.5 hz, 1h), 7.577.52 (m, 2h), 7.427.37 (m, 2h), 7.297.25 (m, 2h), 7.21 (t, j = 7.5 hz, 1h), 6.68 (d, j = 15.5 hz, 1h), 6.07 (dt, j = 15.4, 7.0 hz, 1h), 2.47 (s, 3h), 2.20 (q, j = 6.9 hz, 2h), 1.491.46 (m, 2h), 1.391.33 (m, 10h), 0.94 (t, j = 6.9 hz, 3h) ; c nmr (125.8 mhz, cdcl3) 139.7, 137.5, 133.9, 132.7, 132.2, 130.3, 129.5, 128.1, 128.0, 125.9, 121.6, 117.9, 33.7, 32.2, 30.0, 29.9, 29.7, 29.6, 23.0, 21.9, 14.4 ; b nmr (128.38 mhz, cdcl3) 34.7 ; ir (neat) 3369, 2923, 2852, 1612, 1557, 1450, 969 cm ; hrms (ci) m / z [m ] calcd for c25h32bn 357.2628, found 357.2632. the title compound was obtained as a brown oil in 83% yield (296.3 mg). h nmr (500 mhz, cdcl3) 7.85 (s, 1h), 7.77 (s, 1h), 7.57 (d, j = 1.5 hz, 1h), 7.477.41 (m, 5h), 7.337.31 (m, 1h), 7.16 (d, j = 8.5 hz, 1h), 6.46 (d, j = 16.0 hz, 1h), 6.23 (dt, j = 15.7, 6.9 hz, 1h), 2.272.22 (m, 2h), 1.521.48 (m, 2h), 1.391.28 (m, 10h), 0.92 (t, j = 7.0 hz, 3h), 0.83 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 145.1, 141.8, 139.0, 131.3, 130.0, 129.6, 128.5, 128.4, 127.2, 126.3, 126.0, 125.3, 117.7, 33.4, 32.2, 29.8, 29.8, 29.6, 29.6, 23.0, 14.4 ; b nmr (128.38 mhz, cdcl3) 36.4 ; ir (neat) 3372, 2924, 2853, 1617, 1577, 1456, 962, 762 cm ; hrms (ci) m / z [m ] calcd for c25h32bn 357.2628, found 357.2621. the title compound was obtained as a yellow solid in 76% yield (169.4 mg) : mp 106110 c ; h nmr (500 mhz, cdcl3) 7.75 (s, 1h), 7.60 (s, 1h), 7.48 (d, j = 1.5 hz, 1h), 7.38 (dd, j = 8.3, 1.9 hz, 1h), 7.07 (d, j = 8.4 hz, 1h), 6.62 (dd, j = 15.7, 0.7 hz, 1h), 6.48 (dd, j = 15.7, 1.5 hz, 1h), 6.266.14 (m, 2h), 1.951.93 (m, 6h), 0.86 (s, 3h) ; c nmr (125.8 mhz, cdcl3) 137.4, 137.6, 133.9, 130.1, 130.0, 125.7, 125.4, 124.5, 124.3, 123.1, 116.6, 18.3, 17.8 ; b nmr (128.38 mhz, cdcl3) 37.2 ; ir (neat) 3371, 2957, 2910, 1610, 1557, 1436 cm ; hrms (ci) m / z [m ] calcd for c15h18bn 223.1532, found 223.1532.	conditions have been developed for the palladium - catalyzed cross - coupling of 3-bromo-2,1-borazaronaphthalenes with potassium alkenyltrifluoroborates. twenty - seven alkenyl - substituted azaborines have been synthesized through this method, providing access to a family of 2,1-borazaronaphthalenes with alkenyl substitution at the c3 position.
diabetes, a group of diseases of which type 2 diabetes is the most common, is a rapidly growing health problem worldwide (1,2). type 2 diabetes is a progressive disease in which the advanced stages are characterized by micro- and macrovascular complications (e.g., retinopathy, nephropathy, and neuropathy) and atherosclerosis (3,4). it affects quality of life and ranks ninth as a cause of global mortality (1). physical inactivity and obesity are the most important modifiable risk factors for type 2 diabetes (5,6). some studies suggest that exposure to job strain, the most widely studied form of work stress (7), is also associated with an increased risk of type 2 diabetes (810). an association between job strain and diabetes is biologically plausible (11) because stress response increases secretion of the fight - or - flight hormone cortisol, which stimulates glucose production in the liver and antagonizes the action of insulin in peripheral tissues (1214). whereas some studies have shown an association (810), other studies have found no evidence for job strain as a risk factor for diabetes (1517). a further complication is that lifestyle risk factors for type 2 diabetes tend to cluster in those who also report job strain (1822). dissecting out the effects of job strain from those of an unhealthy lifestyle is challenging as few studies are large enough to determine the association between job strain and type 2 diabetes in analysis stratified by lifestyle factors. to address these limitations, we pooled results from 13 cohort studies and conducted an analysis of individual - participant data on almost 125,000 men and women initially free from diabetes. the size of the data and the number of incident type 2 cases at follow - up exceed those of previous reports. data are drawn from 13 independent cohort studies from finland, france, denmark, sweden, and the u.k. all the studies are part of the individual - participant - data meta - analysis of working populations (ipd - work) consortium (23). details of the study design and participants have been published previously (supplementary data). we included a total of 131,955 participants who were employed at the baseline assessment, which took place between 1986 and 2008, depending on the study. we excluded from the analyses 4,080 (3%) participants with missing values for sex, age, job strain, or diabetes and 3,067 (2%) with a diagnosis of diabetes before or at study baseline. each constituent study in the consortium was approved by the relevant local or national ethics committees, and all participants gave informed consent (supplementary data). job strain was measured with questions from the validated job content questionnaire and demand control questionnaire, which were included in the baseline self - report questionnaire of all studies (24,25). we have previously published a detailed description of the job - strain measure, including its validation and harmonization, as part of the consortium (24). in brief, participants were asked to answer questions about psychosocial aspects of their job. for each participant, mean response scores were calculated for job demand items (i.e., inquiries about whether the participant had to work very hard or had excessive amounts of work, conflicting demands, or insufficient time) and job control items (i.e., inquiries about decision freedom and learning new things at work). the agreement between the harmonized scales used in this study and the complete versions was mostly good or very good (statistic > 0.68) with a few exceptions for which agreement was moderate (between 0.54 and 0.60) (24). we defined high job demands as having a job demand score that was greater than the study - specific median score ; similarly, we defined low job control as having a job control score that was lower than the study - specific median score. we defined the exposure as a binary variable : job strain (high demands and low control) versus no job strain (all other combinations) according to the job strain model (25). as an alternative conceptualization, we defined job strain quadrants : high - strain job (high demands and low control), active job (high demands and high control), passive job (low demands and low control), and low - strain job (low demands and high control). to minimize investigator bias, we validated the job strain measure before extracting data on incident type 2 diabetes, with investigators masked to outcome information (24). the outcome was the first record of type 2 diabetes, diagnosed corresponding to icd-10 code e11. we collected records from hospital admissions and discharge registers and mortality registers with a mention of diagnosis of type 2 diabetes in any of the diagnosis codes. additionally, in the finnish datasets (fps, hessup, and still working), participants were also defined as an incident type 2 diabetes case the first time they appeared in the nationwide drug reimbursement register as eligible for type 2 diabetes medication (27). in the whitehall ii study, type 2 diabetes was ascertained by 2-h oral glucose tolerance test administered every 5 years (11) using world health organization criteria and complemented by self - reports of diabetes diagnosis and medication (28). in the gazel study, we only had icd codes for mortality data so new nonfatal cases were based on self - report from annual questionnaires. the date of incident diabetes was defined as the date of the first record during the follow - up in any of the previously mentioned sources (supplementary table 1). prevalent (existing) type 2 diabetes cases were defined using information from any of the following : hospital records (all studies except for gazel and whitehall ii), baseline medical assessment (whitehall ii), self - report from the baseline questionnaire (copsoq - ii, fps, gazel, hessup, ipaw, slosh, whitehall ii, wolf norrland [wolf n ], and wolf stockholm [wolf s ]), or drug reimbursement register in finland (fps, hessup, and still working). we excluded participants with a diagnosis of either type 1 or type 2 diabetes either before or at the study baseline (icd-10 codes e10e11 or icd-9 and icd-8 code 250) (supplementary table 2). in addition to age and sex, we used data on socioeconomic status (ses), working hours, bmi, leisure - time physical activity, smoking, and alcohol consumption as covariates (that is, confounders or mediators). ses was defined based on occupational title, which was register based (in copsoq - i, copsoq - ii, dwecs, fps, gazel, ipaw, puma, and still working) or self - reported (in whitehall ii, slosh, wolf n, and wolf s). in hessup, ses was based on self - reported highest educational qualification. ses was categorized into low, intermediate, high, and other, with participants who were self - employed or whose job title was missing included in the last category. working hours were divided into categories of 50 kg / m were excluded from the analysis including bmi. we grouped participants into three categories according to their level of leisure - time physical activity : sedentary (physically inactive), highly active (at least 2.5 h of moderate, or at least 1 h 15 min of vigorous, physical activity per week), or moderately active (all levels in between). information on physical activity was not available for participants in copsoq - i (18). tobacco smoking was self - reported and categorized into never, ex-, and current smoking (19). we used responses to questions about the total number of alcoholic drinks consumed per week to classify participants as nondrinkers, moderate drinkers (114 drinks per week for women and 121 drinks per week for men), high - to - intermediate drinkers (1520 drinks per week for women and 2227 drinks per week for men), and heavy drinkers (21 drinks per week for women and 28 drinks per week for men) (20). harmonized data on alcohol consumption were not available for participants in copsoq - i or slosh. for additional adjustment for biological risk markers (representing potential mediators), we included self - reported hypertension or use of antihypertensive medication (fps, hessup, slosh, ipaw, and copsoq - ii), self - reported elevated lipids (hessup), or measured systolic blood pressure, triglycerides, and hdl cholesterol (whitehall ii, wolf n, and wolf s). because shift work has been suggested to elevate the risk of type 2 diabetes (3032), we also identified respondents who worked in shifts or during the night. participants who reported daytime work only were classified as nonshift workers, and those reporting nighttime work (between 6:00 p.m. and 6:00 a.m.) or any form of shift work were classified as shift workers. in addition, data for shift or nighttime working were not available for copsoq - i, copsoq - ii, dwecs, gazel, ipaw, and puma. follow - up time was calculated from baseline assessment until the first record of type 2 diabetes, death, or end of follow - up, whichever came first. job strain was modeled as a binary exposure (job strain vs. no job strain [the reference ]) and in sensitivity analysis as a categorical variable (high strain, active, passive, and low strain [the reference ]). all analyses were adjusted for sex, age, and ses and then further adjusted for lifestyle variables (bmi category, physical activity, smoking, and alcohol consumption). the models adjusted for age, sex, ses, and lifestyle factors were also additionally adjusted for biological risk markers. to address reverse causation, we excluded the first 3 years of follow - up. to minimize the possibility that shift work affected any associations, we repeated the analyses separately in participants who reported working shifts or nights and among those who did not. participants with missing data were excluded from this analysis. as in previous studies from the ipd - work consortium, we also examined risk of diabetes in the four groups created by combining data on job strain and each lifestyle risk factor (33). dichotomized lifestyle risk factors used in these analyses were current smoking (yes vs. no), heavy alcohol use (21 drinks per week for women and 28 drinks per week for men vs. other), obesity (bmi 30 vs. 0.68) with a few exceptions for which agreement was moderate (between 0.54 and 0.60) (24). we defined high job demands as having a job demand score that was greater than the study - specific median score ; similarly, we defined low job control as having a job control score that was lower than the study - specific median score. we defined the exposure as a binary variable : job strain (high demands and low control) versus no job strain (all other combinations) according to the job strain model (25). as an alternative conceptualization, we defined job strain quadrants : high - strain job (high demands and low control), active job (high demands and high control), passive job (low demands and low control), and low - strain job (low demands and high control). to minimize investigator bias, we validated the job strain measure before extracting data on incident type 2 diabetes, with investigators masked to outcome information (24). the outcome was the first record of type 2 diabetes, diagnosed corresponding to icd-10 code e11. we collected records from hospital admissions and discharge registers and mortality registers with a mention of diagnosis of type 2 diabetes in any of the diagnosis codes. additionally, in the finnish datasets (fps, hessup, and still working), participants were also defined as an incident type 2 diabetes case the first time they appeared in the nationwide drug reimbursement register as eligible for type 2 diabetes medication (27). in the whitehall ii study, type 2 diabetes was ascertained by 2-h oral glucose tolerance test administered every 5 years (11) using world health organization criteria and complemented by self - reports of diabetes diagnosis and medication (28). in the gazel study, we only had icd codes for mortality data so new nonfatal cases were based on self - report from annual questionnaires. the date of incident diabetes was defined as the date of the first record during the follow - up in any of the previously mentioned sources (supplementary table 1). prevalent (existing) type 2 diabetes cases were defined using information from any of the following : hospital records (all studies except for gazel and whitehall ii), baseline medical assessment (whitehall ii), self - report from the baseline questionnaire (copsoq - ii, fps, gazel, hessup, ipaw, slosh, whitehall ii, wolf norrland [wolf n ], and wolf stockholm [wolf s ]), or drug reimbursement register in finland (fps, hessup, and still working). we excluded participants with a diagnosis of either type 1 or type 2 diabetes either before or at the study baseline (icd-10 codes e10e11 or icd-9 and icd-8 code 250) (supplementary table 2). in addition to age and sex, we used data on socioeconomic status (ses), working hours, bmi, leisure - time physical activity, smoking, and alcohol consumption as covariates (that is, confounders or mediators). ses was defined based on occupational title, which was register based (in copsoq - i, copsoq - ii, dwecs, fps, gazel, ipaw, puma, and still working) or self - reported (in whitehall ii, slosh, wolf n, and wolf s). in hessup, ses was based on self - reported highest educational qualification. ses was categorized into low, intermediate, high, and other, with participants who were self - employed or whose job title was missing included in the last category. working hours were divided into categories of 50 kg / m were excluded from the analysis including bmi. we grouped participants into three categories according to their level of leisure - time physical activity : sedentary (physically inactive), highly active (at least 2.5 h of moderate, or at least 1 h 15 min of vigorous, physical activity per week), or moderately active (all levels in between). information on physical activity was not available for participants in copsoq - i (18). tobacco smoking was self - reported and categorized into never, ex-, and current smoking (19). we used responses to questions about the total number of alcoholic drinks consumed per week to classify participants as nondrinkers, moderate drinkers (114 drinks per week for women and 121 drinks per week for men), high - to - intermediate drinkers (1520 drinks per week for women and 2227 drinks per week for men), and heavy drinkers (21 drinks per week for women and 28 drinks per week for men) (20). harmonized data on alcohol consumption were not available for participants in copsoq - i or slosh. for additional adjustment for biological risk markers (representing potential mediators), we included self - reported hypertension or use of antihypertensive medication (fps, hessup, slosh, ipaw, and copsoq - ii), self - reported elevated lipids (hessup), or measured systolic blood pressure, triglycerides, and hdl cholesterol (whitehall ii, wolf n, and wolf s). because shift work has been suggested to elevate the risk of type 2 diabetes (3032), we also identified respondents who worked in shifts or during the night. participants who reported daytime work only were classified as nonshift workers, and those reporting nighttime work (between 6:00 p.m. and 6:00 a.m.) or any form of shift work were classified as shift workers. in addition, data for shift or nighttime working were not available for copsoq - i, copsoq - ii, dwecs, gazel, ipaw, and puma. follow - up time was calculated from baseline assessment until the first record of type 2 diabetes, death, or end of follow - up, whichever came first. job strain was modeled as a binary exposure (job strain vs. no job strain [the reference ]) and in sensitivity analysis as a categorical variable (high strain, active, passive, and low strain [the reference ]). all analyses were adjusted for sex, age, and ses and then further adjusted for lifestyle variables (bmi category, physical activity, smoking, and alcohol consumption). the models adjusted for age, sex, ses, and lifestyle factors were also additionally adjusted for biological risk markers. to address reverse causation to minimize the possibility that shift work affected any associations, we repeated the analyses separately in participants who reported working shifts or nights and among those who did not. participants with missing data were excluded from this analysis. as in previous studies from the ipd - work consortium, we also examined risk of diabetes in the four groups created by combining data on job strain and each lifestyle risk factor (33). dichotomized lifestyle risk factors used in these analyses were current smoking (yes vs. no), heavy alcohol use (21 drinks per week for women and 28 drinks per week for men vs. other), obesity (bmi 30 vs. 0.5). further subgroup analyses showed that the association between job strain and type 2 diabetes was similar among shift workers (age-, sex-, and ses - adjusted hr 1.28 [95% ci 1.091.51 ] ; incident cases 779, n = 27,955, six studies), those not working shifts or nights (hr 1.07 [0.941.22 ] ; incident cases 1,937, n = 67,758, seven studies), and in the low - ses group (hr 1.33 [1.181.51 ] ; incident cases 1,376, n = 35,038, 13 studies). no significant association was observed in the intermediate - ses group (hr 1.03 [0.901.18 ] ; incident cases 1,515, n = 55,051, 11 studies), and the association was heterogeneous in the high - ses groups (i = 60%, p = 0.01, hr 1.37 [0.762.47 ] in the random - effects model and 1.09 [0.801.49 ] in the fixed - effect model ; incident cases 725, n = 25,220, eight studies). as expected, all lifestyle risk factors (obesity, physical inactivity, smoking, and heavy alcohol consumption) were associated with an increased diabetes risk. figure 2 shows the risk of diabetes in categories defined by combining measures of job strain with these individual lifestyle risk factors. job strain was associated with a similar excess risk of type 2 diabetes in both participants exposed and unexposed to lifestyle risk factors. associations of job strain and incident type 2 diabetes in healthy and unhealthy lifestyle subgroups. no difference in the association between job strain and incident type 2 diabetes was observed for men and women (age-, sex-, and ses - adjusted hrs 1.19 [95% ci 1.061.34 ] and 1.13 [1.001.28 ], respectively). the association was also similar among employees younger than 50 years (1.13 [0.991.28 ] ; incident cases 1,685, n = 80,798, 13 studies) and those 50 years or older (1.16 [1.041.31 ] ; incident cases 2,018, n = 44,010, 13 studies). there was very little heterogeneity in the study - specific estimates (i = 0%, all p values > 0.5). further subgroup analyses showed that the association between job strain and type 2 diabetes was similar among shift workers (age-, sex-, and ses - adjusted hr 1.28 [95% ci 1.091.51 ] ; incident cases 779, n = 27,955, six studies), those not working shifts or nights (hr 1.07 [0.941.22 ] ; incident cases 1,937, n = 67,758, seven studies), and in the low - ses group (hr 1.33 [1.181.51 ] ; incident cases 1,376, n = 35,038, 13 studies). no significant association was observed in the intermediate - ses group (hr 1.03 [0.901.18 ] ; incident cases 1,515, n = 55,051, 11 studies), and the association was heterogeneous in the high - ses groups (i = 60%, p = 0.01, hr 1.37 [0.762.47 ] in the random - effects model and 1.09 [0.801.49 ] in the fixed - effect model ; incident cases 725, n = 25,220, eight studies). in this pooled analysis of almost 125,000 european adults, job strain was associated with a 1.15-fold increased risk of incident type 2 diabetes, with no evidence of differences in the association by sex. importantly, the excess risk of type 2 diabetes associated with job strain was similar in magnitude among participants with and without unhealthy lifestyle factors : obesity, physical inactivity, smoking, and heavy alcohol use. few studies have examined the association between work - related stress and type 2 diabetes (36). this is the largest prospective study of work - related stress and type 2 diabetes to date that has used job strain as a measure of work stress. previous reports from the ipd - work consortium have shown a robust cross - sectional association between job strain and diabetes that was independent of other cardiometabolic risk factors (37). in the most recent previous meta - analysis, based on four studies with a combined sample size of 92,485 (36), the point estimate (hr 1.08 [95% ci 0.841.32 ]) was lower than in the present analysis. this summary estimate is within the confidence intervals of our study (age-, sex-, and ses - adjusted hr for job strain vs. no job strain 1.15 [1.061.25 ]). some previous studies have reported an association between job strain and diabetes, but only among women (810), whereas other studies have found no association (1517). our results, based on a substantially larger sample (n = 125,000), suggests a modest association between job strain and diabetes in both men and women. diabetes association, such as increased cortisol secretion in response to stress (1214). cortisol stimulates glucose production in the liver and antagonizes the action of insulin in peripheral tissues ; both processes have the potential to contribute to risk of hyperglycemia. in addition, job strain could increase the risk of diabetes indirectly through effects on lifestyle. for example, job strain is associated with an elevated risk of physical inactivity, and longitudinal analyses suggest that higher job strain is associated with a higher risk of obesity (1822). these indirect effects via lifestyle are likely to explain only part of the job strain diabetes association as the association was not removed after adjustment for lifestyle risk factors and was observed among those with and without a healthy lifestyle. the present pooled analysis has a number of strengths, including size (high statistical power even after risk factor stratification), prospective design (reducing the risk of reverse causation bias), and inclusion of well - characterized cohort studies (facilitating an assessment of the independent effects of stress). only the whitehall ii study administered an oral glucose tolerance test, the gold standard, to all participants who had not already been diagnosed with diabetes over the follow - up period. this study was thus able to report on both diagnosed and undiagnosed diabetes, whereas the other studies, based on health records or self - reports, missed undiagnosed type 2 diabetes cases. in whitehall ii, the age-, sex-, and ses - adjusted hr for job strain and diabetes was 1.09, which is in agreement with that in the entire consortium (1.15). furthermore, i statistics suggested that the method of outcome ascertainment was not a source of heterogeneity between the studies. second, we focused on job strain, which is the most widely studied form of work - related stress. however, there are other conceptualizations of work - related stress, such effort - reward imbalance (38), and other work - related stressors such as job insecurity (39) as well as various sources of stress outside work (7). thus, our findings on a single work - related stressor are likely to provide an underestimate of the overall impact of life stress on diabetes risk. furthermore, as job strain and lifestyle were measured only at baseline, changes in these factors might have contributed to an under- or overestimation of the associations. third, reverse causation remains a potential source of bias in studies of type 2 diabetes, which has a long subclinical phase. to reduce this bias this procedure did not attenuate the association, suggesting that reverse causation is likely to explain little, if any, of the observed association. lastly, our analyses are based on data from observational studies and, as such, preclude direct causal inference. we can not exclude the possibility that the results were affected by residual confounding caused by imprecisely measured covariates or some other unmeasured exposures. in conclusion, we show a modest but robust association between job strain and the development of type 2 diabetes irrespective of lifestyle risk factors such as obesity and physical inactivity. cluster - randomized controlled trials focused on job strain reduction, with work units or work places as the entity for randomization, are needed to determine whether stress management could be an effective means to reduce type 2 diabetes risk in working populations. given the likely sample size requirement of such a trial (as well as the fact that randomized trials frequently produce smaller effect sizes than observational studies) (40), the most cost - effective way to proceed might be to conduct an intervention with surrogate biomarkers of diabetes risk, such as fasting or postload glucose.	objectivethe status of psychosocial stress at work as a risk factor for type 2 diabetes is unclear because existing evidence is based on small studies and is subject to confounding by lifestyle factors, such as obesity and physical inactivity. this collaborative study examined whether stress at work, defined as job strain, is associated with incident type 2 diabetes independent of lifestyle factors.research design and methodswe extracted individual - level data for 124,808 diabetes - free adults from 13 european cohort studies participating in the ipd - work consortium. we measured job strain with baseline questionnaires. incident type 2 diabetes at follow - up was ascertained using national health registers, clinical screening, and self - reports. we analyzed data for each study using cox regression and pooled the study - specific estimates in fixed - effect meta-analyses.resultsthere were 3,703 cases of incident diabetes during a mean follow - up of 10.3 years. after adjustment for age, sex, and socioeconomic status (ses), the hazard ratio (hr) for job strain compared with no job strain was 1.15 (95% ci 1.061.25) with no difference between men and women (1.19 [1.061.34 ] and 1.13 [1.001.28 ], respectively). in stratified analyses, job strain was associated with an increased risk of diabetes among those with healthy and unhealthy lifestyle habits. in a multivariable model adjusted for age, sex, ses, and lifestyle habits, the hr was 1.11 (1.001.23).conclusionsfindings from a large pan - european dataset suggest that job strain is a risk factor for type 2 diabetes in men and women independent of lifestyle factors.
dynamic contrast - enhanced magnetic resonance imaging (dce - mri) is an important diagnosis tool of breast disease because of its good soft tissue resolution and time - signal intensity curve ; it has a higher sensitivity and often presents malignant breast nodules which are missed by physical examination, mammography and ultrasound (1 - 3). we present an interesting case of an asymptomatic 30-year - old female asian patient with a small suspicious breast nodule which was not detected by breast palpation, mammography, and color doppler ultrasound, but was detected by dce - mri. this nodule was surgically removed and pathologically confirmed as hemangioma, a kind of benign vascular tumor which is rarely seen in the breast. we present an asymptomatic 30-year - old female asian patient who gave birth 2 years ago. ten months ago, the patient underwent breast palpation, mammography, and ultrasound (for the first time). the results present a nodule (1.6 1.2 0.9 cm) locating on the right breast at 7 o'clock position with an oval shape, clear boundary, internal hypoechoic, and calcifications., mr scanning (ge signa hdxt 3.0 t) was performed according to the recommendation of a clinician. dce - mri did not detect enhancement of the mass (breast fibroadenoma with calcification). surprisingly, a new small nodule (0.7 0.5 0.4 cm) was found at the upper outer quadrant of the right breast. mri presented an oval mass located on the mammary gland, anterior to the superficial pectoralis fascia with a well - defined margin. 1a), while a t2-weighted image showed the mass area of high signal (fig. the region of interest was obtained from the enhancing area of the lesion to generate a time - signal intensity curve. ultrasound was then carefully performed on the patient (for the second time), showing an oval nodule with a well - defined margin and internal hypoechoic (fig. two experienced radiologists made a surface marker for the lesion based on the dce - mri and ultrasound presentation. in the operating room, the surgeon confirmed the lesion again by ultrasound (for the third time) before the surgery. the lesion was pathologically confirmed as breast capillary hemangioma, which is a benign vascular tumor and rarely seen in the breast (fig. immunohistochemical staining showed strong positive cd34 expression and negative ck expression of the lesion, which supported our diagnosis (fig. ultrasound is a very useful tool for checking breast diseases, especially in young women with mammary gland hyperplasia. while it has been reported that hemangiomas always show an oval or lobular shape with well circumscribed or microlobulated margins on sonography (4) (which were also seen in our case), these characteristics are not sufficiently specific. while hemangiomas often locate in a superficial position (subdermal or in the subcutaneous tissues) (5, 6) (which is similar to our case), this location makes it difficult to visualize the mass on regular craniocaudal and mediolateral oblique views, tangential views are necessary to prove the superficial nature of the mass. however, benign hemangiomas in the breast have also been described as displaying high vascularity. mri and dce - mri are frequently used to check for breast disease in asian women because of the common occurrence of mammary gland hyperplasia and smaller size of female breasts. it is well known that mri could detect inconspicuous lesions because of its good soft tissue resolution (7). breast hemangiomas have been reported to show homogeneous hyperintensity on t2-weighted images as in our case. in a gd - enhanced dynamic study, vorela al. reported that hemangiomas present early intensive enhancement followed by a plateau (8), which differs from our case (washout type curve, indicating malignant breast lesion), and the images and curve render it difficult to confirm the diagnosis. the difference of vascular morphology and numbers might be the main reason for the different dce - mri presentation and curve type. angiosarcoma is rare but the most malignant of all breast neoplasm, with a mean survival time of about two years. this tumor presents as an ill - defined, unencapsulated, soft, spongy mass with dilated vascular channels. hemorrhage and necrosis are common, especially in larger tumors. histologically, the endothelial cells of angiosarcoma are usually large, plump, and single layered. hemangiomas are benign vascular tumors of two common types (capillary or cavernous) that are based on the size of the vessels involved. in this case, microscope imaging showed that the hyperplastic vascular tumor has a regular morphology and clear boundary, has no interstitial infiltrates, no vascular branching structure, and the papilla structure could be found in the lumens. endothelial cell atypia, papillary tufting, mitotic figures, necrosis, or other features of atypical vascular lesion or angiosarcoma were not identified. it has been reported that breast hemangioma could be found in 11% of postmortem specimens of the female (10). if the dce - mri presents a small mass (usually 2 cm) with a clear boundary, homogeneous enhancement, superficial location, and even the mass presents a washout type curve, alerting the radiologists that hemangioma could be a possible diagnosis.	breast capillary hemangioma is a type of benign vascular tumor which is rarely seen. little is known about its presentation on dynamic contrast - enhanced magnetic resonance imaging (dce - mri). here, we describe a case of suspicious breast lesion detected by dce - mri and pathologically confirmed as capillary hemangioma. our case indicates that a small mass with a superficial location, clear boundary, and homogeneous enhancement on dce - mri indicates the possible diagnosis of hemangioma, whereby even the lesion presents a washout type curve.
in ophthalmologic practice, neodymium : yttrium - aluminum - garnet (nd : yag) laser is mainly applied for treatment of posterior capsular opacity.1 it has also been used for a variety of indications, including peripheral iridotomy,2 anterior vitreolysis,3 anterior capsule relaxing incisions,4 anterior capsulotomy in white intumescent cataracts,5 goniopuncture,6 removal of residual cortex,7 and treatment of endocapsular hematoma.8 however, to our best knowledge, the treatment of organized hyphema anterior to intraocular lens (iol) using nd : yag laser has never been reported.. however, surgical intervention can often be difficult due to the risk of rebleeding, and other treatment options should be considered. herein we present a case of successful treatment of pupil - occluding organized hyphema anterior to the iol by direct application of nd : yag laser to the hematoma. a 78-year - old asian female underwent cataract surgery in the right eye (oculus dexter [od ]). she had a history of ischemic heart disease and hypertension and, therefore, was taking aspirin. cataract surgery was performed with topical anesthesia and clear corneal incision, and was uneventful until iol implantation and removal of viscoelastics. however, she had intraoperative floppy iris syndrome and iris prolapse occurred through the main wound at the conclusion of the surgery, which led to intracameral hemorrhage. although copious irrigation of the anterior chamber combined with aspiration of the blood clot was done, bleeding was not completely controlled, and eventually, hyphema filled half of the intracameral space. postoperatively, oral prednisolone (30 mg a day for 3 days) and topical prednisolone acetate (1%, six times a day) were prescribed. one week later, her bcva was hand motion, and intraocular pressure (iop) measured with goldmann applanation tonometry (gat) was 15 mmhg. although hyphema had decreased, organized hyphema anterior to the iol had occluded the pupil (figure 1a). one week later, her bcva was hand motion, and there was no change in the organized hyphema anterior to the iol. using nd : yag laser (3.04.0 mj), the organized hemorrhage on the iol was lysed. one week later, her bcva was 20/400, and iop was 15 mmhg by gat. however, endocapsular hematoma that occluded two - thirds of the pupil was observed (figure 1b). nd : yag laser posterior capsulotomy (1.02.0 mj) was performed to disperse the blood clot from the visual axis. two weeks later, her bcva improved to 30/60, and iop was 14 mmhg by gat. there was no blood occluding the visual axis, and hyphema was completely absorbed (figure 1c). written informed consent was obtained from the patient for publication of this case report and any accompanying images. as this is a single case report and only retrospective review of medical records was needed, ethics approval was waived by the institutional review board of kangwon national university hospital. studies showed that phacoemulsification of uncomplicated cataract with iol implantation with topical anesthesia and clear corneal incision can be safe even in patients taking anticoagulants or antiplatelet drugs.9 however, in conditions that may increase the risk of bleeding, such as small pupil, floppy iris syndrome, iris neovascularization, or pseudoexfoliation or interruption of antiplatelet or anticoagulant therapy, the risk of postoperative hematoma should be considered.9 although aspirin was stopped and coagulation panels were normal in our patient, inadvertent intracameral hemorrhage occurred associated with iris prolapse. complete removal of the hemorrhage was impossible, which led to blood collection in the anterior chamber and capsular bag. although the hemorrhage was partly absorbed, remnant organized hyphema anterior to the iol occluded the pupil and led to substantial decrease in vision. however, considering that the patient had a risk for iris prolapse, additional surgical manipulation could exacerbate the hyphema. however, the organized hyphema anterior to the iol persisted for 2 weeks, and reduced her vision significantly by occluding the pupil. there was also a possibility that the organized hemorrhage anterior to the iol had formed a persistent fibrin membrane, necessitating intervention to improve the condition. eventually, the remaining organized hemorrhage anterior and posterior to the iol was removed using nd : yag laser. we chose lysis of the hematoma anterior to the iol by direct application of nd : yag laser because it can deliver strong energy to a small target without the risk of damage to the adjacent tissue. as in the present case, intraoperative hyphema can not be completely prevented, and in these circumstances, secondary operation may often be difficult due to the risk of rebleeding. surgical intervention of intracameral hemorrhage can sometimes be dangerous also in patients with increased bleeding tendency, such as hemophilia or liver diseases. in these patients, nd : yag laser can be a safe option for vision recovery. nd : yag laser was also used for the treatment of postoperative endocapsular hematoma.8 however, in these cases, the laser was used to make posterior capsular opening and to drain the blood to the vitreous cavity, as we did for the treatment of endocapsular hematoma.8 for the treatment of organized hyphema anterior to the iol, we directly applied the nd : yag laser for the lysis of the hematoma, and thus used higher energy compared to the energy level used for posterior capsulotomy. although nd : yag laser posterior capsulotomy is relatively safe, complications including retinal detachment, macular edema, optic disc edema, uveitis, glaucoma, macular hole, and endophthalmitis have been reported.10 in the present case, the energy of the laser was concentrated on the blood clot and the possibility of damage to other tissues was low. however, as relatively stronger energy was used, we believe attention should be paid to the possibility of complications. in conclusion, pupil - occluding organized hyphema anterior to the iol can occur as a complication of cataract surgery, although it may be rare. direct lysis of the hematoma using nd : yag laser can be a useful treatment option in this condition, particularly when surgical intervention is difficult due to the risk of rebleeding.	we report, to our best knowledge, the first case of treatment of pupil - occluding postoperative organized hyphema anterior to the intraocular lens (iol) using neodymium : yttrium - aluminum - garnet (nd : yag) laser. a 78-year - old asian female underwent uneventful cataract operation. she had been taking aspirin, which she discontinued 1 week before surgery. iris prolapse occurred at the end of the surgery, which led to intracameral bleeding. two weeks later, her best - corrected visual acuity was hand motion. although hyphema had decreased, pupil - occluding organized hematoma had formed anterior to the iol. the blood clot anterior to the iol was removed using nd : yag laser. one week later, although the hematoma anterior to the iol resolved, endocapsular hematoma was observed, which was dispersed with nd : yag laser posterior capsulotomy. two weeks later, her best - corrected visual acuity improved to 20/60. there was no complication associated with nd : yag laser. in conclusion, pupil - occluding organized hyphema anterior to the iol can occur as a complication of cataract surgery, in which nd : yag laser can be a useful treatment option.
dedicated research by numerous scientific groups into the causes and treatment of the human demyelinating disease multiple sclerosis (ms), the most common disabling neurological condition of european, north american, and other temperate climates, has been ongoing for many decades. ms affects relatively young individuals, with a female to male ratio of approximately 2 : 1. the disease is considered to involve central nervous system (cns) autoantigen - directed t lymphocytes acting in concert with a genetically determined susceptibility and exposure to environmental induction factors. progress has been made to advance understanding of the disease process and offer effective methods of control. however, there remains a lack of fundamental knowledge on the primary aetiology of ms and a paucity of treatments to alleviate symptoms and ultimately improve quality of life for the patient. the development and refinement of the inducible animal disease experimental autoimmune encephalomyelitis (eae) has provided a reliable model for the study of ms offering pathological and neurological features of striking similarity to the human condition. the principal characteristics in common include immunoregulatory defects, neurological disabilities, blood - brain barrier (bbb) damage with associated vasogenic oedema, inflammatory cell invasion of the cns parenchyma and, in the chronic models, demyelination and macroscopic plaque formation. however, the premise that eae is strictly a model for ms must remain, not least because of the obvious species differences and time - scale of disease appearance and progression, but also because of factors such as the divergence in identity of the causative agents and the unpredictable patterns of clinical deficits experienced by patients. animal counterparts of human disease, whether spontaneous or inducible, have inherent limitations and eae is no exception. however, the model does provide an extensively validated and useful in vivo system of immune cell - mediated demyelination complete with quantifiable neurological deficits. in particular, the model provides the opportunity to evaluate potential new therapies for ms treatment and explore novel approaches to drug design, identify new targets, and add to the growing number of drugs in clinical trials. the search for compounds with the ability to modify the onset and development of eae have invariably focused on immunomodulatory agents. however, over the last few years, a group of established compounds have emerged, with the ability to dramatically improve the course of eae but without apparent immunosuppressive activity. the compounds interact with members of the neuronal ionotropic glutamate receptor family comprising the n - methyl - d - aspartate (nmda), -amino-3-hydroxy-5-isoxazolepropionic acid (ampa), and kainate receptors (figures 1(a)1(c)). the 3 types of receptors are ligand - gated ion channels, named according to their specific agonists, which control the most rapid synaptic events in the nervous system through receptor - channel complex - mediated events. our original studies of 1994 [4, 5 ] were the first to implicate the nmda receptor in the pathogenesis of eae. over the intervening years, there has been compelling evidence, reviewed below, to confirm an important role for the nmda receptor in the disease. additional investigations have strongly indicated that ampa receptors play a part in the development of eae and, of particular interest, more recent unpublished studies have shown altered receptor expression in cns tissues from ms patients (t. smith, personal communication). the amino acid glutamate is the main agonist of the receptors and has been implicated in the pathogenesis of neuroinflammatory disease [6, 7 ]. hence, the discovery of nmda / ampa receptor involvement in both eae and ms offers a plausible association between the receptors, the amino acid, and development of both diseases. olney, in the late 1960s, was the first to recognise that the ubiquitous neurotransmitter, glutamate, when present in excess, has the potential to be excitotoxic. glutamate formation is regulated by the enzyme glutamate dehydrogenase which catalyses the reaction of -oxoglutarate with ammonia. the agonist concentration can be abnormally increased by accelerating the reversible formative reaction that is controlled by pyridine nucleotide coenzyme activity. glutamine synthetase controls the incorporation of ammonia into glutamate to form glutamine and the activity of the enzyme can be dramatically increased or decreased in the presence of excess divalent cations, including magnesium (mg). glutamate is stored in synaptic vesicles and released by calcium (ca)-dependent exocytosis. sodium - dependent, plasma membrane transporter proteins eaac1 (eaat3) and eaat4, present mainly in neurons, and glt-1 (gaat2) and glast (gaat1), expressed predominantly in glial cells, facilitate cellular uptake of glutamate and accumulation in synaptic vesicles. several studies have demonstrated glutamate involvement in the pathology of eae, and also ms, offering the clear potential for aberrant ionotropic receptor activation. in particular, the glutamate antagonist amantidine has been shown to reduce the relapse rate in individuals with ms. also, stover have reported elevated glutamate levels in the cerebrospinal fluid from ms patients. interestingly, the elevation was similar to concentrations recorded in myelopathy and, perhaps more surprisingly, greater than noted during cerebral ischaemia. however, and in contrast to the previous findings, klivenyi found no differences between cerebrospinal fluid glutamate concentrations in ms and control samples despite elevated levels in both groups. enhanced concentrations of the agonist may result from malfunctioning of activated astrocytes normally efficient at controlling excess glutamate through regulation of the metabolising enzymes glutamate dehydrogenase and glutamine synthetase, which become down - regulated during inflammatory conditions such as eae [14, 15 ]. the amount of cns glutamate may also be increased in eae by abnormal changes in neuronal and glial glutamate transporter levels which, under pathological conditions, are either inoperative or acting reversibly to raise extracellular concentrations of the agonist. glutamate leakage from the serum across the compromised bbb during eae plus infiltrating inflammatory leukocytes and activated resident microglia with the potential to synthesise and release glutamate would provide a continuous, local supply of the agonist. also, microglia are known to generate reactive oxygen and nitrogen species that impair glutamate uptake mechanisms. the constant availability of glutamate would induce upregulation of its receptors and, ultimately, the synthesis of mediators responsible for neuronal dysfunction [12, 1619 ]. indeed, a recent study in eae found that prophylactic administration of riluzole, an inhibitor of glutamate - dependent neurotransmission, reduced neurological severity, inflammation, demyelination, and axonal damage strongly suggesting a broad role for the enhanced presence of glutamate in the pathology of the disease. in a novel approach to account for the increase in cns glutamate concentrations during neuroinflammation, rose have suggested a mechanism that would operate through the actions of two enzymes, cyclooxygenase-2 (cox-2) and inducible nitric oxide synthase (inos), both of which have been located in ms lesions. cox-2-derived prostanoids, which exist at high concentrations in eae and ms cns tissues [2224 ], stimulate glutamate release from cns - derived cells [25, 26 ]. additionally, nitric oxide (no), from inos, can increase cox-2, plus reactive oxygen species (ros), to react with no to produce peroxynitrite (onoo) that inactivates the glutamate transporters [30, 31 ]. in addition, onoo directly damages myelin, oligodendrocytes, and axons, and therefore plays a predominant role in the pathogenesis of eae. the evidence is unequivocal as to the consequences of excitotoxic glutamate levels in the cns of patients with neuroinflammatory - based disease and that target tissues require protection from the sustained biochemically - mediated attack. interestingly, in eae, work by schori supports a t - cell - dependent, self - protective immune mechanism that may, at least in part, reduce the effects of enhanced glutamate levels. however, the need for greater control of the excitotoxic actions of ionotropic receptor agonists is apparent and has not diminished. indeed, over the past decade much effort has been diverted to identifying compounds that can negate glutamate - mediated neurotoxicity incurred as a consequence of conditions such as stroke and head injury. results to date have been largely negative and evidence for a neuroprotective role of glutamate antagonists in neurodegenerative diseases is lacking. similarly, despite efforts to develop compounds that act by altering the metabolism of glutamate, no such drugs have been produced. the rationale is now strong for assessing compounds designed to limit the possible damaging effects of glutamate in diseases such as ms and, in particular, to employ the animal counterpart eae as the in vivo test system of choice. the nmda receptor is most abundant in the cortex, basal ganglia, and sensory pathways of the nervous system, and has also been identified in a variety of nonneuronal and peripheral locations. in particular, the receptor has been found on the neurovasculature and mast cells derived from the cns [3742 ]. the receptor consists of several subunits, comprising the ubiquitous nr1 subunit and a variety of combinations of nr2a to nr2d and the more recently identified nr3 subunit [39, 43, 44 ]. each subunit has 4 membrane domains, an extracellular amino terminal region and an intracellular carboxy group tail. the domains 1, 3, and 4 transverse the membrane and domain 2 appears to form the reentrant loop which lines the ion channel (figure 2). the channel pore is normally blocked by mg to prevent ion flux but, on appropriate ligand stimulation, membrane depolarisation occurs and the mg blockade is removed to cause a functional opening of the receptor channel (figure 1(a)). the nmda receptor is of particular interest to pharmacologists as there are a number of ligand binding and modulatory sites that offer potential therapeutic targets for control and points of intervention (figure 1(a)). functional nmda receptor complexes are constructs of the nr1 and nr2/nr3 subunits containing the glycine and glutamate recognition sites, respectively [4547 ]. agonists, including nmda and glutamate, bind to the glutamate recognition site, whereas competitive antagonists such as selfotel may occupy a single region, distinct from the agonist site, but coupled to provide a competitive interaction. interestingly, selfotel has been effectively used in vivo to block nmda - induced bbb permeability increases. glycine and d - serine act as coagonists, through the glycine site, to prevent receptor desensitisation and are prerequisites for the generation of enhanced inward flow of current at the receptor. histamine and the polyamines (pas), including spermine and spermidine, act as receptor modulators to both potentiate and inhibit nmda - induced responses through distinct sites [4951 ]. the receptor can also be modulated by sigma site ligands at a position distinct from the channel - blocking site. a clearer understanding of sigma site function in glutamate - mediated responses is required before agents, directed at the target, can be designed to offer therapeutic efficacy. the current extent of nmda receptor modulatory sites is summarised in figure 1(a). nmda receptors have been extensively studied and show special pharmacological properties that are thought to play a role in pathophysiological mechanisms. for example, the receptor is highly permeable to ca and other cations, including sodium (na) and potassium (k), and is readily blocked by physiological concentrations of mg when the cell is normally polarised. the ca permeability of the receptor is controlled by an asparagine residue in the nr1 subunit within the channel pore loop structure of the second membrane domain. the residue also determines the voltage - dependent mg blockade of the nmda receptor channel. depolarisation of the receptor leads to loss of mg from the channel pore and an influx of ca with subsequent activation of enzyme systems we, and others, have shown to be pertinent to the inflammatory processes involved in eae, including no and pa production [55, 56 ]. indeed, bolton first showed elevated no and pa levels in cns tissues from eae - diseased rats prompting the suggestion of an important role for the nmda receptor in the pathogenesis of the disease and, by implication, in ms. the open channel can be blocked by the uncompetitive nmda receptor antagonist (+) mk-801 (dizocilpine maleate) (figures 1(a) and 3), thereby limiting the flow of ca into the cell and curbing activation of enzyme systems. our subsequent studies using (+) mk-801 confirmed a role for the nmda receptor in eae through the prevention of bbb breakdown and neurological deficits and strongly suggests the involvement of glutamate in the disease. a recent investigation by sharp has described the use of (+) mk-801 to confirm nmda receptor involvement in an in vitro model of bbb damage, and our studies with the drug have indicated the existence of the receptor on immortalised bend 3 brain endothelial cells. in addition, zhu and liu used (+) mk-801 to attenuate glutamate - induced expression of p - glycoprotein on cns - derived microvessel endothelial cells and further verify the existence of nmda receptors on neuroendothelium. the precise mechanism of action for (+) mk-801 in eae is unclear. in vitro studies by us have shown that the compound does not interfere with mitogen - driven t cell proliferation or affect the inflammatory response made by macrophages (unpublished data). however, ongoing studies examining the downstream ca - dependent events triggered as a result of nmda receptor activation may offer some insight into the actions of the drug in models of neuroinflammation. preliminary work has shown that treatment of bend 3 cells with (+) mk-801 prevents glutamate - induced release of onoo. treatment of eae - sensitised animals with (+) mk-801 also reduces the disease - associated increase in cns levels of the pa putrescine (figure 4) [56, 63 ]. pas, formed by the rate - limiting ca - dependent enzyme ornithine decarboxylase, act as cell membrane perturbators and vasodisruptors in non - immune - mediated cns diseases [64, 65 ]. pas and onoo, along with other ros, including superoxide and hydroxyl radicals, closely influence neurovascular changes that are typical during the development and progression of eae and ms. there is a requirement to clarify pa - mediated events at neurovascular sites with the onset and development of the disease. one approach has been to examine the role of the pas in eae by employing enzyme - specific drugs that interrupt the formation of putrescine, spermine, and spermidine, plus compounds that antagonise the pa site on the nmda receptor. results indicate a complex series of responses to treatment that are dependent upon the compound, dose, and frequency of administration. interestingly, the importance of the ornithine decarboxylase - pa pathway in other cns conditions, including stroke, epilepsy, alzheimer 's disease, and schizophrenia, is being realised and will undoubtedly lead to a determined effort to understand the significance of the agents in disease pathogenesis. studies by paul and bolton together with earlier experiments by wallstrom, using the relatively uncompetitive aminoadamantane nmda receptor antagonist memantine (1-amino-3, 5-dimethyl - adamantane) (figure 3), confirmed that pharmacological modulation of receptor function during eae results in disease suppression and restoration of neurovascular function. importantly, the work by paul and bolton indicates, through the use of specific dosing regimes, that nmda receptor involvement in eae is at, or just prior to, symptom onset and bbb breakdown, rather than earlier, during the induction phase of disease or later at the height of neurological deficits. furthermore, a significant effect was noted on neuroinflammatory infiltrates which appears distinct from ampa / kainite antagonist activity. memantine, unlike (+) mk-801, has been reported to differentiate between transient physiological activation and sustained pathological stimulation of the nmda receptor with actions preferentially directed towards the latter state. the apparent discriminatory profile ascribed to the pharmacology of memantine on abnormal nmda receptor activity makes the drug particularly attractive for use during the onset of clinical episodes in human cns diseases. indeed, memantine has been reported to provide symptomatic relief to ms patients. one current theory suggests that the compound, like mg, occupies the receptor channel and rapidly exits the pore under strong, physiological synaptic depolarisation and in the presence of glutamate fluxes of millimolar concentrations. in contrast, and under pathological conditions where sustained micromolar concentrations of glutamate preside, memantine, unlike mg, will maintain channel block during prolonged depolarisation. consequently, total blockade of the receptor, by compounds including (+) mk-801, leads to numerous side effects. specific prevention of the pathological activation of nmda receptors with drugs such as memantine reduces unwanted activity and thereby improves clinical tolerance, offering a useful feature in the treatment of neurodegenerative diseases including ms. another approach towards improving specificity and reducing the unwanted side effects of drug therapy might be to target particular modulatory sites on the nmda receptor. in addition, an alternative to blocking nmda receptor action completely would be to suppress an exaggerated receptor response. therefore, targeting inhibitory modulatory sites, such as the pa or neurosteroid binding positions or the poorly defined sigma site (figure 1(a)), offers the potential to down - regulate rather than completely inhibit nmda receptor - mediated events. alternatively, it may be that the subunit - specific modulatory sites mediate features of neuroinflammatory pathology and thereby become primary targets through which to achieve disease control. indeed, the nr2b subunit has been recognised as a particular therapeutic target for several neurological conditions and initial studies by wheeler have shown an increased expression of the nr2a and nr2b subunits in cns tissues from eae - diseased rats [72, 73 ]. postsynaptic ampa receptors are considered to mediate rapid glutamergic neurotransmission with low ca permeability. the receptor consists of four subunits, glur1 to glur4, which are widely distributed throughout the cns [74, 75 ] and each of which can be expressed in two variants originally termed flip and flop (figure 1(b)). ampa receptors are invariably colocalised with nmda receptors indicating a close functional relationship between the 2 ligand - gated cation channel - bearing receptors. indeed, ampa activation causes cellular depolarisation and nmda channel opening with ca influx. pharmacological studies have provided strong evidence for ampa receptor involvement in several cns conditions including stroke, traumatic brain injury, and parkinson 's disease. kainate receptors are closely related to ampa receptors and are involved in both pre- and postsynaptic neurotransmission. the receptor class is comprised of 5 subunits falling into 2 families, glur5 to glur7 and ka1 plus ka2 (figure 1(c)). each subunit family shares 70% sequence homology with its members, but only 40% with nonfamily subunits. weaker identities are shown with ampa (3035%) and nmda (1020%) receptor subunits, although some studies have suggested that glur5 is part of the ampa family [7577 ]. the ampa and kainate positioning of subunits in their respective receptor complex is similar to the arrangement present in the nmda receptor. the amino terminal portion of each subunit is extracellular ; there are 4 hydrophobic sections, 3 of which are membrane spanning, plus a reentrant membrane loop that contributes to the pore lining. the cytoplasmic carboxy terminus, in common with the nmda receptor, contains sites for phosphorylation, with a minimum of 12 in the ampa subunits and a suggested involvement in the regulation of channel function [78, 79 ]. fewer modulatory sites have been identified for ampa and kainate receptors compared to the nmda receptor (figures 1(b) and 1(c)). however, information on the endogenous mechanisms for regulating non - nmda ionotropic receptor function is increasing and a similar capacity for pharmacological modulation of subtype activity can be anticipated. there is scant information on the subtype selectivity of ampa receptor antagonists (figure 3) and the standard competitive drugs nbqx (2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]-quinoxaline-2,3-dione) and cnqx (6-cyano-7-nitroquinoxaline-2,3-dione) are not selective between non - nmda ionotropic receptors. nbqx and cnqx have been investigated, with some success, in models of global ischaemia, cns trauma, and parkinson 's disease [8183 ] although drug effects mediated through kainate receptor involvement are suspected and therefore can not be excluded [8486 ]. one cardinal feature of ms and the more chronic models of eae is the demyelination of central nerve fibres. restoration of normal nerve function in ms is dependent, at least in part, upon recruitment of myelin - forming oligodendrocytes to lesioned areas. limited remyelination is possible in acute lesions but virtually nonexistent in chronic states due to lack of oligodendrocyte viability and recruitment to damaged areas [87, 88 ]. the oligodendrocyte has been reported to express, exclusively, ampa and kainate receptors, thereby making the cell a target for attack by excitotoxic glutamate in eae [8991 ]. however, investigations by wosik indicate a lack of ampa receptors on human oligodendrocytes and a resistance to agonist - mediated toxicity. furthermore, the work suggests that ampa expression is limited to astrocytes. despite conjecture over the cellular expression of receptor types in brain tissue, the administration of kainate to the optic nerve causes degenerative toxic lesions, nerve damage in association with inflammation, and demyelination, all of which are strongly suggestive of an ms - related pathology. interestingly, administration of cnqx prevents kainate - induced lesions whereas the ampa receptor antagonist gyki 53655 (1-[4-aminophenyl]-3-methylcarbamyl-7, 8-methylenedioxy-3,4-dihydro-5h-2,3-benzodiazepine) had no significant effect indicating a kainate - specific action and implicating receptor involvement in early ms pathology. recent investigations in acute and chronic - relapsing eae have demonstrated the effectiveness of nbqx together with mpqx ([1,2,3,4-tetrahydro-7-morpholinyl 1,2,3-dioxo-6-(trifluoromethyl)quinoxa lin-1-yl]methylphosphonate) and the noncompetitive antagonists gyki 52466 (1-(4-amino - phenyl) 4-methyl-7,8-methylene dioxy-5h-2,3-benzodiaze - pine) and gyki 53773 ((-)1-(4-aminophenyl) 4-methyl-7,8-methylene - dioxy 4,5-dihydro 3-methylcarbamoyl 2,3-benzodiazepine) (figure 3) in reducing the neurological symptoms of the disease [19, 94, 95 ]. interestingly, earlier related work demonstrated that nbqx had anti - oedematous effects at neurovascular sites via a proposed action on glial cells. eae studies using the competitive antagonists to modify the course of disease can not exclude drug effects on kainate receptor - mediated events. in contrast, the noncompetitive antagonists do differentiate between the two receptors and therefore indicate a specific ampa involvement in disease development. more recently, a series of ampa receptor antagonists, with structures based on 2,3-benzodiazepine, have proved effective in reducing the symptoms and morphological changes associated with eae [97, 98 ]. the studies with nbqx have highlighted the ability of competitive receptor antagonists to reduce eae - mediated neuronal death and oligodendrocyte loss despite the uncertainty of ampa or kainate involvement [94, 95 ]. however, the extent of oligodendrocyte depletion may be dependent on additional endogenous factors. for example, testosterone has been shown to amplify both ampa- and kainate - induced toxicity to oligodendrocytes in vitro, suggesting the existence of a steroidal modulatory site on non - nmda receptors. the studies described by pitt and groom also considered the possibility that the competitive and noncompetitive antagonists may operate in eae through an immunosuppressive action. results showed that competitive antagonists, such as nbqx, did not affect t - cell proliferation rates or reduce perivascular inflammatory cuffs. however, noncompetitive antagonists did suppress mitogen - induced t cell proliferation, thus offering an alternative explanation for the compounds abilities to modify eae and indicating ampa receptor involvement in immune - mediated inflammation. glutamate excitotoxicity, together with neuroinflammatory factors in both eae and ms, may be important codeterminants in oligodendrocyte death. for example, inflammatory mediators, such as interleukin 1 and tumour necrosis factor- (tnf-) can promote in vitro oligodendrocyte apoptosis and changes in the glutamate buffering system of astrocytes. moreover, the cytokine - induced effects can be blocked by nbqx and cnqx [100, 101 ]. interestingly, research into the regulation of gene expression during eae has identified a reduction in the important plasma membrane caatpase2, necessary for cation homeostasis and expressed exclusively in the grey matter, which occurs coincidently with the development of neurological signs. the studies also found that application of kainate to spinal cord slice cultures significantly lowered the mrna levels of caatpase. collectively, the results implicate glutamate, particularly via kainate receptors, in the suppression of neuronal plasma membrane caatpase 2 and abnormal ca levels. perhaps of greater significance is the observation that nbqx can suppress alterations in glutamate transporter expression during eae. the study found protein and mrna levels of eaac1 to be dramatically increased, while transporters glt-1 and glast were down - regulated together with a concomitant reduction in the incidence of disease. nbqx, administered semiprophylactically from day 7 postinoculation, suppressed the changes in the expression of transporters suggesting the activation of non - nmda receptors. undoubtedly, the studies indicate an important role for the ampa receptor in eae and, possibly, in ms. however, the investigations can not exclude the possible contribution of kainate receptors in the development of disease pathology. the prospect of ampa / kainate receptor involvement in experimental and human neuroinflammatory conditions offers new targets to focus treatments for the demyelinating diseases with an emphasis on the preservation of oligodendrocyte function. postsynaptic ampa receptors are considered to mediate rapid glutamergic neurotransmission with low ca permeability. the receptor consists of four subunits, glur1 to glur4, which are widely distributed throughout the cns [74, 75 ] and each of which can be expressed in two variants originally termed flip and flop (figure 1(b)). ampa receptors are invariably colocalised with nmda receptors indicating a close functional relationship between the 2 ligand - gated cation channel - bearing receptors. indeed, ampa activation causes cellular depolarisation and nmda channel opening with ca influx. pharmacological studies have provided strong evidence for ampa receptor involvement in several cns conditions including stroke, traumatic brain injury, and parkinson 's disease. kainate receptors are closely related to ampa receptors and are involved in both pre- and postsynaptic neurotransmission. the receptor class is comprised of 5 subunits falling into 2 families, glur5 to glur7 and ka1 plus ka2 (figure 1(c)). each subunit family shares 70% sequence homology with its members, but only 40% with nonfamily subunits. weaker identities are shown with ampa (3035%) and nmda (1020%) receptor subunits, although some studies have suggested that glur5 is part of the ampa family [7577 ]. the ampa and kainate positioning of subunits in their respective receptor complex is similar to the arrangement present in the nmda receptor. the amino terminal portion of each subunit is extracellular ; there are 4 hydrophobic sections, 3 of which are membrane spanning, plus a reentrant membrane loop that contributes to the pore lining. the cytoplasmic carboxy terminus, in common with the nmda receptor, contains sites for phosphorylation, with a minimum of 12 in the ampa subunits and a suggested involvement in the regulation of channel function [78, 79 ]. fewer modulatory sites have been identified for ampa and kainate receptors compared to the nmda receptor (figures 1(b) and 1(c)). however, information on the endogenous mechanisms for regulating non - nmda ionotropic receptor function is increasing and a similar capacity for pharmacological modulation of subtype activity can be anticipated. there is scant information on the subtype selectivity of ampa receptor antagonists (figure 3) and the standard competitive drugs nbqx (2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]-quinoxaline-2,3-dione) and cnqx (6-cyano-7-nitroquinoxaline-2,3-dione) are not selective between non - nmda ionotropic receptors. nbqx and cnqx have been investigated, with some success, in models of global ischaemia, cns trauma, and parkinson 's disease [8183 ] although drug effects mediated through kainate receptor involvement are suspected and therefore can not be excluded [8486 ]. one cardinal feature of ms and the more chronic models of eae is the demyelination of central nerve fibres. restoration of normal nerve function in ms is dependent, at least in part, upon recruitment of myelin - forming oligodendrocytes to lesioned areas. limited remyelination is possible in acute lesions but virtually nonexistent in chronic states due to lack of oligodendrocyte viability and recruitment to damaged areas [87, 88 ]. the oligodendrocyte has been reported to express, exclusively, ampa and kainate receptors, thereby making the cell a target for attack by excitotoxic glutamate in eae [8991 ]. however, investigations by wosik indicate a lack of ampa receptors on human oligodendrocytes and a resistance to agonist - mediated toxicity. furthermore, the work suggests that ampa expression is limited to astrocytes. despite conjecture over the cellular expression of receptor types in brain tissue, the administration of kainate to the optic nerve causes degenerative toxic lesions, nerve damage in association with inflammation, and demyelination, all of which are strongly suggestive of an ms - related pathology. interestingly, administration of cnqx prevents kainate - induced lesions whereas the ampa receptor antagonist gyki 53655 (1-[4-aminophenyl]-3-methylcarbamyl-7, 8-methylenedioxy-3,4-dihydro-5h-2,3-benzodiazepine) had no significant effect indicating a kainate - specific action and implicating receptor involvement in early ms pathology. recent investigations in acute and chronic - relapsing eae have demonstrated the effectiveness of nbqx together with mpqx ([1,2,3,4-tetrahydro-7-morpholinyl 1,2,3-dioxo-6-(trifluoromethyl)quinoxa lin-1-yl]methylphosphonate) and the noncompetitive antagonists gyki 52466 (1-(4-amino - phenyl) 4-methyl-7,8-methylene dioxy-5h-2,3-benzodiaze - pine) and gyki 53773 ((-)1-(4-aminophenyl) 4-methyl-7,8-methylene - dioxy 4,5-dihydro 3-methylcarbamoyl 2,3-benzodiazepine) (figure 3) in reducing the neurological symptoms of the disease [19, 94, 95 ]. interestingly, earlier related work demonstrated that nbqx had anti - oedematous effects at neurovascular sites via a proposed action on glial cells. eae studies using the competitive antagonists to modify the course of disease can not exclude drug effects on kainate receptor - mediated events. in contrast, the noncompetitive antagonists do differentiate between the two receptors and therefore indicate a specific ampa involvement in disease development. more recently, a series of ampa receptor antagonists, with structures based on 2,3-benzodiazepine, have proved effective in reducing the symptoms and morphological changes associated with eae [97, 98 ]. the studies with nbqx have highlighted the ability of competitive receptor antagonists to reduce eae - mediated neuronal death and oligodendrocyte loss despite the uncertainty of ampa or kainate involvement [94, 95 ]. however, the extent of oligodendrocyte depletion may be dependent on additional endogenous factors. for example, testosterone has been shown to amplify both ampa- and kainate - induced toxicity to oligodendrocytes in vitro, suggesting the existence of a steroidal modulatory site on non - nmda receptors. the studies described by pitt and groom also considered the possibility that the competitive and noncompetitive antagonists may operate in eae through an immunosuppressive action. results showed that competitive antagonists, such as nbqx, did not affect t - cell proliferation rates or reduce perivascular inflammatory cuffs. however, noncompetitive antagonists did suppress mitogen - induced t cell proliferation, thus offering an alternative explanation for the compounds abilities to modify eae and indicating ampa receptor involvement in immune - mediated inflammation. glutamate excitotoxicity, together with neuroinflammatory factors in both eae and ms, may be important codeterminants in oligodendrocyte death. for example, inflammatory mediators, such as interleukin 1 and tumour necrosis factor- (tnf-) can promote in vitro oligodendrocyte apoptosis and changes in the glutamate buffering system of astrocytes. moreover, the cytokine - induced effects can be blocked by nbqx and cnqx [100, 101 ]. interestingly, research into the regulation of gene expression during eae has identified a reduction in the important plasma membrane caatpase2, necessary for cation homeostasis and expressed exclusively in the grey matter, which occurs coincidently with the development of neurological signs. the studies also found that application of kainate to spinal cord slice cultures significantly lowered the mrna levels of caatpase. collectively, the results implicate glutamate, particularly via kainate receptors, in the suppression of neuronal plasma membrane caatpase 2 and abnormal ca levels. perhaps of greater significance is the observation that nbqx can suppress alterations in glutamate transporter expression during eae. the study found protein and mrna levels of eaac1 to be dramatically increased, while transporters glt-1 and glast were down - regulated together with a concomitant reduction in the incidence of disease. nbqx, administered semiprophylactically from day 7 postinoculation, suppressed the changes in the expression of transporters suggesting the activation of non - nmda receptors. undoubtedly, the studies indicate an important role for the ampa receptor in eae and, possibly, in ms. however, the investigations can not exclude the possible contribution of kainate receptors in the development of disease pathology. the prospect of ampa / kainate receptor involvement in experimental and human neuroinflammatory conditions offers new targets to focus treatments for the demyelinating diseases with an emphasis on the preservation of oligodendrocyte function. we have reviewed the evidence for ionotropic glutamate receptor involvement in eae and speculated on a role for the receptors in ms. the finding of both nmda and non - nmda receptor involvement in the pathology of eae is substantiated. therefore, it is our belief that therapeutic targeting of both receptor groups in models of eae represents a viable proposition for the development of new treatments for ms. the observation that a variety of nmda, ampa, and kainate receptor antagonists are beneficial in eae corroborates the considerable involvement of glutamate in the pathology of the disease. aberrant glutamate transporter mechanisms in resident cells of the cns together with altered ionotropic receptor or subunit receptor expression during eae may collectively contribute to excess glutamate levels in target tissues and the gross disturbance to normal homeostasis and nerve function. figure 5 summarises the involvement of glutamate in eae and, by inference ms, highlights the ways through which excitotoxic levels of the amino acid could be achieved. direct discharge from resident and infiltrating cells or indirect release resulting from the actions of inflammatory mediators would serve to raise cns glutamate concentrations. the consequences of damage to oligodendrocytes, neurons, and the bbb, plus inflammatory cytokine release from microglia, contribute considerably to the pathology of eae and could account, at least in part, for some of the major central disturbances observed in ms. the control of glutamate release and metabolism may offer a viable therapeutic approach to limiting the subsequent damage associated with excitotoxicity. suppression of agonist - activated ionotropic receptor function has proved effective in controlling eae, but efficacy in ms remains largely uninvestigated. regulation of abnormal receptor function rather than total blockade of activity may effectively reduce the results of enhanced cns glutamate levels and allow homeostatic mechanisms to operate thereby reducing unwanted side effects. a clear delineation between the receptor type targeted and the ensuing benefits to limit the disease process appears to exist. axonal sparing and oligodendrocyte protection arises from the use of non - nmda receptor competitive antagonists whereas the restriction of bbb dysfunction and reduction of inflammation can be ascribed to drug effects on the nmda receptor. however, an exclusive action for the compounds at their respective target sites can not be guaranteed. therefore, coadministration of ionotropic receptor antagonists, with different specificities, offers the real prospect of inhibiting several fundamental parameters of experimental and human demyelinating disease. indeed, a recent study by kanwar has indirectly addressed our suggestion by treating eae with nbqx in conjunction with a monoclonal antibody directed against mucosal addressin cell adhesion molecule-1 and the n - terminal tripeptide of insulin - like growth factor. unremitting disease was ameliorated and oligodendrocyte survival and remyelination were increased. furthermore, cns inflammation, apoptosis, and axonal damage were reduced. finally, there is a requirement for an increased selectivity of antagonists towards specific receptor subtypes, either through targeting a specific subunit or by targeting a modulatory site, if the true therapeutic potential of ionotropic receptor inhibition is to be realised. a concerted effort to search for drugs with possible efficacy in ms that operate at nonimmunological sites or do not have exclusive, immunosuppressive properties however, compounds designed to antagonise the agonist actions on nmda and ampa / kainate receptors administered either alone or in combination with other therapies may offer the real prospect of treatment for patients with ms and related disorders of the cns. modulation of ionotropic glutamate receptor function. (a) nmda receptor, (b) ampa receptor, and (c) kainate receptor. the main endogenous modulatory sites for the glutamate ionotropic receptors are shown and the sites of key exogenous pharmacological agents are in italics. (+) stimulatory / potentiating action, () inhibitory action, (ss) subunit - specific action. additional modifying agents (not shown), where the action has not been specified through a binding site on the receptor, are (a) no, ethanol, histamine (via polyamine site) ; (b) arachidonic acid (), no ; (c) ethanol (), arachidonic acid (). the diagram is intended as a summary overview and provides an indication of modulation at these receptors. the discovery of new modulatory agents is ongoing, particularly for the ampa and kainate receptors, where significantly less is known compared to the nmda receptor. abbreviations : h, proton ; no, nitric oxide ; p, phosphorylation site ; pcp, phencyclidine. (a) each subunit comprises 4 hydrophobic regions of which 1, 3, and 4 are transmembrane domains, while region 2 forms a reentrant loop at the intracellular surface. (b) receptor subunit organization : the ion channels are formed from four subunits, which orientate allowing the second membrane domain to form the ion channel pore. putrescine levels detected in the cns of normal and acute eae rats 13 days postinoculation (pi), with and without mk801 treatment. mk801 was administered intraperitoneally for 6 days from day 7 pi at a concentration of 0.3 mg / kg body weight in sterile phosphate buffered saline. putrescine levels at day 13 pi are significantly increased in all tissues compared to normal (# p <.001 ; student t test). mk801 significantly reduced elevated putrescine at day 13 pi in all cns areas (p < 0.01, p <.001 ; student t test). schematic diagram summarising the known and proposed role of glutamate and associated relevant mediators in the development of neuroinflammatory and neurodegenerative pathology during eae. abbreviations : eae, experimental autoimmune encephalomyelitis ; no, nitric oxide ; onoo, peroxynitrite ; ros, reactive oxygen species ; tnf-, tumour necrosis factor alpha.	multiple sclerosis (ms) is a chronic demyelinating disease of the human central nervous system (cns). the condition predominantly affects young adults and is characterised by immunological and inflammatory changes in the periphery and cns that contribute to neurovascular disruption, haemopoietic cell invasion of target tissues, and demyelination of nerve fibres which culminate in neurological deficits that relapse and remit or are progressive. the main features of ms can be reproduced in the inducible animal counterpart, experimental autoimmune encephalomyelitis (eae). the search for new ms treatments invariably employs eae to determine drug activity and provide a rationale for exploring clinical efficacy. the preclinical development of compounds for ms has generally followed a conventional, immunotherapeutic route. however, over the past decade, a group of compounds that suppress eae but have no apparent immunomodulatory activity have emerged. these drugs interact with the n - methyl - d - aspartate (nmda) and -amino-3-hydroxy-5-isoxazolepropionic acid (ampa)/kainate family of glutamate receptors reported to control neurovascular permeability, inflammatory mediator synthesis, and resident glial cell functions including cns myelination. the review considers the importance of the glutamate receptors in eae and ms pathogenesis. the use of receptor antagonists to control eae is also discussed together with the possibility of therapeutic application in demyelinating disease.
effective risk stratification is important for management of patients with acute myocardial infarction (ami) (1, 2). complete blood cell (cbc) is a simple, easily and widely performed laboratory test. among several cbc markers, the predictive value of white blood cell (wbc) and hemoglobin (hb) for short- and long - term survival in patients with ami has been relatively well established (3, 4, 5, 6, 7, 8), although some studies have refuted this association (9, 10, 11). although thromboembolic events are among the major causes of mortality in patients with ami (12, 13), limited evaluation of platelet distribution width (pdw), a progressive platelet activation marker, has been conducted in patients with coronary artery disease (cad), with conflicting results (14, 15, 16, 17). simple combination of these cbc markers, all connected in some way with ami prognosis, has never been tested in risk assessment for ami. we attempted to determine whether the combined use of wbc, hb, and pdw had prognostic value in patients with ami. this observational study included 1,365 consecutive patients with ami who were enrolled in the korea acute myocardial infarction registry (kamir) from the authors ' single center between november 2005 and february 2009. kamir is a korean, prospective, open, observational, multicenter on - line registry of ami supported by the korean society of cardiology since november 2005. pdw levels were collected retrospectively, because they had not been entered into the kamir database. ami was diagnosed by characteristic clinical presentation, serial changes on ecg suggesting infarction, and an increase in cardiac enzymes (19). st - segment elevation myocardial infarction (stemi) was defined by new st elevation in 2 contiguous leads, measuring > 0.2 mv in leads v1 - 3, or 0.1 mv in all other leads. peripheral blood samples for laboratory tests, except for lipid profile, were collected at admission. peripheral venous blood specimens were sampled in vacutainer tubes containing k - ethylenediaminetetraacetic acid and applied immediately to an automated hematologic analyzer, k-4500 (toa medical electronics, kobe, japan) for wbc count, hb concentration, and pdw. the estimated glomerular filtration rate (egfr) was calculated according to the cockcroft - gault formula. in addition to laboratory findings, baseline demographic characteristics, initial vital signs, and cardiovascular risk factors were evaluated. hypertension was defined as a previous medical diagnosis of hypertension, use of antihypertensive medications or blood pressure140/90 mmhg. history of hyperlipidemia was defined as the diagnosis previously made by a physician or treatment with lipid - lowering medications. we assessed in - hospital and 12-month mortalities. at 12 months after the index ami, follow - up data were obtained by review of the medical records and/or telephone interview with the patient or family members. n - terminal pro - b - type natriuretic peptide (nt - probnp) was log - transformed to reduce the effect of skewed distribution of the data. receiver - operating characteristics (roc) curve analysis was used for determination of the cut - off values for prediction of in - hospital mortality. comparisons were made using the student 's t - test for continuous variables and the chi - square test for categorical variables. multivariate logistic regression analysis and cox proportional hazard model were used for determination of independent parameters for in - hospital and 12-month mortalities, respectively. sequential models were developed in order to examine the incremental prognostic value of the parameters. incremental factors added to the model at each step were considered significant when the difference in the log - likelihood associated with each model corresponded to p<0.05. spss version 15.0 for windows (spss inc, chicago, il, usa) was used in performance of statistical analysis. kamir was approved by the institutional review board of kyungpook national university hospital (no. kamir was approved by the institutional review board of kyungpook national university hospital (no. of the consecutive 1,365 patients, 15 patients were on hemodialysis due to end stage renal disease and 18 patients who had underlying malignancy were excluded, leaving 1,332 patients in the final analysis. the mean age of patients was 6412 yr and 901 (67.6%) were men. mean levels of wbc, hb, and pdw were 10.94.110/l, 13.51.9 g / dl, and 52.27.4%, respectively (fig. wbc count showed positive correlation with the killip class (r=0.148), levels of creatinine (r=0.081), peak cardiac troponin i (ctni) (r=0.189), and hb (r=0.174), but showed negative correlation with age (r=-0.130) and left ventricular ejection fraction (lvef) (r=-0.091) (table 2). hb level showed negative correlation with age (r=-0.442), killip class (r=-0.242), levels of creatinine (r=-0.289), and log nt - probnp (r=-0.427), but showed positive correlation with lvef (r=0.137) and wbc count (r=0.174). pdw showed weak positive correlation with killip class (r=0.091) and levels of creatinine (r=0.075) and log nt - probnp (r=0.096). during the index admission, there were 59 (4.4%) in - hospital deaths. in the univariate analysis, age, heart rate, killip class3, levels of wbc, pdw, blood urea nitrogen, peak ctni, uric acid, and nt - probnp were significantly higher ; and systolic blood pressure (sbp), previous history of hyperlipidemia, levels of hb, sodium, and egfr were significantly lower in patients with in - hospital death. echocardiographic data were not available in 95 patients, including 34 (58%) who died before undergoing echocardiographic examination. percutaneous coronary intervention (pci) was performed less frequently at index hospitalization among patients with in - hospital death and left anterior descending artery location of infarct - related artery was significantly higher in patients with in - hospital death. however, regarding gender, body mass index, current smoking status, pre - hospital medication, history of hypertension, diabetes mellitus, and ischemic heart disease, levels of creatine kinase - mb, cholesterol, and high - sensitivity c - reactive protein, no significant differences were observed between the groups. wbc count (12.75.510/l vs 10.84.010/l, p=0.013) and pdw (54.27.6% vs 52.27.4%, p=0.037) were significantly higher in the group with in - hospital death than in the group without. however, hb level was significantly lower in patients with in - hospital death (12.42.2 g / dl vs 13.51.9 g / dl, p<0.001). area under the roc curve for prediction of in - hospital death was 0.602 (95% ci, 0.517 - 0.687) for wbc count, 0.669 (95% ci, 0.595 - 0.742) for hb, and 0.581 (95% ci, 0.507 - 0.655) for pdw. the best cut - off levels of wbc, hb, and pdw by roc curve analysis were 14.510/l, 12.7 g / dl, and 51.2%, respectively. patients with high killip class and high serum levels of creatinine and nt - probnp had significantly lower hemoglobin level and higher wbc and pdw (table 3). patients were categorized into four cbc groups ; group 0 (n=346, 26.0%), 1 (n=622, 46.7%), 2 (n=324, 24.3%), and 3 (n=40, 3.0%) according to the sum of values defined by the cut - off levels of wbc (1, 14.510/l ; 0, < 14.510/l), hb (1, < 12.7 g / dl ; 0, 12.7 g / dl), and pdw (1, 51.2% ; 0, < 51.2%). cbc group showed positive correlation with age (r=0.189, p<0.001), killip class (r=0.243, p<0.001), levels of creatinine (r=0.238, p<0.001), and log nt - probnp (r=0.278, p<0.001), but showed negative correlation with lvef (r=-0.124, p<0.001). significant differences in in - hospital death were observed among cbc group 0 (1.2%), 1 (2.7%), 2 (9.0%), and 3 (22.5%) (p<0.001) (fig. logistic regression analysis, after adjusting for multiple clinical prognostic factors, wbc14.510/l (odds ratio [or ], 2.077 ; 95% confidence interval [ci ], 0.674 - 6.398, p=0.203), hb<12.7 g / dl (or, 1.285 ; 95% ci, 0.374 - 4.415, p=0.691), and pdw51.2% (or, 1.697 ; 95% ci, 0.495 - 5.819, p=0.401) were not independent predictors for in - hospital death when they were entered separately into the model (table 4). however, when the sum of the three cbc markers was entered into the model, cbc group2 (or, 3.604 ; 95% ci, 1.040 - 12.484, p=0.043) was an independent predictor for in - hospital death in addition to sbp (or, 0.962 ; 95% ci, 0.943 - 0.981, p<0.001), killip class3 (or, 3.432 ; 95% ci, 1.053 - 11.181, p=0.041), left anterior descending artery infarction (or, 4.194 ; 95% ci, 1.046 - 16.812, p=0.043), log nt - probnp (or, 3.853 ; 95% ci, 1.461 - 10.162, p=0.006), and peak ctni (or, 1.004 ; 95% ci, 1.001 - 1.006, p=0.012). in addition, cbc group2 had incremental prognostic value (chi - square=4.3, p=0.038) (fig. 52 patients were lost, leaving 1280 patients in the analysis for 12-month mortality, and there were 106 (8.3%) all cause deaths. kaplan - meier curves showed a significant gradual increase in the risk of 12-month mortality according to the sum of the cbc markers (3.0%, 5.4%, 14.7%, and 46.2% of groups 0, 1, 2, and 3, p<0.001) (fig. when patients with in - hospital death were excluded, 12-month mortality was still significantly different among the cbc groups (1.8%, 2.6%, 6.0%, and 30.0% of group 0, 1, 2, and 3, p<0.001) (fig. 4b). in the cox - proportional hazard model, cbc group2 (hazard ratio, 2.067 ; 95% ci, 1.017 - 4.204, p=0.045) was an independent predictor for 12-month mortality (table 5). in the current study, the combination of wbc, hb, and pdw was useful in prediction of in - hospital death in patients with ami, although they did not have independent value, individually. in addition, it had an important incremental prognostic value to the combination of conventional risk factors, peak ctni and nt - probnp, which are strong prognostic markers in ami, both stemi and non - stemi (1, 20). the prognostic impact of the combined use of cbc markers remained significant over 12 months after index admission. cbc, a cheap and simple hematologic marker, has been used for risk stratification of patients with ami (3, 4, 5, 6, 7, 8). among several cbc markers, the predictive values of wbc and hemoglobin in relation to short- and long - term survival in patients with ami has been relatively well established (3, 4, 5, 6, 7, 8). inflammation has been shown to be an important risk factor for development of cardiovascular events and several studies have also reported an association of elevated wbc count with increased risk of short- and long - term mortality in patients with ami (3, 4, 5, 6, 7). although the mechanism responsible for these associations is unknown, several hypotheses have been postulated, including a leukocyte - mediated diminished microcirculatory perfusion (21) and greater thrombus formation at the site of the atherosclerotic plaque (22), leukocyte - mediated no reflow (23), indirect cardiotoxicity mediated through proinflammatory cytokines (24), and negative inotropic effects on the myocardium via the nitric oxide synthesis pathway (25). anemia, also a common comorbidity in patients with ami, is associated with increased mortality (6, 7, 8). in a recent study with stemi patients, lower hemoglobin level 12.5 g / dl, similar to the cut - off level of hemoglobin (12.65 g / dl) in our study, was an independent predictor for in - hospital mortality (8). the relationship between low hemoglobin and increased mortality could basically be explained by lower oxygen delivery to myocardial tissue (26, 27). however, negative studies for the prognostic value of cbc markers in ami patients have also been reported (9, 10, 11). in stemi patients who underwent primary pci, wbc count did not show statistical association with an increased incidence of one - month and/or 12-month death (9, 10). hemoglobin was not an independent predictor for 12-month death in patients with stemi who underwent pci (11). in our previous study with ami patients, wbc and hemoglobin were not associated with six - month major adverse cardiac events (28). in the current study, after adjusting for multiple confounding variables, an elevated wbc count and lower hemoglobin were also not independent predictors for in - hospital and 12-month death. patients with high wbc count and/or low hemoglobin had concomitantly low lvef, high killip class, high creatinine, and high nt - probnp. unlike wbc and hemoglobin, limited evaluation of pdw has been conducted in patients with cad, with conflicting results (14, 15, 16, 17). in a small study reported by ihara. (16), lower pdw was observed in patients with angiographic coronary artery stenosis. (17), significantly higher pdw was observed in patients with ami and unstable angina, compared to those with stable cad and the control group. thromboembolic events are a major cause of mortality in patients with ami (12, 13). reliable markers of platelet activation, such as thrombin - antithrombin complex, -thromboglobulin, and soluble platelet p - selectin have been investigated. however activation of platelets causes morphologic changes, including both spherical transformation and pseudopodia formation (29). therefore, pdw could be an important, simple, effortless, and cost effective tool that can be used more extensively for prediction of impending acute events. in the current study, pdw of patients with in - hospital death was significantly higher than that of those without in - hospital death. the combination of wbc and hb was studied with regard to the combined use of cbc markers for risk stratification (6, 7). in the anin myocardial infarction registry, a u - shaped relationship of hb levels and mortality was observed for patients with higher leukocytosis, whereas, in patients with lower wbc count, higher mortality was related solely to the lowest hb quintile (6). another study from the korean acute myocardial infarction registry reported that patients with anemia and high neutrophil / lymphocyte (n / l) had higher mortality at six months after stemi, compared to patients with no anemia and low however, to the best of our knowledge, a triple combination of cbc markers, all connected in some way with ami prognosis, had never been used in risk assessment for ami. in the current study, combined use of wbc, hemoglobin, and pdw, which are readily available cbc data in the early in - hospital period, showed good correlation with age, killip class, nt - probnp, and lvef which are well known as prognostic markers in patients with ami. furthermore, a triple combination of cbc markers was useful for prediction of in - hospital and 12-month mortality in patients with ami. first a single cut - off level for hemoglobin (12.7 g / dl) was used for prediction of in - hospital death. in general, however, because best cut - off level of hemoglobin by roc curve analysis did not differ between genders, we used a single cut - off value for hemoglobin. however, gurm. reported that total wbc count was a better prognostic marker for long - term prognosis than the wbc components (30). in conclusion, combination of wbc, hb, and pdw, a cheap and simple hematologic marker, is useful in early risk stratification of patients with ami.	the aim of this study was to assess the prognostic value of combined use of white blood cell (wbc), hemoglobin (hb), and platelet distribution width (pdw) in patients with acute myocardial infarction (ami). this study included 1,332 consecutive patients with ami. patients were categorized into complete blood cell (cbc) group 0 (n=346, 26.0%), 1 (n=622, 46.7%), 2 (n=324, 24.3%), and 3 (n=40, 3.0%) according to the sum of the value defined by the cut - off levels of wbc (1, 14.5103/l ; 0, < 14.5103/l), hb (1, < 12.7 g / dl ; 0, 12.7 g / dl), and pdw (1, 51.2% ; 0 : < 51.2%). in - hospital death occurred in 59 (4.4%) patients. patients who died during index hospitalization had higher wbc and pdw and lower hb. the patients could be stratified for in - hospital mortality according to cbc group ; 1.2%, 2.7%, 9.0%, and 22.5% in cbc groups 0, 1, 2, and 3 (p<0.001), respectively. in multivariate logistic regression analysis, cbc group2 (odds ratio, 3.604 ; 95% confidence interval, 1.040 - 14.484, p=0.043) was an independent predictor for in - hospital death. the prognostic impact of the combined use of cbc markers remained significant over 12 months. in conclusions, combination of wbc, hb, and pdw, a cheap and simple hematologic marker, is useful in early risk stratification of patients with ami.
	the potential health and ecological effects of endocrine disrupting chemicals has become a high visibility environmental issue. the 1990s have witnessed a growing concern, both on the part of the scientific community and the public, that environmental chemicals may be causing widespread effects in humans and in a variety of fish and wildlife species. this growing concern led the committee on the environment and natural resources (cenr) of the national science and technology council to identify the endocrine disruptor issue as a major research initiative in early 1995 and subsequently establish an ad hoc working group on endocrine disruptors. the objectives of the working group are to 1) develop a planning framework for federal research related to human and ecological health effects of endocrine disrupting chemicals ; 2) conduct an inventory of ongoing federal research programs ; and 3) identify research gaps and develop a coordinated interagency plan to address priority research needs. this communication summarizes the activities of the federal government in defining a common framework for planning an endocrine disruptor research program and in assessing the status of the current effort. after developing the research framework and compiling an inventory of active research projects supported by the federal government in fiscal year 1996, the cenr working group evaluated the current federal effort by comparing the ongoing activities with the research needs identified in the framework. the analysis showed that the federal government supports considerable research on human health effects, ecological effects, and exposure assessment, with a predominance of activity occurring under human health effects. the analysis also indicates that studies on reproductive development and carcinogenesis are more prevalent than studies on neurotoxicity and immunotoxicity, that mammals (mostly laboratory animals) are the main species under study, and that chlorinated dibenzodioxins and polychlorinated biphenyls are the most commonly studied chemical classes. comparison of the inventory with the research needs should allow identification of underrepresented research areas in need of attention.imagesfigure 1
a 61-year - old immunocompetent woman presented with a chronic headache for 10 months that had been progressively worsening for 18 days with emesis. the patient was a peasant with no particular exposure to avian (especially pigeon) feces or woodworking. the headache got worse, with several nonbloody, nonbilious vomiting episodes, and the patient developed an ataxic gait 18 days before hospital admission. a comprehensive physical examination of her body at admission did not show any pertinent findings. she could not complete the hand alternating movement test, her right side of finger nose test was positive, and her heel - knee - tibia test was suspiciously positive, but romberg s sign was negative. the cryptococcal antigen latex agglutination test of csf was negative the first time, but it was positive the second time ; the titer was 1:32. a plain magnetic resonance imaging (mri) scan of the head demonstrated a 262116 mm nodule as long or equal to t1 and long t2 signal in the right cerebellar hemisphere with no adjacent edema, and the nodule was significantly enhanced as nodular with a thick and annular wall after contrast administration (figure 1c e). there was no midline shift, and the compression of adjacent structures was not apparent (figure 1a and b). the patient was considered to have a metastatic tumor or glioma in the right cerebellar hemisphere. the cerebellar nodule was removed by neurosurgery via a suboccipital craniotomy in the left lateral position. an 80 mm long straight incision was made 20 mm outside the right side of the occipital bone of the lower lateral hairline. intraoperatively, the lower part of the transverse sinus and the surface layer of cerebellum were exposed. subcortical dissection was performed until a 403020 mm firm nodule with moderate vascular supply and distinct border was identified, located at the upper portion of the right cerebellar hemisphere. the blood vessels that supply for the nodule were cut off by electrocoagulation, and then en bloc resection of the nodule along the border was performed. a large number of mononuclear cells and multinucleated giant cells were seen, with diffused distribution. there was a small amount of bacteria circular structure and mesenchyme hyalinization, with a large number of lymphocytes and a few plasma cell infiltrations ; local tissue necrosis with neutrophil infiltration was also found in the frozen section tissues (figure 1f). immunohistochemical staining yielded cd68 (+), cytokeratin (), epithelial membrane antigen (), glial fibrillary acidic protein (), ki-67 (), neurofilament(), neuron - specific enolase (), soluble - protein 100 (), vimentin (+), cluster of differentiation 1a (), and thallus periodic acid - schiff stain (+). the neuropathology evidenced a right cerebellar cryptococcal granuloma. a 61-year - old immunocompetent woman presented with a chronic headache for 10 months that had been progressively worsening for 18 days with emesis. the patient was a peasant with no particular exposure to avian (especially pigeon) feces or woodworking. the headache got worse, with several nonbloody, nonbilious vomiting episodes, and the patient developed an ataxic gait 18 days before hospital admission. a comprehensive physical examination of her body at admission did not show any pertinent findings. she could not complete the hand alternating movement test, her right side of finger nose test was positive, and her heel - knee - tibia test was suspiciously positive, but romberg s sign was negative. the cryptococcal antigen latex agglutination test of csf was negative the first time, but it was positive the second time ; the titer was 1:32. a plain magnetic resonance imaging (mri) scan of the head demonstrated a 262116 mm nodule as long or equal to t1 and long t2 signal in the right cerebellar hemisphere with no adjacent edema, and the nodule was significantly enhanced as nodular with a thick and annular wall after contrast administration (figure 1c e). there was no midline shift, and the compression of adjacent structures was not apparent (figure 1a and b). the patient was considered to have a metastatic tumor or glioma in the right cerebellar hemisphere. the cerebellar nodule was removed by neurosurgery via a suboccipital craniotomy in the left lateral position. an 80 mm long straight incision was made 20 mm outside the right side of the occipital bone of the lower lateral hairline. intraoperatively, the lower part of the transverse sinus and the surface layer of cerebellum were exposed. subcortical dissection was performed until a 403020 mm firm nodule with moderate vascular supply and distinct border was identified, located at the upper portion of the right cerebellar hemisphere. the blood vessels that supply for the nodule were cut off by electrocoagulation, and then en bloc resection of the nodule along the border was performed. a large number of mononuclear cells and multinucleated giant cells were seen, with diffused distribution. there was a small amount of bacteria circular structure and mesenchyme hyalinization, with a large number of lymphocytes and a few plasma cell infiltrations ; local tissue necrosis with neutrophil infiltration was also found in the frozen section tissues (figure 1f). immunohistochemical staining yielded cd68 (+), cytokeratin (), epithelial membrane antigen (), glial fibrillary acidic protein (), ki-67 (), neurofilament(), neuron - specific enolase (), soluble - protein 100 (), vimentin (+), cluster of differentiation 1a (), and thallus periodic acid - schiff stain (+). the neuropathology evidenced a right cerebellar cryptococcal granuloma. cns cryptococcosis is a kind of subacute or chronic deep mycosis caused by cryptococcus neoformans.1 cryptococcus neoformans mainly spreads via the respiratory route and infects predilection sites in cns via hematogenous spread. it usually infects patients with immunosuppression, especially aids patients, as well as patients with obvious consumptive disease and patients who consumed extensive immunosuppressants and extensive broad - spectrum antibiotic therapy. however, the morbidity of cryptococcosis in immunocompetent patients has increased in recent years.2 the main pathologic changes of cns cryptococcosis were meningitis, meningoencephalitis, vasculitis, and rarely, granulomas. furthermore, intracranial cryptococcal granulomas were significantly more common in the cerebral hemisphere than in the cerebellar hemisphere. there are two recognized varieties of cryptococcus neoformans : cryptococcus neoformans var neoformans and cryptococcus neoformans var gattii, which rarely infected immunocompromised patients while usually infecting immunocompetent patients.3,4 cerebral cryptococcoma is the most common form of expression in immunocompetent individuals.5,6 there are only a few cases of isolated cerebellar cryptococcoma that were reported in immunocompetent patients.710 the clinical feature of cns cryptococcosis is highly variable and nonspecific, which is related to immune status and the types of cryptococcus.11 because of its insidious onset, progressive intracranial hypertension may be the initial symptom, characterized by headache, nausea, and vomiting, and then followed with other symptoms, such as cranial nerve damage and focal neurological signs. the imaging diagnosis of immunocompetent patients with cerebellar cryptococcomas is a challenge. in general, csf, including pressure measurement, routine tests, biochemistry indicators, and examination of ink stain of csf, is essential to the diagnosis. cryptococcal antigens latex agglutination test of cerebrospinal fluid and blood examination contribute to the diagnosis, but the definitive diagnosis of cryptococcosis must be confirmed by histopathologic examination. in the current patient, the diagnosis of cerebellar cryptococcoma was confirmed by histopathology, whereas the csf and cryptococcal antigen latex agglutination tests were both negative. different sites, pathological types, and stages of cns cryptococcosis may show different computed tomographic (ct) and mri findings.12 early stages of cryptococcosis may not show any abnormal ct findings, but a few nonspecific manifestations, including diffuse cerebral atrophy, hydrocephalus, and diffuse cerebral edema, can be found.3,13 cryptococcoma can appear as a localized tumor - like mass when the fungus has involved brain parenchyma. cryptococcus tumors typically show long t1 and long t2 signals on mri, as well as circular enhancement.14 although certain characteristic imaging findings have been described, the differential diagnosis is still difficult because of the overlap of imaging findings with granulomatous tumors and abscesses, especially the tuberculous granuloma. early diagnosis and early initiation of treatment are associated with a good prognosis the therapeutic goal is to control the initial infection and lifelong antifungal therapy for patients with hiv coinfection and to cure the cryptococcus infection completely for patients without hiv coinfection. the lack of specificity of clinical manifestations and the low detection rate of cryptococcus in csf bring difficulties to its diagnosis. in this reported case, cns cryptococcosis manifested as an intracranial lesion, which was misdiagnosed as a tuberculoma or a cerebral tumor.	central nervous system (cns) cryptococcosis is an opportunistic fungal infection that typically occurs in patients with reduced immunological function, such as patients with aids, patients receiving organ transplants, or patients receiving corticosteroid and immunosuppressive therapy. cns cryptococcosis rarely occurs in immunocompetent patients. cns cryptococcosis is characterized by meningitis and encephalitis and occasionally forms isolated granulomas. isolated cerebellar cryptococcoma is a rare condition, especially in immunocompetent patients, and the misdiagnosis rate is high. a definite diagnosis must be based on pathology. to raise awareness of this disease, the clinical data of a patient with cryptococcomas in the right side of the cerebellum are reported.
lynx samples were collected as part of a large study assessing the health of the free - ranging lynx population in sweden from 1989 through 1999 and providing baseline data on diseases and parasites of the eurasian lynx. complete necropsy was performed on all animals shipped to the national veterinary institute, uppsala, sweden, during the study period. samples of lung, kidney, and spleen were collected during necropsy and stored at 20c until analysis. the main causes of death were traffic accidents and sarcoptic mange (sarcoptes scabiei infestation) (27). infections with common feline viruses were rare (28), and seropositivity to toxoplasma gondii was more common among lynx from central sweden than from farther north, where the climate is more harsh and the human population less dense (29). a total of 263 lynx, sampled during 19951999, were included in the present study. we differentiated 3 main geographic regions according to human population density (figure 1). northern sweden, apart from the mountain chain bordering norway, is part of the circumpolar northern coniferous forest belt, the taiga. it is sparsely populated by humans ; human populations increase toward the south and gradually decline farther from the coast. the southernmost part of sweden belongs to the broad - leaf forest region of central europe but is mainly cultivated. although their main prey are roe deer, they also prey on reindeer and sheep. furthermore, debilitated lynx affected by sarcoptic mange and orphans searching for easily accessible food are often found around human settlements. interactions between diseased lynx and domestic cats and dogs have been documented (30,31). geographic origin of 263 eurasian lynx (lynx lynx) collected in sweden during 19951999 and tested for orthopoxvirus (opv)specific dna (open circles). opv dna was amplified by pcr from 24 animals (9% ; red circles). light blue areas represent sparsely populated (50 inhabitants / km). most (245) animals submitted were apparently healthy (225 killed by hunters, 19 died of traumatic injury, 1 died of acute circulatory failure during anesthesia), 15 had sarcoptic mange, and 3 died of starvation caused by noninfectious or unclear etiology. body condition was evaluated by body weight, fat deposits in the abdomen and on the heart, and appearance of the bone marrow of the femur. the animals were categorized into 3 groups : normal body condition (normal to fat, n = 230), poor body condition (low amount of fat in abdomen and on heart, n = 15), and emaciated (no abdominal fat, serous atrophy of fat deposits on heart and of bone marrow, n = 18, i.e., all diseased animals). for 228 animals, age was determined by counting cementum annuli of a canine tooth (matson s laboratory, milltown, mt, usa) ; for the others, it was estimated on the basis of body size and weight, tooth wear, development of genital organs, and, if appropriate, morphologic characteristics and ossification of the skull. investigated lynx with exact age known were 013 years of age (mean 2.5 years 2.6 sd). on the basis of sexual maturity, and thus reflecting social behavior, 3 age classes were established : juvenile (2 years for females and > 3 years for males ; n = 104). numbers of males and females were equal in the juvenile and adult age classes, but within subadults there were more males (n = 68) than females (n = 27). according to the time of death, animals were grouped into seasons relevant to the biology of the lynx : delivery (may july, n = 6) ; small kittens and lactation (august october ; n = 7) ; large kittens hunting with the dam (november january, n = 24) ; and breeding season, separation of kittens from dam, and main hunting season (february april, n = 224). from each sampled lynx, 25 mg lung (n = 262), 25 mg kidney (n = 263), and 10 mg spleen (n = 261) were cut in small pieces and homogenized in phosphate - buffered saline. dna was extracted by using a qiaamp tissue kit (qiagen gmbh, dsseldorf, germany) ; mean dna concentration was 39.7 g / ml 23.8 sd for lung, 99.0 g / ml 46.4 sd for kidney, and 101.0 g / ml 49.0 sd for spleen. pcr primers (medprobe as, oslo, norway) from the thymidine kinase gene (tk) were used as described (21), generating an expected pcr amplicon of 339 bp. dna from vaccinia virus (western reserve ; vr-119), cowpox virus (brighton ; vr-302) (both from american type culture collection, rockville, md, usa), and cowpox virus isolated from a felid with clinical disease (16) was used as positive control. reaction volume was 50 l and contained 5 l of the dna eluate from the lynx tissue, 1 l of each primer (25 m), 4 l dntp (10 mmol / l), 5 l mgcl2 (25 mmol / l), 5 l geneamp 10x gold buffer (applied biosystems, oslo, norway), 2.5 u amplitaq gold polymerase (applied biosystems), and 19 l water. the tubes were placed in a preheated block (95c) and held for 5 min before cycling. five cycles of denaturation (95c for 30 s), annealing (53c for 2 min), and primer extension (72c for 30 s) were followed by 35 cycles with 30 s annealing time. pcr amplicons were analyzed by using the gibco brl horizon 11 - 14 gel electrophoresis system (life technologies, paisly, scotland) in a 2% agarose gel (ultrapure agarose gel ; life technologies) with tae buffer (0.04 m tris - acetate, 1.0 mmol / l edta) and ethidium bromide for dna staining. fifteen l of pcr product mixed with 3 l 6x loading buffer (0.25% [wt / vol ] bromphenol blue and 40% [wt / vol ] sucrose) were loaded in each well. the gels were run in tae buffer at 150 v for 1.5 h and examined by using a gel doc 2000 documentation system (bio - rad laboratories, oslo, norway). primers and dntp were removed from amplicons by using exosapit reagent (amersham pharmacia, uppsala, sweden), adding 1 l/5l pcr product, incubating 45 min at 37c, and by conducting enzyme inactivation for 20 min at 80c. cycle sequencing was conducted in both directions by using big dye 3.1 reagents (abi bigdye terminator version 3.1, applied biosystems). two microliters edta (125 mmol / l), 2 l sodium acetate (3 mol / l), and 50 l of ethanol were added to the 20 l sequencing product. electrophoresis of the cycle sequencing extension products was conducted in an abi prism 377 dna analyzer (applied biosystems). raw sequence data were edited by chromas pro software version 1.41 (technelysium pty ltd., tewantin, queensland, australia) and bioedit sequence alignment editor version 7.0.4 (www.mbio.ncsu.edu/bioedit/bioedit.html). multiple sequence alignment the phylogenetic tree was generated with the neighbor - joining method by using mega 3.1 (www.megasoftware.net). the reliability of the phylogenetic relationship in the tree topology was statistically evaluated from 1,000 bootstrap replicates. the lynx orthopoxvirus dna sequences obtained in this study have been submitted to the genbank nucelotide sequence database (table). evidenced by pcr targeting a part of the thymidine kinase gene (tk). amplicons that were sequenced are underlined. age determined by sectioning of teeth. lung, 11 (4.2%), kidney, 13 (4.9%), spleen, 11 (4.2%) of 263 samples positive. animals with sarcoptic mange (sarcoptes scabiei) ; all were emaciated (other orthopoxvirus - positive animals except no. statistical calculations regarding differences in prevalence were performed by using ncss 2007 statistical software (www.ncss.com). statistical significance of differences was analyzed by using the 2-tailed fisher exact test ; level of significance was set at p 50 inhabitants / km). most (245) animals submitted were apparently healthy (225 killed by hunters, 19 died of traumatic injury, 1 died of acute circulatory failure during anesthesia), 15 had sarcoptic mange, and 3 died of starvation caused by noninfectious or unclear etiology. body condition was evaluated by body weight, fat deposits in the abdomen and on the heart, and appearance of the bone marrow of the femur. the animals were categorized into 3 groups : normal body condition (normal to fat, n = 230), poor body condition (low amount of fat in abdomen and on heart, n = 15), and emaciated (no abdominal fat, serous atrophy of fat deposits on heart and of bone marrow, n = 18, i.e., all diseased animals). for 228 animals, age was determined by counting cementum annuli of a canine tooth (matson s laboratory, milltown, mt, usa) ; for the others, it was estimated on the basis of body size and weight, tooth wear, development of genital organs, and, if appropriate, morphologic characteristics and ossification of the skull. investigated lynx with exact age known were 013 years of age (mean 2.5 years 2.6 sd). on the basis of sexual maturity, and thus reflecting social behavior, 3 age classes were established : juvenile (2 years for females and > 3 years for males ; n = 104). numbers of males and females were equal in the juvenile and adult age classes, but within subadults there were more males (n = 68) than females (n = 27). according to the time of death, animals were grouped into seasons relevant to the biology of the lynx : delivery (may july, n = 6) ; small kittens and lactation (august october ; n = 7) ; large kittens hunting with the dam (november january, n = 24) ; and breeding season, separation of kittens from dam, and main hunting season (february april, n = 224). from each sampled lynx, 25 mg lung (n = 262), 25 mg kidney (n = 263), and 10 mg spleen (n = 261) were cut in small pieces and homogenized in phosphate - buffered saline. dna was extracted by using a qiaamp tissue kit (qiagen gmbh, dsseldorf, germany) ; mean dna concentration was 39.7 g / ml 23.8 sd for lung, 99.0 g / ml 46.4 sd for kidney, and 101.0 g / ml 49.0 sd for spleen. pcr primers (medprobe as, oslo, norway) from the thymidine kinase gene (tk) were used as described (21), generating an expected pcr amplicon of 339 bp. dna from vaccinia virus (western reserve ; vr-119), cowpox virus (brighton ; vr-302) (both from american type culture collection, rockville, md, usa), and cowpox virus isolated from a felid with clinical disease (16) was used as positive control. reaction volume was 50 l and contained 5 l of the dna eluate from the lynx tissue, 1 l of each primer (25 m), 4 l dntp (10 mmol / l), 5 l mgcl2 (25 mmol / l), 5 l geneamp 10x gold buffer (applied biosystems, oslo, norway), 2.5 u amplitaq gold polymerase (applied biosystems), and 19 l water. the tubes were placed in a preheated block (95c) and held for 5 min before cycling. five cycles of denaturation (95c for 30 s), annealing (53c for 2 min), and primer extension (72c for 30 s) were followed by 35 cycles with 30 s annealing time. pcr amplicons were analyzed by using the gibco brl horizon 11 - 14 gel electrophoresis system (life technologies, paisly, scotland) in a 2% agarose gel (ultrapure agarose gel ; life technologies) with tae buffer (0.04 m tris - acetate, 1.0 mmol / l edta) and ethidium bromide for dna staining. fifteen l of pcr product mixed with 3 l 6x loading buffer (0.25% [wt / vol ] bromphenol blue and 40% [wt / vol ] sucrose) were loaded in each well. the gels were run in tae buffer at 150 v for 1.5 h and examined by using a gel doc 2000 documentation system (bio - rad laboratories, oslo, norway). primers and dntp were removed from amplicons by using exosapit reagent (amersham pharmacia, uppsala, sweden), adding 1 l/5l pcr product, incubating 45 min at 37c, and by conducting enzyme inactivation for 20 min at 80c. cycle sequencing was conducted in both directions by using big dye 3.1 reagents (abi bigdye terminator version 3.1, applied biosystems). two microliters edta (125 mmol / l), 2 l sodium acetate (3 mol / l), and 50 l of ethanol were added to the 20 l sequencing product. electrophoresis of the cycle sequencing extension products was conducted in an abi prism 377 dna analyzer (applied biosystems). raw sequence data were edited by chromas pro software version 1.41 (technelysium pty ltd., tewantin, queensland, australia) and bioedit sequence alignment editor version 7.0.4 (www.mbio.ncsu.edu/bioedit/bioedit.html). the phylogenetic tree was generated with the neighbor - joining method by using mega 3.1 (www.megasoftware.net). the reliability of the phylogenetic relationship in the tree topology was statistically evaluated from 1,000 bootstrap replicates. the lynx orthopoxvirus dna sequences obtained in this study have been submitted to the genbank nucelotide sequence database (table). age determined by sectioning of teeth. lung, 11 (4.2%), kidney, 13 (4.9%), spleen, 11 (4.2%) of 263 samples positive. animals with sarcoptic mange (sarcoptes scabiei) ; all were emaciated (other orthopoxvirus - positive animals except no. statistical calculations regarding differences in prevalence were performed by using ncss 2007 statistical software (www.ncss.com). statistical significance of differences was analyzed by using the 2-tailed fisher exact test ; level of significance was set at p 1 internal organs of the lynx is evidence of a virus infection and indicates that the host most likely has entered a viremic phase. no specific pathologic lesions in skin or lungs were recorded, but only a limited number of skins were submitted with the carcasses. this finding calls for increased efforts to document possible clinical signs, pathologic changes, and the effects of cowpox virus infection in lynx, taking into account the clinical signs and organ lesions documented in domestic cats and in large felid species in zoos (11,12). wild rodents such as bank voles, wood mice, and field voles have been suggested as the main cowpox virus reservoirs in finland (23), great britain (19,22), and norway (in addition to lemmings and common shrews) (20,21). cowpox virus is considered to be endemic in these species in certain regions, circulating in several host species at the same time. in a previous examination of stomach contents of norwegian lynx, which are part of the common lynx population inhabiting norway and sweden, remnants of small rodents were found in 8% of the individuals (38), demonstrating that rodents are part of their prey. domestic cats may also act as source of infection for lynx, as a potential prey species. lynx from areas with high human density, and thus a presumably larger domestic cat population, were more often infected than lynx from less populated areas, indicating a possible relationship between domestic cats and lynx. a similar association with human presence has already been observed with regard to seropositivity to t. gondii in some lynx in this study (29). in contrast, previous studies (1999) of other pathogens in some of the lynx (n = 70) included in this study suggested that contacts between lynx and domestic cats are uncommon ; seroprevalence of antibodies against feline parvovirus was low ; and feline leukemia virus antigen and antibodies against feline coronavirus, calicivirus, herpesvirus, and feline immunodeficiency virus were absent (28). assuming that lynx are susceptible to these feline pathogens, this difference may be explained by the fact that domestic cats are commonly vaccinated against these viral infections but not against t. gondii and opvs. nevertheless, these results may suggest only limited contact between domestic cats and lynx, pointing at wild rodents and possibly shrews as the direct source of the opv infection for lynx. among the 24 animals that were opv - positive by pcr, 5 (21%) had sarcoptic mange, whereas the total prevalence of mange among the 264 animals included in this study was 6.4% ; this finding indicates a possible relationship between opv infection and infestation with s. scabiei. the severe epidermal lesions and breakage of the skin barrier might predispose the animals to infection by opvs, such as cowpox virus, and such viruses might contribute to the severity of sarcoptic mange in lynx. alternatively, altered behavior in diseased lynx could influence exposure to infection. in contrast to healthy lynx, those with mange are commonly found in human settlements, where they sometimes prey on domestic animals, including cats and dogs (30). we found no seasonal differences in opv prevalence, although one could assume that autumn, with peak populations of rodents and shrews, would be when contact rate between these animals and lynx is highest. it could also be expected that the presence of opvs such as cowpox virus would fluctuate between years, reflecting the fluctuations of rodent populations, but we found no significant differences in prevalence between the years in this study. these findings could be the result of the low number of samples that were included from the nonhunting seasons and from some of the years represented in the study. cowpox virus infection is a zoonosis, capable of being transmitted from rodents to humans, often by domestic cats. cowpox virus infection in humans is usually characterized by single lesions on the infection site (face, hands, arms), but it sometimes spreads and cause secondary lesions and complications and can be especially severe in immunocompromised persons (6,16,23). to our knowledge, however, only 2 verified cowpox cases in humans have been recorded from sweden (16,26). in conclusion, our results support the hypothesis that carnivores that prey on opv reservoir species can be used as indicator species for the presence of such viruses in the ecosystem (25). this study also provides further evidence that opv, presumably cowpox virus, is widely distributed in ecosystems in sweden. this finding may be relevant for vaccination strategies (39), especially when considering the use of opvs as vectors in genetically recombinant vaccines and their ability to undergo spontaneous genetic recombinations with virus relatives when replicating in the same cells of a host. moreover, our findings indicate that the role of opvs, such as cowpox virus, as potential human pathogens may increase, considering their broad distribution in ecosystems, the cessation of smallpox vaccination of humans, and an increasing number of immunocompromised persons. thus, targeted surveillance of rodent species, and the carnivores that prey on them, is necessary for monitoring the emergence or reemergence of these viruses as potential human pathogens. further genetic studies are needed to determine whether the detected virus in the free - ranging lynx population in scandinavia is indeed cowpox virus.	cowpox virus, which has been used to protect humans against smallpox but may cause severe disease in immunocompromised persons, has reemerged in humans, domestic cats, and other animal species in europe. orthopoxvirus (opv) dna was detected in tissues (lung, kidney, spleen) in 24 (9%) of 263 free - ranging eurasian lynx (lynx lynx) from sweden. thymidine kinase gene amplicon sequences (339 bp) from 21 lynx were all identical to those from cowpox virus isolated from a person in norway and phylogenetically closer to monkeypox virus than to vaccinia virus and isolates from 2 persons with cowpox virus in sweden. prevalence was higher among animals from regions with dense, rather than rural, human populations. lynx are probably exposed to opv through predation on small mammal reservoir species. we conclude that opv is widely distributed in sweden and may represent a threat to humans. further studies are needed to verify whether this lynx opv is cowpox virus.
between 1983 and 1989, 1,441 subjects with type 1 diabetes were enrolled in the dcct. a total of 249 subjects were recruited as adolescents aged 1319 years : 32% were 1314 years old, 37% were 1516 years old, and 31% were 1719 years old. all adolescents had to be at least tanner stage ii in pubertal development, which is the stage at which the first signs of puberty are visible on physical examination. we chose age 19 as the upper age limit, rather than age 18 as used in other studies on the dcct cohort (15,16), because the sample size was considerably larger when the age limit was extended to the final year of adolescence. the primary prevention cohort (n = 149) had diabetes for 15 years, no retinopathy, and urinary albumin excretion 9.5%), and frequency of severe hypoglycemia (0, 15, and > 5 reported events) on the standardized quantitative score for each of the eight cognitive domains. each model adjusted for baseline age, sex, education, length of follow - up, visual acuity, self - reported sensory loss attributable to peripheral neuropathy, and the number of interval cognitive tests taken (to control for practice effects). results are presented as the average increase or decrease in the standardized score from the dcct baseline within or between groups or as the per unit change in a quantitative covariate. nominally significant results (p 9.5%), and frequency of severe hypoglycemia (0, 15, and > 5 reported events) on the standardized quantitative score for each of the eight cognitive domains. each model adjusted for baseline age, sex, education, length of follow - up, visual acuity, self - reported sensory loss attributable to peripheral neuropathy, and the number of interval cognitive tests taken (to control for practice effects). results are presented as the average increase or decrease in the standardized score from the dcct baseline within or between groups or as the per unit change in a quantitative covariate. nominally significant results (p 5 episodes) (fig. 1b), and metabolic control (tertiles of mean a1c values) (fig. 1c). neither original treatment assignment nor cumulative number of hypoglycemic events influenced performance in any cognitive domain. performance in both sexes improved over time, and this effect was more pronounced in male participants (p < 0.01). higher values of a1c were associated with modest declines in psychomotor and mental efficiency (p < 0.01). degree of self - reported symptoms of depression at year 12, as indexed by the scl-90r (median t score 46.0 ; scores 63 reflect possible depressive disorder), was not associated with poorer performance on any of the eight domains. analyses were repeated using the broader definition of hypoglycemia, which includes episodes in which the patient has incapacity sufficient to require assistance. the results with the broad definition were similar to those obtained using the narrow definition (i.e., restricted to seizure or coma). the timing of severe hypoglycemic events did not affect performance on any of the eight cognitive domains. we found that 47 patients reported their first episode of coma or seizure during adolescence (ages 1319) and 43 patients reported having lost consciousness between one and five times before their dcct baseline evaluation. patients who experienced dka (n = 26) during the dcct (ages 1319 years) declined in cognitive performance on the learning domain, whereas the patients without dka improved. further, patients with dka episodes during the dcct improved less on the spatial information domain than patients with no dka events (data not shown). our previous report on the entire dcct / edic cohort showed no detrimental effects of intensive treatment or severe hypoglycemic episodes on cognitive performance (4). however, because of the potential vulnerability of the developing brain (14), we evaluated whether intensive treatment during adolescent years posed threats to long - term cognitive functioning. severe hypoglycemic episodes during childhood are a major concern, especially given the findings that cognitive deficits may be more common in those in whom type 1 diabetes is diagnosed during childhood (9,12,22). moreover, children may be more sensitive than adults to even modestly lower glucose levels, with cognitive deterioration at 3.3 versus 2.5 mmol / l in adults (23). intensive treatment is not associated with risk for long - term cognitive dysfunction, even in the subset of patients who entered the dcct during adolescence. as with the findings from the entire cohort, we found that higher a1c values were associated with poorer performance on measures of psychomotor and mental efficiency, which require the integration of motor and cognitive processes. higher a1c values were also associated with somewhat slower performance on a simple measure of motor speed (fig. 1), but, unlike our earlier results with the entire cohort, that effect failed to reach statistical significance in this cohort, possibly due to smaller sample size. although we collected data on retinopathy, which are associated with higher a1c levels, the effect that this complication has on cognitive ability is beyond the scope of the article and will be addressed separately. despite no discernible ill effects on cognition as a result of severe hypoglycemia, brain abnormalities due to serious hypoglycemia for example, children with a history of severe hypoglycemia have shown some abnormalities in brain structure and function (22). slow - wave electroencephalographic activity is increased in this patient population, especially in the frontal regions (24), which govern executive function and attention. finally, recent evidence has suggested that severe hypoglycemia in children alters gray matter density (22), analogous to what has been reported in young adults with type 1 diabetes (25). thus, although no notable cognitive deficits were observed, these early brain changes may serve as a marker of future cognitive impairments (25). although our conclusion from this study is that severe hypoglycemic episodes have no long - term effect on cognition, even when experienced during adolescence, several study limitations need to be considered. first, we included patients between the ages of 13 and 19 years ; therefore, we can not determine whether diabetes or hypoglycemia during early childhood is associated with cognitive deficits later in life. accordingly, we have limited information on the effects of diabetes on the very young brain. second, the results must be generalized cautiously not only because the sample size of our cohort is relatively small but also because of the careful selection criteria applied to subjects recruited into the dcct. third, our study imposed restrictions on the number and severity of dka and hypoglycemic episodes that patients could experience during the several years before the dcct. these exclusionary factors restrict our ability to comment on whether long - term cognition is further affected in patients who experienced these particularly serious metabolic consequences of diabetes. in summary, our study indicates that with regard to long - term cognitive function, intensive treatment is safe for patients who had the diagnosis of diabetes as children, despite the increased threat of severe hypoglycemic episodes. nevertheless, we need to remain cognizant of the dangers of acute hypoglycemia, which can lead to comas, accidents, injuries, death, family stress, loss of school or work time, and loss of commitment to the goals of intensive treatment. thus, continued research is needed to develop new therapies and technologies that will minimize or eliminate this major obstacle to achievement of optimal control of type 1 diabetes.	objective the purpose of this study was to evaluate whether severe hypoglycemia or intensive therapy affects cognitive performance over time in a subgroup of patients who were aged 1319 years at entry in the diabetes control and complications trial (dcct).research design and methods this was a longitudinal study involving 249 patients with type 1 diabetes who were between 13 and 19 years old when they were randomly assigned in the dcct. scores on a comprehensive battery of cognitive tests obtained during the epidemiology of diabetes interventions and complications follow - up study, 18 years later, were compared with baseline performance. we assessed the effects of the original dcct treatment group assignment, mean a1c values, and frequency of severe hypoglycemic events on eight domains of cognition.resultsthere were a total of 294 reported episodes of coma or seizure. neither frequency of hypoglycemia nor previous treatment group was associated with decline on any cognitive domain. as in a previous analysis of the entire study cohort, higher a1c values were associated with declines in the psychomotor and mental efficiency domain (p < 0.01) ; however, the previous finding of improved motor speed with lower a1c values was not replicated in this subgroup analysis.conclusionsdespite relatively high rates of severe hypoglycemia, cognitive function did not decline over an extended period of time in the youngest cohort of patients with type 1 diabetes.
there has been a remarkable increase in use of multidetector computed tomography (mdct) since its introduction. mdct has greater diagnostic capability and enables extended clinical applications, but it also has the potential to lead to an increase in radiation dose owing to the routine use of thinner sections, the extended volume of acquisition, and multiple - phase acquisitions. according to the literature, currently, ct represents about 7% of all radiologic examinations in the world but contributes more than 40% of the collective effective dose. the theoretic risk to patients for radiation - indeed cancer from ct examination is not negligible [24 ]. in particular, the radiation dose from hepatic ct examinations has notably increased because multiple - phase dynamic - enhanced ct scan was routinely performed in patients who are suspected of having hepatic tumors. the estimated risk of cancer death for those undergoing ct is 12.5/10,000 population for each pass of the ct scan through the abdomen. therefore, concerns regarding a reduction in radiation dose have been recently raised during abdominal ct acquisitions. although decreasing tube current is the most means of reducing ct radiation dose [69 ], this alteration also reduces the contrast - to - noise ratio (cnr), which may affect the diagnostic outcome of the examination. this is especially true in abdominal studies, where low - contrast areas are severely affected by the cnr. some studies [1115 ] suggest that scanning with low tube voltage is possible to reduce dose without markedly affecting image quality ; however, there are few reports on the effect of low tube voltage on abdominal image quality and low - contrast detectability (lcd). thus, the purpose of this study was to investigate the effect of low tube voltage with 80 kv on image noise (sd, standard deviation of ct number), cnr, radiation dose, and lcd at abdominal mdct. the institutional review board approved this study, and informed consent was obtained from all participating radiologists. we used a phantom (catphan 500 ; phantom laboratory, cambridge, ny) with an additional annulus provided by the manufacturer to simulate the x - ray absorption of a standard abdomen (giving a total test object diameter of 30 cm). the phantom contains a ctp515 module, which consists of a 40 mm thick and 200 mm diameter slice of tissue equivalent background material containing a series of cylinders of various diameters to measure low - contrast performance. the cylinders varied in diameters from 2.0 to 15.0 mm and deviated from nominal contrast levels by 0.3%, 0.5%, and 1.0% (figure 1). in our study, in order to avoid a partial volume effect, only the 15.0 mm diameter object with a contrast difference of 1.0% (having an attenuation difference with the background of 10 hu) was chosen to be analyzed. the phantom was scanned three times for each protocol with a 16-section mdct scanner (lightspeed ; ge medical systems). the scanning parameters were configuration of 16 (detectors) 1.5 mm (detector collimation), rotation time of 0.75 second, section thickness of 5.0 mm, section interval of 5.0 mm, pitch of 0.659, scan field of view of 50 cm, reconstruction algorithm (kernel) b30f (medium - sharp), and pixel matrix size of 512 512. scanning was performed at the standard tube voltage of 120 kv and at the low tube voltage of 80 kv, with corresponding tube current - time product settings at 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, and 650 mas, respectively. we used the ct dose index volume (ctdivol) based on the manufacturer 's data for estimation of radiation dose. the ctdivol obtained at standard tube voltage protocol was compared with that obtained at low tube voltage protocol. for each scanning technique, we measured the ct number of the low - contrast object in 15 mm diameter and the background of the module. the region of interest used to perform the measurements was kept at 100 mm. cnrs were calculated as follows : cnr = (roim roib)/sdb, where roim and roib are the ct numbers of the low - contrast object in a 15 mm diameter region of interest and of the background region of interest, respectively, and sdb is the standard deviation of the attenuation values of the background. the measurement was repeated three times on each image, giving nine measurements for each acquisition condition. from these nine measurements, a mean cnr was calculated for each set of acquisition conditions. for the subjective assessments of lcd, we evaluated the images obtained at 120 kv and 300 mas and the images obtained at 80 kv and 150650 mas. two experienced observers who were blind to each set of scanning parameters were asked to review independently the images. the visualization of each object was graded on a 3-point scoring scale by each observer : a score of 3.0 was obtained when the object was clearly visible and appeared as a perfect circle, a score of 2.0 was obtained when the object was not clearly visible, and a score of 1.0 was obtained when the object could not be detected. a total of 36 images (twelve sets of three images each) were respectively assessed by each observer. the final score of lcd of each acquisition sets was calculated by averaging the results of the two observers. the time for reading the images was not limited, and each observer could freely adjust the window levels and window widths on the monitor screen. we used a two - tailed student 's t test to evaluate differences in sd, cnr, and ctdivol between scanning performed with 80 kv and scanning performed with 120 kv. both the relationship between sd and tube current - time product settings and the relationship between cnr and ctdivol were investigated using the linear regression analysis and pearson correlation coefficient (r). for subjective assessment, the mann - whitney u test was used to analyze differences in subjective scores between standard setting (120 kv, 300 mas) and low tube voltage settings (80 kv, 150650 mas). kappa values less than 0.20 indicated poor agreement ; 0.210.40, fair agreement ; 0.410.60, moderate agreement ; 0.610.80, good agreement ; 0.811.00, excellent agreement. all statistical analyses were performed with a commercially available software package (spss, version 15.0), and a p value of less than 0.05 was considered to be statistically significant. the ctdivol obtained from each set of acquisition conditions is shown in table 1. at equal tube current - time product settings, the ctdivol obtained at 80 kv was approximately 32% of that at 120 kv. compared with the ctdivol obtained at 120 kv and 300 mas, the relative ctdivols obtained at 80 kv were 16% at 150 mas, 21% at 200 mas, 27% at 250 mas, 32% at 300 mas, 37% at 350 mas, 42% at 400 mas, 47% at 450 mas, 53% at 500 mas, 58% at 550 mas, 63% at 600 mas, and 68% at 650 mas. the results of ct numbers, image noise, and cnr at each scanning technique are listed in table 2. as expected, the image noise was inversely correlative to tube current. at identical tube current, the lowest and the highest noise were seen at 120 kv and 80 kv, respectively (figure 2). compared with the noise obtained with 120 kv at 300 mas, the noise obtained with 80 kv at 150650 mas was significantly higher (p 0.05) (table 2). the subjective scores of lcd assigned by two observers are shown in table 3. the mean score of the three images assigned at 120 kv and 300 mas was 2.83 0.41. at 80 kv, the mean score (1.00 0.00 at 150250 mas, 1.83 0.41 at 300 mas, 2.00 0.00 at 350 mas, 2.33 0.52 at 400500 mas) was significantly lower than that at 120 kv and 300 mas (p = 0.001 at 150350 mas ; p = 0.019 at 400500 mas) (table 3). however, there was no statistically significant difference between the mean score at 120 kv and 300 mas and the mean score assigned at 80 kv and the other tube current settings that we investigated (p = 0.138 at 550 mas ; p = 0.317 at 600 mas ; p = 1.0 at 650 mas) (table 3). using cohen kappa statistics, the interobserver agreement in regard to subjective assessment of lcd was good (= 0.67). improvement in mdct technology now allows ct examinations to be easily and fast performed, leading to a possible increase of the radiation dose to patients. in particular, the radiation exposure and risk of cancer death from hepatic ct examinations have notably increased because multiple - phase dynamic - enhanced ct scan is routinely performed. managing patient dose is therefore a major concern in abdominal mdct examinations. in our study, we used the ct dose index volume (ctdivol) based on the manufacturer 's data for estimation of radiation dose. ctdi, expressed in terms of air kerma in milligray, was obtained at the periphery (ctdip) and at the centre (ctdic) of a special 100 mm long pencil - shaped ionisation chamber. the weighted ctdiw is obtained as the sum of one - third of ctdic and two - thirds of ctdip. the ctdivol, which is ctdiw divided by the pitch, represents the average volume dose (air kerma) within a specified ct dosimetry phantom. ctdivol is a good measure of ct radiation dose for applications where the patient table is incremented during the scan. adoption of ctdivol as the intensity of the radiation dose would facilitate accurate comparisons of radiation doses used for different tube voltages. for instance, in our study, it is easy for us to compare the difference of radiation dose between 80 kv and 120 kv tube voltage. results of our study showed that it was possible to reduce radiation exposure substantially by decreasing the tube voltage from 120 kv to 80 kv. however, it has limitations. because the ctdivol is an averaged dose to a homogeneous cylindrical phantom, the measurements are only an approximation of patient dose. another limitation is that ctdivol phantom does not provide a sufficiently long scatter path relative to the typical length of a human ; hence, patient dose may be underestimated with ctdivol. therefore, the results of radiation dose based on the ctdivol in our study could not be accurate represented patient dose. furthermore, the difference of the radiation dose between the central and peripheral cavities of the phantom also could not be discerned by using the ctdivol as estimation of radiation dose. in present study, our findings showed that there was a direct correlation between the cnr and the ctdivol, which was consistent with previous studies [16, 20 ]. although the mean cnr was decreased when ct acquisition was performed at a tube voltage of 80 kv and an identical tube current setting, cnr improved substantially when identical ctdivol was used. compared with cnr obtained at 120 kv and 300 mas, there was no statistically significant difference at 80 kv and 550 mas, 600 mas, and 650 mas (p > 0.05). this suggest that image quality including cnr acquired at 80 kv with tube current higher than 550 mas is equivalent to that acquired at 120 kv and 300 mas. furthermore, the relative radiation dose obtained at 80 kv and 550 mas, 600 mas, and 650 mas was 58%, 63%, and 68% of that at 120 kvp and 300 mas, respectively. therefore, we postulate that scanning with a low tube voltage as low as 80 kv is feasible in abdominal ct examination without loss of diagnostic accuracy when the tube current is higher than 550 mas allowing reduction in the radiation dose by 32% to 42%. lcd is one of the most important factors in abdominal ct, especially when looking for small lesions in abdominal organs such as liver, spleen, pancreas, or kidneys. this lcd is not only relevant for unenhanced series but also contrast enhanced series, as contrast between normal and abnormal tissue may be only slightly increased by iodine. reported that in enhanced hepatic ct, tumor - to - liver contrast was 540 hu. in present study, the object with a contrast difference of 1.0% (having an attenuation difference with the background of 10 hu) was chosen to be analyzed. when the two experienced observers subjectively assessed the visibilities of low - contrast images, the subjective scores of lcd assigned at 80 kv and 550, 600, and 650 mas did not differ significantly from those assigned at 120 kv and 300 mas. furthermore, the mean score of lcd obtained at 80 kv and 650 mas was slightly higher than that at 120 kv and 300 mas. these results suggested that a reduction from 120 kv to 80 kv also could result in up to 42% dose reduction without compromising lcd.. showed that a 35% reduction in the radiation dose could be achieved when scanning was performed at 90 kv rather than at 120 kv without degradation of lcd. our findings agree with funama and suggest that lower tube voltage can be used in abdominal ct thereby achieving dose reduction while maintaining acceptable image quality. in this study, we found no statistically significant difference in both cnr and lcd at 120 kv and 300 mas compared with those at 80 kv and 550650 mas. this is probably because cnr and lcd are parallel to each other, which is consistent with verdun 's result. he found that there was a significant correlation between the mean cnr measurements and the subjective scores of lcd (r = 0.95, p < 0.05). the main drawback of the low tube voltage technique is the increase in image noise caused by the reduced photon flux. in our study, we found that the noise values obtained with 80 kv at the 150650 mas settings were significant higher than that obtained with 120 kv and 300 mas (p < 0.001). as previously reported [10, 20 ], we found that there was an inversely correlative relationship between the image noise and the tube current. in another word, the increased noise will be obtained when the strategy of lower tube current or lower tube voltage is implemented. image noise, however, has a greater effect on the quality of abdominal images because the abdominal region is inherently of lower contrast. therefore, for ct scanning with low tube voltage, higher tube current settings are required to compensate for the lower number of photons. several articles in the last years have been reported that noise reduction filters [2325 ] as well as reconstruction methods, such as adaptive statistical iterative reconstruction [2628 ] could effectively help to reduce the noise on ct images with radiation dose reduction without compromise of image quality. first, this ct scanning with low tube voltage at 80 kv was only performed in a phantom study, and the phantom did not consider variability of body composition, therefore, whether this result is suitable to clinical using needs to be further confirmed. however, marin. showed that a technique with low tube voltage at 80 kv could be applied to improve the conspicuity of malignant hypervascular liver tumors while significantly reducing patient radiation dose. secondly attenuation of the incident x - ray beam in ct depends on the size of body portion being evaluated ; that is, greater exposure is required in corpulent patients to attain image quality equal to that in slimmer patients. although studies in patients were not part of this investigation, previous studies with a phantom suggest that the technique is effective for dose reduction of abdominal ct for relatively light weight patients whose body weight is less than 80 kg. another important aspect is that many patients present with high - attenuation implants, which can dramatically decrease image quality when low kv protocols are used routinely. finally, we only used the ctdivol provided by the manufacturer to estimate the radiation dose. although the agreement between the values provided by the manufacturer and the measured values was good, with differences of less than 10%, there were some limitations as mentioned above. in this ct phantom study we have shown that although image noise is increased at low tube voltage, it is possible to reduce radiation dose by up to 42% without degradation of cnr and lcd by reducing tube voltage from 120 to 80 kv and increasing tube current to more than 550 mas. as an effective technique of reducing ct radiation dose, scanning with low tube voltage would benefit patients with relatively light weight, especially those who may need to undergo mdct examinations for long - term followup or high - risk screening.	purpose. to investigate the effect of low tube voltage (80 kv) on image quality, radiation dose, and low - contrast detectability (lcd) at abdominal computed tomography (ct). materials and methods. a phantom containing low - contrast objects was scanned with a ct scanner at 80 and 120 kv, with tube current - time product settings at 150650 mas. the differences between image noise, contrast - to - noise ratio (cnr), and scores of lcd obtained with 80 kv at 150650 mas and those obtained with 120 kv at 300 mas were compared respectively. results. the image noise substantially increased with low tube voltage. however, with identical dose, use of 80 kv resulted in higher cnr compared with cnr at 120 kv. there were no statistically significant difference in cnr and scores of lcd between 120 kv at 300 mas and 80 kv at 550650 mas (p > 0.05). the relative dose delivered at 80 kv ranged from 58% at 550 mas to 68% at 650 mas. conclusion. with a reduction of the tube voltage from 120 kv to 80 kv at abdominal ct, the radiation dose can be reduced by 32% to 42% without degradation of cnr and lcd.
the patient, a 55-year - old man, sought treatment at a hospital in the southeast of scotland in april 2012 for nausea, vomiting, anorexia, jaundice, headache, and abdominal pain. at admission, the patient s blood sample was positive for anti - hev igm and igg and raised levels of alanine aminotransferase (alt) (4,023 iu / l) and bilirubin (91 mol / l ; reference range 317 mol / l). over the next 10 days, alt declined steadily to 404 iu / l and returned to normal levels 1 month later. the patient s abdominal pain had largely resolved by 6 weeks after seeking treatment, but fatigue and lethargy persisted for > 8 weeks. there was no evidence that the patient was immunocompromised or was co - infected with other hepatitis viruses. the patient had unexplained jaundice at age 10 years and a history of alcohol abuse as an adult. virus rna was extracted from 140 l of serum and sequenced at limiting dilution in the hvr and orf2 regions as described (6). analysis of the orf2/orf3 overlap region used the primers 5-cgggtggaatgaataacatgt-3 (outer sense nucleotides 50985118 relative to m73218), 5-gcrgtyarcggcgmrgccccagctg-3 (outer antisense, 54815457), 5-tytgcctatgctgcccgcgccaccg-3 (inner sense, 51845209) and 5-ggcgctgggmytggtcrcgccaag-3 (inner antisense, 54265403). genbank accession numbers for the orf2 and orf2/3 sequences described here are jx516004jx516053 and for the hvr sequences are jx270882jx270902. nucleotide sequence analysis of the hvr from individual virus genomes sampled by limiting dilution of cdna revealed 2 distinct genotype 3 populations (populations a and b, figure). members of these populations all differed by the same 18 nucleotide substitutions, of which 13 were synonymous and 5 nonsynonymous. two populations (a and b) were also observed among 25 orf2 sequences (the 2 populations differed by 5 synonymous substitutions) and 26 orf2/orf3 sequences (the 2 populations differed by a single substitution, which was nonsynonymous in orf2 but synonymous in orf3). phylogenetic tree of hepatitis e virus hypervariable region variants. a neighbor - joining tree of hypervariable region sequences was constructed by using mega 4 (15). a and b indicate sequences for virus populations a and b. sequences from the patient studied here (patient 21) belonging to virus population a are indicated by a solid square, and those belonging to virus population b are indicated by a solid circle. 1 of the virus populations represented 80%90% of the sequences : in hvr, 17 sequences were population a and 4 population b ; in orf2, 21 sequences were population a, 2 population b, and 1 mixed ; and in orf2/3, 20 sequences were population a, 4 population b, and 2 mixed. the 3 mixed sequences contained ambiguities at positions where the 2 populations differed, implying that templates from both populations were present in the limiting dilution reaction. the expected number of such mixed - template reactions can be calculated by using the poisson distribution and the number of pcr - negative replicate reactions, which were 47% (hvr), 50% (orf2), and 43% (orf2/3). thus, 17.5%, 15.4%, and 20.8%, respectively, of reactions would be expected to contain > 1 template, of which, given the ratio of populations a and b in each region, 31%, 17.2%, and 28%, respectively, would contain templates from different populations, equivalent to 5.4%, 2.6%, and 5.8%, respectively, of positive reactions, compared with the 4.2% (3/71) observed. all other substitutions in these sequence sets were unique to a single sequence. a trivial explanation for the observation of mixed populations in this sample would be contamination during sample collection, processing, or analysis. however, this appears unlikely, given the low frequency of hev infection in the united kingdom, the lack of pcr products in negative controls, and the distinctness of the sequences obtained from those previously studied in our laboratory. infection of the patient in this study with 2 hev variants could arise for several different reasons. first, the person could have been exposed to a homogeneous source of hev, which then diversified during infection. assuming that infection occurred 1 month before the first symptoms and 6 weeks before sampling, a divergence of hvr sequences at 18 (7.4%) of 243 nucleotide positions equates to a rate of nucleotide substitution from a presumed ancestor of both populations of 0.32/site / year. not only were these substitutions mostly synonymous, and therefore unlikely to represent selection during infection, but the inferred rate of nucleotide substitution would be several orders of magnitude higher than those previously reported for hev (0.0014/site / year) (7) or for the hcv hvr (0.0043/site / year) (8). in addition, the divergence between the 2 virus populations contrasts with the homogeneity observed among 7 other acutely infected patients and between viruses transmitted from 1 person or host to another (6). a second possibility is that this person had a subclinical chronic hev infection and then became superinfected with a second virus that induced jaundice. no previous blood samples were available for testing, but a prior subclinical chronic infection seems unlikely, given the absence of immunosuppression ; the presence of anti - hev igm and igg ; the decline and increase, respectively, in these antibody titers in a sample collected 7 days later ; and the decline in alt and bilirubin levels in the weeks following the patient s hospital visit. last, the clinical features and our immunologic and virologic findings are consistent with the patient having been infected from a single source containing > 1 variant, such as pig - derived figatellu sausage (9). alternatively, he might have been multiply infected from different sources within in a short period and before the development of protective immunity. follow - up interview revealed no occupational exposure to animals, apart from a domestic dog, and no recent foreign travel ; he spent 3 days in a beach resort in southeastern scotland 12 days before onset of symptoms. the patient ate supermarket - bought prewashed salad vegetables and fresh fruit and drank only bottled or tap water. the patient ate fish, shellfish, chicken, pork, bacon, sausages, ham, brussels pat, and eggs but not venison or pig liver. hev has been found on the hands and gloves of 17% of persons selling pork products (10). this study suggests that mixed infection with hev can occur in immunocompetent persons with no obvious high - risk exposure to hev - infected food or sewage. only about 300 cases of acute hev are reported each year in england and wales (11), and the prevalence of hev pcr - positive blood donors in england is 0.014% (12). however, the seroprevalence of anti - hev igg is 1,000 higher (16%) (13), and in this context, the identification of sources of autochthonous hev infection remains an important goal. our results are also relevant to the suggestion that hev may undergo recombination (14). the case described here suggests that the conditions required for virus recombination may occasionally arise in immunocompetent persons.	we detected 2 hepatitis e virus (hev) strains in an acutely infected immunocompetent patient. two populations of genotype 3 virus were observed in the hypervariable regions and open reading frames 2 and 3, indicating multiple infection with hepatitis e virus. persons with mixed infections may provide the opportunity for virus recombination.
mediastinal abscess is a rare yet emergent infection of the thoracic cavity connective tissue, resulting from multiple potential sources : esophageal perforation (boerhaave s syndrome), odontogenic and peritonsillar abscess (descending necrotizing mediastinitis), cardiovascular or thoracic surgical procedures, trauma or rarely due to hematogenous spread (1,2). clinical diagnostic signs include dysphagia, dyspnea, fever, and chest pain with the potential for septic shock, as the disease progresses (3). water - soluble barium contrast swallow, transcutaneous ultrasound, chest radiograph, and ct are frequently utilized tools for visualization and prognosis (3,4). the foremost aim in management of mediastinal abscess is early debridement and drainage (5). while thoracotomy remains the gold standard of treatment, this approach carries significant risk of complication in certain populations (3,5). minimally invasive options such as, video - assisted thoracoscopic surgery (vats), ct guided percutaneous drainage, cervical drainage (irrigation / suction) and trans - esophageal endoscopic ultrasound (eus) represent safe and effective management strategies, in certain cases (4 - 6). herein is reported a novel application of laparoscopy as an additional alternative approach to drainage of anterior inferior mediastinal abscess. a 54-year - old female with a history significant for alcohol abuse, surgical correction of annular pancreas 20 years ago and chronic pancreatitis presented to a rural emergency department with a 1-week history of worsening severe epigastric pain, nausea and vomiting. she was in baseline health until 2 months prior when she first noticed episodic epigastric pain that was stabbing in nature. the patient was transferred to our institution for surgical evaluation based on an external department ct raising concern of possible diaphragmatic herniation versus fluid collection. re - scan of the abdomen confirmed a fluid collection of 8 - 10 cm in the extra - peritoneal space of the intra - abdominal wall, extending superiorly to the anterior mediastinum. subsequent laparoscopy revealed dense omental adhesions encapsulating an abscess cavity, which tracked into the inferior mediastinum. purulent material was evacuated, fluid was sampled for bacterial culture and a blake drain was placed. the patient was afebrile without leukocytosis throughout the hospital course and was discharged with clinic follow - up after 3 days of post - operative evaluation. at the one - month post - operative clinic visit, symptoms had resolved. development of mediastinal abscess is inclusive of multiple etiologies (1,2). given the location and encapsulation of the abscess in the presented case, descending mediastinitis from an oropharyngeal source seems unlikely. similarly, the patient remained afebrile without leukocytosis throughout the hospital course thereby precluding hematogenous spread. most recent procedural history includes an unremarkable esophagogastroduodenoscopy and mrcp 2 years prior to presentation. abscess fluid cultures from drainage revealed prevotella intermedia, haemophilus parainfluenzae and streptococcus spp. at the time of procedure, the patient was not known to be immunosuppressed or receiving long - term corticosteroids. taken together, these findings do not provide a definitive etiology for abscess development, though a role for recent instrumentation in the context of chronic inflammation and extensive abdominal surgical history may be suspected. the management of mediastinal abscess is three - fold : early diagnosis, surgical drainage and appropriate antibiotic therapy (5). location and extent of abscess and are both important factors in this determination. transcervical drainage has been recommended by prior studies for management of mediastinal abscess at or above the level of the carina whereas thoracotomy or sub - xipoid incision is recommended for extensive sub - carinal abscesses (6). recently, several investigators have shown video - assisted thoracoscopic surgery to be a successful alternative to open methods in mediastinitis management from multiple etiologies (3). moreover, the effectiveness of non - invasive techniques has been demonstrated in cases of sub - carinal abscess (7). such therapies may be preferable to more aggressive methods due to reduced risk of complication, less pain and more rapid recovery (5). successful employment of minimally invasive technique has also been shown recently in the management of boerhaave s syndrome (8,9). in this context, laparoscopy is utilized for primary repair of esophageal tear, gastrostomy, feeding jejunostomy, esophagomyotomy and harvesting of omentum (8). while local debridement of the posterior mediastinum is possible utilizing laparoscopic approach, thoracoscopy and vats are often additionally utilized due to delays in diagnosis, extensive pleural contamination and post - procedural complication (9). percutaneous drainage is an alternative to surgical management of mediastinal abscess in subacute or well - defined fluid collections (10). moreover, it represents the preferred course of treatment for intra - abdominal abscess and is a viable alternative to open drainage (10). despite these factors, laparoscopy was undertaken in the present case due to the need for adequate inspection of the abdomen to exclude sources of pathology causal to abscess. the treatment for mediastinal abscess remains controversial. while invasive technique may still represent the gold standard of therapy (6), we report the successful treatment of anterior inferior mediastinal abscess by laparoscopy in a 54 year - old female, without further complication. this method represents a novel approach to the management of abscess in this location without subsequent need for thoracoscopy or thoracotomy.	mediastinal abscess can be managed through a variety of both invasive and minimally invasive methods, as determined by location and spread of infection. thoracotomy remains the gold standard in treatment and is often employed. in the present case, a 54-year - old female with chronic pancreatitis presented with a 1-week history of severe epigastric pain, nausea and vomiting. on ct scan, a large abscess was discovered in the anterior inferior mediastinum. she was taken to the operating room and underwent laparoscopic drainage of the abscess. laparoscopy represents a novel approach in management of anterior inferior mediastinal abscess.
patient 1, the index case - patient, was a 66-year - old tunisian man with a 4-year history of untreated diabetes mellitus. during march 20april 27, 2013, he visited his daughter (patient 2) in qatar for 5 weeks (figure 1), 1 week of which they spent on a muslim pilgrimage to mecca, kingdom of saudi arabia. on april 18, results of a physical examination (including chest radiograph) for a visa extension in qatar were unremarkable. on the day of arrival back in tunisia (april 28), the patient experienced chills, followed by arthralgia, dry cough, and fever. the daughter reported that her father had had no direct contact with camels during his stay in qatar or saudi arabia. one of his children (patient 3, a nurse) gave him acetaminophen and aspirin for 3 days and then intravenously administered dexamethasone (4 mg) twice a day for 2 days. on may 6, patient 1 experienced worsened dyspnea and he sought care at the centre hospitalier - universitaire fattouma bourguiba hospital (monastir, tunisia) emergency department, where he received a fifth injection of dexamethasone. the patient was first admitted to the pulmonary ward, where he received amoxicillin - clavulanate (1 g) 3 times daily ; however, on may 8, respiratory failure and peripheral signs of shock necessitated admission to the intensive care unit (icu), where he was positioned prone and given noradrenalin infusion and mechanical ventilation with additional nitric oxide. clinical course of disease for patients with confirmed middle east respiratory syndrome coronavirus infection, tunisia, 2013. rh, regional hospital ; dh, district hospital ; rrt - pcr, real - time reverse transcription pcr. mini (< 10 ml fluid injected) bronchoalveolar lavage recovered a liquid of low cellularity ; cultures for bacteria and fungi were negative. the lavage fluid was then tested in the tunisia national reference laboratory (tnrl) for mers - cov by using real - time reverse transcription pcr (rrt - pcr) upe (region upstream of the e gene), open reading frame (orf) 1a, and orf1b assays. on may 10, patient 1 died of multiple organ failure. because nasopharyngeal swab samples from his 2 adult children were positive for mers - cov, the case of patient 1 was reported to the world health organization as probable mers - cov infection (4). on august 5, 2013, the centers for disease control and prevention (cdc) tested a serum sample collected from the index - patient on may 9. independent rrt - pcrs were positive for mers - cov (5) ; targets were upe (cycle threshold [ct ] 30.27) and nucleocapsid protein (n)2 (ct 30.46). sequences of the full n and spike (s) protein coding regions were submitted to genbank (accession nos. kf811035 and kf811036, respectively). nucleotide / predicted amino acid sequence identities with published mers - cov sequences for the n and s gene coding regions ranged from 99.2%100% to 99.0%100% and from 99.4%99.9% to 99.4%99.8%, respectively. phylogenetic relationships between this virus (designated tunisia - qatar_2013) and other published mers - cov sequences showed clustering with geographically diverse sequences from saudi arabia and the united arab emirates (figure 2). midpoint - rooted phylogenetic trees of the full - length nucleocapsid (n) (panel a) and spike (s) (panel b) open - reading frames (orfs) of isolates obtained from index case - patient with middle east respiratory syndrome coronavirus (mers - cov) infection, tunisia, 2013. serum and available nucleotide sequences from genbank and public health england (http://www.hpa.org.uk/webw/hpaweb&hpawebstandard/hpaweb_c/1317136246479) and the institut fr virologie (http://www.virology-bonn.de/index.php?id = 46). the estimated neighbor - joining trees were constructed from nucleotide alignments by using mega version 6.06 (http://www.megasoftware.net). numbers in parentheses denote the number of additional sequences from viruses isolated from humans that are identical to the listed sequence. bootstrap support values (1,000 replicates) 75% were plotted at the indicated internal branch nodes. patient 2 was the 30-year - old daughter who had accompanied the index case - patient to mecca. she remained in qatar until she attended her father s funeral in tunisia on may 11, 2013, when she reported sore throat, cough, and fever. on may 13, a nasopharyngeal swab sample collected on may 16 was positive for mers - cov by rrt - pcr performed at the tnrl : upe ct 27.5, orf1a ct 27.46, and orf1b ct 37.55. testing at cdc detected a ct of 28.46 for upe and negative results for n2 and n3 (5). patient 3 was the 34-year - old son of the index case - patient, a nurse in the icu where his father had been admitted. he had not traveled outside the country during the incubation period, and his first contact with the index case - patient was after his father s return to tunisia and illness onset. he cared for his father at home during the initial phase of illness and thereafter in the pulmonology department and icu. patient 3 reported a sore throat on the day after his father s funeral. a nasopharyngeal swab sample obtained on may 16 was positive for mers - cov by rrt - pcr performed at tnrl : upe ct 21.56, orf1a ct 27.6, and orf1b ct 31.39. at cdc, the nasopharyngeal swab sample was positive for mers - cov by 3 independent rrt - pcrs (5) : ct 21.67 for upe, 34.51 for n2, and 32.32 for n3. nasopharyngeal and/or throat swab samples were collected a mean of 5 weeks after contact from the other 2 (not ill) children of patient 1, his spouse, and the spouse of patient 3. health care workers who had been in contact with the index case - patient in the pulmonology ward (n = 2) or icu (n = 6) and who had reported sore throat, hyperthermia, and/or diarrhea (1 worker) were also investigated. all respiratory samples from contacts were negative for mers - cov by rrt - pcr. the fact that the diagnosis for the index case - patient was made by pcr of a serum sample collected 10 days after symptom onset and tested several weeks later highlights the value of testing serum samples for mers - cov rna. this finding also provides valuable information about viremia in mers cov infected patients, contributing to our understanding of the natural history of mers - cov infection and kinetics of virus shedding (7). given the incubation period of the disease (up to 15 days), the father most likely acquired his infection in qatar (8,9). patient 3, who had not traveled outside tunisia, could have been exposed during the 11 days he cared for his father at home and in the hospital. the history of patient 2 is less clear ; she might have acquired the virus from the same source as her father in qatar, or she might have been secondarily infected by contact with him before he left qatar, given that her illness began almost 12 days after her father s. patient 1 was severely ill at the time of icu admission ; in <3 days, his condition rapidly evolved to multiple organ system failure and death. although we can not account for the diabetes or corticosteroid contributions to his disease severity, we can speculate that they might have worsened his outcome. other mers cov patients who have died had concurrent conditions (2), and corticosteroids are thought to worsen the outcomes for patients with influenza a(h1n1) virus infection (10). the contact tracing results shed light on the potential for person - to - person transmissibility of mers - cov. only 2 family members who had been in close and prolonged contact with the index case - patient became infected. infection was not acquired by the case - patient s wife, his 2 children who did not live with him, or the icu workers who had short - term close contact with him. however, these results should be interpreted cautiously because only nasopharyngeal swab samples obtained 5 weeks after contact with the index case - patient were tested. in addition, serologic testing, which was not performed in the present investigation, could have shed more light on person - to - person mers - cov transmissibility. chest radiograph of the middle east respiratory syndrome index case - patient, taken at the time of admission to intensive care unit (may 8, 2013), tunisia.	in 2013 in tunisia, 3 persons in 1 family were infected with middle east respiratory syndrome coronavirus (mers - cov). the index case - patient s respiratory tract samples were negative for mers - cov by reverse transcription pcr, but diagnosis was retrospectively confirmed by pcr of serum. sequences clustered with those from saudi arabia and united arab emirates.
we investigated 383 consecutive classical ptmc patients from the department of pathology, kangbuk samsung hospital, sungkyunkwan university school of medicine (seoul, korea) from january 1, 2010 to december 31, 2011. the correlations between multifocality and clinicopathological characteristics including age, sex, tumor size, extrathyroidal extension, lymph node metastasis, ptnm stage, and us findings were evaluated by reviewing medical charts, pathology records, and glass slides and based on the seventh edition ajcc tnm classification system. patients had undergone either total thyroidectomy (n = 237) or lobectomy (including hemithyroidectomy) (n = 146) with lymph node dissection. the decisions to perform lymph node dissection and the extent of dissection were made by the surgeon based on ata management guidelines and various risk - evaluation systems. the mean age of patients was 45.4 10.4 years, the mean tumor size of the largest dominant tumor was 0.65 0.20 cm, and the sex ratio (male : female) was 1:3.85. by ajcc stage grouping, there were 295 cases in stage i, 0 cases in stage ii, 86 cases in stage iii, and two cases in stage iv. the study protocol was approved by the institutional review board (irb) of kangbuk samsung hospital (irb no. kbc12202), who confirmed informed consents. sections were cut to a thickness of 0.2 cm for postoperative pathological examination. all tumors detected during inspection were histologically examined. for evaluation of extrathyroidal extension, peritumoral parenchyma with thyroidal in addition, one section with normal - appearing parenchyma was included from each lobe. mptmcs were defined as tumors with an intertumoral distance greater than 0.5 cm in the ipsilateral lobe or contralateral lobe of the primarytumor. the number of tumors ranged from two to six (mean, 2.36 0.70), and we designated ptmcs other than the largest tumor as supplemental tumors. the first group included suspicious mptmcs, the second group included uptmcs accompanied by benign or indeterminate nodules, and the third group included suspicious uptmcs. within the second and third groups, incidental mptmcs were defined as those discovered on pathological examination, which were not detected on preoperative us. us evaluation was performed by radiologists at our institution, and results were reported according to standard criteria. relevant articles were obtained by searching pubmed and medline databases up to january 15, 2015. searches were performed using the keywords papillary thyroid carcinoma and multifocal. search results were then scanned according to the following inclusion and exclusion criteria : (1) ptc investigated in human tissue, (2) available information regarding tumor recurrence in uptmc and mptmc, (3) case reports or non - original articles were excluded, and (4) non - english language publications were excluded. the following data were extracted from each of the eligible studies : the first author s name, year of publication, number of patients analyzed, and number of patients with tumor recurrence. the significance of tumor multifocality and correlations with clinicopathological parameters were determined by either chi - squre test or the fisher exact test (two - sided). the relationship between tumor multifocality and tumor size was analyzed using a two - tailed student s t test. linear regression analysis was conducted to investigate correlations between primary tumor size and tumor multifocality, number of tumor, and supplemental tumor size. in addition, multivariate logistic regression analysis was performed to identify the most influential variables associated with tumor multifocality. to perform the meta - analysis, comprehensive meta - analysis ver. odds ratio (or) with a 95% confidence interval (ci) was calculated by fixed - effect and random - effect models and used to evaluate the correlation between tumor multifocality and recurrence. heterogeneity between studies was evaluated with the q test, i, and p - values. we investigated 383 consecutive classical ptmc patients from the department of pathology, kangbuk samsung hospital, sungkyunkwan university school of medicine (seoul, korea) from january 1, 2010 to december 31, 2011. the correlations between multifocality and clinicopathological characteristics including age, sex, tumor size, extrathyroidal extension, lymph node metastasis, ptnm stage, and us findings were evaluated by reviewing medical charts, pathology records, and glass slides and based on the seventh edition ajcc tnm classification system. patients had undergone either total thyroidectomy (n = 237) or lobectomy (including hemithyroidectomy) (n = 146) with lymph node dissection. the decisions to perform lymph node dissection and the extent of dissection were made by the surgeon based on ata management guidelines and various risk - evaluation systems. the mean age of patients was 45.4 10.4 years, the mean tumor size of the largest dominant tumor was 0.65 0.20 cm, and the sex ratio (male : female) was 1:3.85. by ajcc stage grouping, there were 295 cases in stage i, 0 cases in stage ii, 86 cases in stage iii, and two cases in stage iv. the study protocol was approved by the institutional review board (irb) of kangbuk samsung hospital (irb no. sections were cut to a thickness of 0.2 cm for postoperative pathological examination. all tumors detected during inspection in addition, one section with normal - appearing parenchyma was included from each lobe. mptmcs were defined as tumors with an intertumoral distance greater than 0.5 cm in the ipsilateral lobe or contralateral lobe of the primarytumor. the number of tumors ranged from two to six (mean, 2.36 0.70), and we designated ptmcs other than the largest tumor as supplemental tumors. the first group included suspicious mptmcs, the second group included uptmcs accompanied by benign or indeterminate nodules, and the third group included suspicious uptmcs. within the second and third groups, incidental mptmcs were defined as those discovered on pathological examination, which were not detected on preoperative us. us evaluation was performed by radiologists at our institution, and results were reported according to standard criteria. relevant articles were obtained by searching pubmed and medline databases up to january 15, 2015. searches were performed using the keywords papillary thyroid carcinoma and multifocal. search results were then scanned according to the following inclusion and exclusion criteria : (1) ptc investigated in human tissue, (2) available information regarding tumor recurrence in uptmc and mptmc, (3) case reports or non - original articles were excluded, and (4) non - english language publications were excluded. the following data were extracted from each of the eligible studies : the first author s name, year of publication, number of patients analyzed, and number of patients with tumor recurrence. statistical analyses were conducted using spss ver. 18.0 software (spss inc., chicago, il, usa). the significance of tumor multifocality and correlations with clinicopathological parameters were determined by either chi - squre test or the fisher exact test (two - sided). the relationship between tumor multifocality and tumor size was analyzed using a two - tailed student s t test. linear regression analysis was conducted to investigate correlations between primary tumor size and tumor multifocality, number of tumor, and supplemental tumor size. in addition, multivariate logistic regression analysis was performed to identify the most influential variables associated with tumor multifocality. to perform the meta - analysis, comprehensive meta - analysis ver. odds ratio (or) with a 95% confidence interval (ci) was calculated by fixed - effect and random - effect models and used to evaluate the correlation between tumor multifocality and recurrence. heterogeneity between studies was evaluated with the q test, i, and p - values. we initially investigated the correlations between tumor multifocality and clinicopathological parameters in 383 resected classical ptmcs. tumor multifocality was noted in 103 of 383 ptmcs (26.9%), and nodal metastasis occurred at significantly higher rates in mptmcs than in uptmcs (p=.003). patients who underwent total thyroidectomy showed a higher incidence of tumor multifocality than patients who underwent lobectomy (p 1 cm) and mptmc. our results and earlier studies suggest that tumor multifocality in ptmcs, regardless of tumor size, is a useful predictive factor of aggressive tumor behavior, such as lymph node metastasis and tumor recurrence. it might be appropriate to manage patients with mptmc differently than patients with uptmc having no unfavorable factors. incidental ptmc is defined as ptmc incidentally discovered and confirmed by gross and/or microscopic examination from surgical specimens resected for the evaluation of other disease entities. however, no clear definition for incidental mptmc has been previously provided. in this study, we defined incidental mptmc as supplemental tumors not detected on preoperative us that were discovered on pathological examination. although these supplemental tumors are important for diagnosis of multifocality, the characteristics of supplemental tumors have not been previously described. in the present study, however, the lower limit of tumor size detectable on us is unclear, and more controlled and careful histological examination for entirely submitted cases is needed to identify tumor multifocality. the mean size of incidentally discovered supplemental tumors was 0.27 0.15 cm, which was significantly smaller than non - incidental tumors (p<.001). 2). the smaller size explains the lack of detection during preoperative us. however, there was no difference in nodal metastasis rate between incidental and non - incidental mptmc. this finding suggests that tumor multifocality itself, rather than the manner of detection, is related to lymph node metastasis. in addition, the detection rate of incidental mptmc might influence the rate of mptmc. if these supplemental tumors are missed in pathological examination, cases are diagnosed as uptmc and not mptmc. therefore, detailed examination for multiple tumors using preoperative us study and adequate postoperative pathological examination is essential in the diagnosis of ptmc. we previously reported that evaluation of tumor multifocality based on total surface area is useful to distinguish aggressive mptmc from less aggressive mptmc and uptmc. on that basis, we concluded that careful detection of multifocal tumors is important despite discrepant reporting on multifocality. the discrepancies largely seem to result from different manners of pathological examination and the number of sections analyzed. although radiological tools have improved, tumors smaller than 0.3 cm are not easily detected via radiological examination. in the present study, only four of 383 ptmcs were smaller than 0.3 cm. among them, two cases were 0.2 cm on histological examination, after appearing larger than 0.3 cm on preoperative us. suspicious nodules on preoperative us in the remaining two cases were confirmed as benign nodules on histological examination. another two incidentally detected nodules (0.2 cm and 0.1 cm) were diagnosed as ptmc. in addition, it is difficult to confirm ptmc in preoperative fine - needle aspiration for tumors smaller than 0.5 cm, which require thin sections in postoperative gross examination for non - detected small nodules. because it is difficult to cut 0.2-cm - thick slices of specimens prior to fixation, thin sectioning after proper fixation is required for assessment of tumor multifocality. in conclusion, this study demonstrates that tumor multifocality in ptmc is significantly correlated with lymph node metastasis. our meta - analysis revealed a positive correlation between tumor multifocality and tumor recurrence in ptmc. therefore, careful attention should be paid to detection of mptmc, which behaves differently from uptmc, on preoperative and postoperative examination.	background : the clinicopathological characteristics and conclusive treatment modality for multifocal papillary thyroid microcarcinoma (mptmc) have not been fully established.methods:a retrospective study, systematic review, and meta - analysis were conducted to elucidate the clinicopathological significance of mptmc. we investigated the multiplicity of 383 classical papillary thyroid microcarcinomas (ptmcs) and the clinicopathological significance of incidental mptmcs. correlation between tumor recurrence and multifocality in ptmcs was evaluated through a systematic review and meta-analysis.results:tumor multifocality was identified in 103 of 383 ptmcs (26.9%). on linear regression analysis, primary tumor diameter was significantly correlated with tumor number (r2=0.014, p=.021) and supplemental tumor diameter (r2=0.117, p=.023). of 103 mptmcs, 61 (59.2%) were non - incidental, with tumor detected on preoperative ultrasonography, and 42 (40.8%) were diagnosed (incidental mptmcs) on pathological examination. lymph node metastasis and higher tumor stage were significantly correlated with tumor multifocality. however, there was no difference in nodal metastasis or tumor stage between incidental and non - incidental mptmcs. on meta - analysis, tumor multifocality was significantly correlated with tumor recurrence in ptmcs (odds ratio, 2.002 ; 95% confidence interval, 1.475 to 2.719, p<.001).conclusions : our results show that tumor multifocality in ptmc, regardless of manner of detection, is significantly correlated with aggressive tumor behavior.
focal osteoporotic bone marrow defect (fobmd) is a radiolucent area corresponding to the uncommon presence of hematopoietic tissue found in the jaws, usually at former extraction sites1,2. radiographi - cally, it is localized, poorly demarcated radiolucency that varies in size, trabeculae, and border definition2 - 4. since fobmd is occasionally included in the differential diagnosis of radiolucent lesions of the jaws, knowledge of the radiographic, clinical, and histopathological characteristics in association with accurate examination are mandatory to distinguish it from other most common intrabony defect lesions such as odontogenic cysts or traumatic bone cyst (tbc), aneurismal bone cyst, central giant cell granuloma, tumors, and primary or metastatic malignancies2. placement of dental implant has become a quite predictable procedure ; nonetheless, there are risks associated with the surgical phase. well - known intraoperative complications and accidents related to surgery include nerve damage, thermal damage, hemorrhage, damage of adjacent tooth, lack of primary stability, and displacement of implants. the displacement of implants occurs intraoperatively or within a short period because of insufficient surgical technique or anatomical variances of the jaws. deficiency of initial stability of implants can result from the low density of trabecular bone, thinness of the cortical bone, and osteopenia or osteoporosis ; problems related to the use of surgical technique by an inexperienced operator, such as inadequate planning, overworking of the implant drilling, mishandling, and incorrect manipulation, have also been suggested as possible factors5,6. the investigators concluded that the medullar component in the posterior mandible may be similar to that found in the maxilla, and that it could facilitate the displacement of dental implants during surgery5. in this report of 3 rare cases, the displacement of implants into the mandible corpus and its management are presented together with a review of literature on fobmd. from november 2011 to november 2012, three women were referred to the department of oral and maxillofacial surgery, gachon university gil medical center for the management of displacement of dental implants into the posterior mandibular body during implant placement surgery. the lesions in our cases were asymptomatic, with no ex pansion of the cortical jawbone detected. a provisional diagnosis of odontogenic cyst or tumor was done, and fobmd was considered a differential diagnosis based on age, site, and clinical and radiographic findings. two patients were referred by private dental clinic, with the other patient referred by another department - dental center - of gachon university gil medical center. all three cases of implant removal surgery were performed by one operator. a 51-year - old woman with no medical history was referred by a private dental clinic for the management of displacement of implant fixture in the right mandible posterior area on november 7, 2011. two days earlier, an implant accidentally fell into the mandible body of the # 46 area during implant surgery. cone - beam computed tomography (cbct) imaging of the jaws showed 2.00.7 cm radiolucency with quite ill - defined and irregular borders located from the premolar area to the right midbody of the # 46, # 47 edentulous regions and indicated the implant location to be near the mandibular inferior border area.(fig. a) block anesthesia of the right mandibular nerve was administered together with infiltration anesthesia of the surrounding tissues. a mucoperiosteal flap was raised, and a periosteal elevator was placed under the periosteum. bone osteotomy (1.00.5 cm) was performed at the lateral corpus of the mandible with fissure bur and osteotomes. the implant (4.0 mm wide, 8.0 mm long) was exposed and carefully removed without any damage to the mandibular neurovascular bundle. she complained of hypoesthesia of the right lower lip and chin area during the 9 months ' follow - up.(fig. b) a 60-year - old woman with medical history of hypertension was referred by another department of our dental center for the management of displacement of implant fixture in the left mandible posterior area on july 16, 2012. thirty minutes earlier, one implant accidentally fell into the mandible body of the # 36 area during implant surgery.(figs. b) note, however, that the # 37 implant (4.5 mm wide, 8.0 mm long) placed simultaneously with # 36 had good initial stability. cbct imaging of the jaws showed radiolucency with quite ill - defined and irregular borders located from the premolar area to the left midbody of the # 36, # 37 edentulous regions and indicated the implant location to be near the mandibular canal.(fig. b) the removal of implant fixture followed the same procedure as that of case 1. c) three months later, replacement of the # 36 implant (4.0 mm wide, 10 mm long) was performed with autogenic and xenogenic bone graft.(fig. six months after the # 37 implant placement and three months after the # 36 implant replacement, secondary surgery was performed, followed by the delivery of prosthetics ; healing proceeded uneventfully without any postoperative complication. a 51-year - old woman with medical history of myoma uteri was referred by a private dental clinic for the management of displacement of implant fixture in the left mandible posterior area on november 28, 2012. three hours earlier, one implant accidentally fell into the mandible body of the # 36 area during implant surgery.(figs. b) the cbct image of the jaws showed radiolucency with quite ill - defined and irregular borders located from the premolar area to the left midbody of the # 36, # 37 edentulous regions and indicated the implant location to be near the mandibular inferior border. a 51-year - old woman with no medical history was referred by a private dental clinic for the management of displacement of implant fixture in the right mandible posterior area on november 7, 2011. two days earlier, an implant accidentally fell into the mandible body of the # 46 area during implant surgery. cone - beam computed tomography (cbct) imaging of the jaws showed 2.00.7 cm radiolucency with quite ill - defined and irregular borders located from the premolar area to the right midbody of the # 46, # 47 edentulous regions and indicated the implant location to be near the mandibular inferior border area.(fig. a) block anesthesia of the right mandibular nerve was administered together with infiltration anesthesia of the surrounding tissues. a mucoperiosteal flap was raised, and a periosteal elevator was placed under the periosteum. bone osteotomy (1.00.5 cm) was performed at the lateral corpus of the mandible with fissure bur and osteotomes. the implant (4.0 mm wide, 8.0 mm long) was exposed and carefully removed without any damage to the mandibular neurovascular bundle. she complained of hypoesthesia of the right lower lip and chin area during the 9 months ' follow - up.(fig. a 60-year - old woman with medical history of hypertension was referred by another department of our dental center for the management of displacement of implant fixture in the left mandible posterior area on july 16, 2012. thirty minutes earlier, one implant accidentally fell into the mandible body of the # 36 area during implant surgery.(figs. b) note, however, that the # 37 implant (4.5 mm wide, 8.0 mm long) placed simultaneously with # 36 had good initial stability. cbct imaging of the jaws showed radiolucency with quite ill - defined and irregular borders located from the premolar area to the left midbody of the # 36, # 37 edentulous regions and indicated the implant location to be near the mandibular canal.(fig. b) the removal of implant fixture followed the same procedure as that of case 1. c) three months later, replacement of the # 36 implant (4.0 mm wide, 10 mm long) was performed with autogenic and xenogenic bone graft.(fig. six months after the # 37 implant placement and three months after the # 36 implant replacement, secondary surgery was performed, followed by the delivery of prosthetics ; healing proceeded uneventfully without any postoperative complication. a 51-year - old woman with medical history of myoma uteri was referred by a private dental clinic for the management of displacement of implant fixture in the left mandible posterior area on november 28, 2012. three hours earlier, one implant accidentally fell into the mandible body of the # 36 area during implant surgery.(figs. b) the cbct image of the jaws showed radiolucency with quite ill - defined and irregular borders located from the premolar area to the left midbody of the # 36, # 37 edentulous regions and indicated the implant location to be near the mandibular inferior border. fobmd of the jaws has been reported as an unusual radiolucency often detected fortuitously in the posterior mandible of a middle - aged woman2,7 - 9. radiographically, this radiolucency varies in size from several millimeters to centimeters in diameter, and the shape and borders are ill - defined with fine central trabeculation1,3,9. various theories include the following : aberrant bone regeneration after tooth extraction ; persistence of fetal marrow, and ; marrow hyperplasia in response to increased demand for erythrocytes9. some osteoporotic bone marrow defects (obmds) are multifocal ; others are bilateral, and few of them are symptomatic1 - 4. no clinical and radiological expansion of the jaw is noted9. according to the study conducted by shankland and bouquot4, the bilateral occurrence of obmd within the jaws affected 3% of patients. previous obmds documented in english literature frequently indicate that the radiographic appearance may be confused with other intraosseous patho - logic conditions1 - 4,7,8. mandibular cysts or tumors often have bilateral radiolucency with indistinct margins. based on age, site, and clinical and radiographic findings microscopically, the existence of hematopoietic marrow composed of erythroid, monocytic, granulocytic, and lympho - cytic series as well as megakaryocytes associated with fatty marrow is required for the diagnosis of this lesion4,7 - 9. fobmd was considered a differential diagnosis based on age, site, and clinical and radiographic findings without biopsy for all our cases. radiologically, cortical bone density and thickness were at normal levels ; the endosteal cortical margin was even, and there were no cystic epithelial walls.(figs. 1 - 3) the cancellous bone marrow was almost absent, and the pattern with hematopoietic marrow tissue was filled roughly. our cbct imaging showed the weakness of bone marrow density near the mandibular canal of the molar area.(figs. a) thus, we diagnosed our 3 patients as fobmd based on age, site, and clinical and radiographic findings. tbcs10 of the jaws are asymptomatic cystic lesions detected incidentally on radiographs or during surgery. the lesion is mainly diagnosed among young patients most frequently during the second decade of life, with men affected somewhat more frequently. expansion of the cortical plate of the jaw bone is often noted, usually buccally, resulting in intraoral and extra - oral swelling. tbc, or extension of the empty bone cavity, will stop expanding once the cortical bone has been reached. a characteristic of tbc is the " scalloping effect " when extending between the roots of the vital teeth10. preoperatively, we could not detect any radiolucency to confirm diagnosis of tbc radiographically. a histological examination of tbc revealed normal - looking bone spicules with parts of vascular connective tissue. nonetheless, fobmd frequently occurs in an edentulous region of a middle - aged woman, and a radiolucent appearance is usually not observed on the preoperative panoramic radiograph2. based on the panoramic view of our patients, we could not detect any cystic lesion on the mandible (figs. b), finding that the cancellous bone marrow was almost nonexistent based on cbct imaging.(figs. a) whether the lesion is tbc or obmd, the radiolucent appearance of a possible lesion was more likely not observed on the preoperative panoramic radiograph5. bender and seltzer11 showed that the removal of cancellous bone from the posterior region of cadaver mandibles did not alter the radiographic appearance of the trabeculae. it was only when the endosteal surface of the cortical bone was removed was the radiographic image altered. vlasiadis.12 demonstrated that later postmenopausal date and number of tooth loss were related to higher degree of osteoporosis. studies of subjects with osteoporosis have shown no differences in the survival of implants compared with healthy subjects ; a review of the published data did not find evidence that osteoporosis could be a risk factor for osseointegrated dental implants13. the # 37 (4.5 mm wide, 8.0 mm long) implant placed simultaneously with # 36 (4.5 mm wide, 10.0 mm long) had good initial stability.(figs. c) note, however, that only the # 36 implant fell down into the mandible body.(fig. 2b) three months later, the replacement of the # 36 implant (4.0 mm wide, 10 mm long) was performed with autogenic and xenogenic bone graft. six months after the # 37 implant placement and three months after the # 36 implant replacement (fig. 2e), delivery of prosthetics was carried out, and healing proceeded uneventfully without any postoperative complication. the displacement of an implant will occur intraoperatively or within a short period due to the poor use of surgical technique by an inexperienced operator or anatomical variances. failure of initial implant stability can result from low trabecular bone density, decreased thickness of the cortical bone, anatomical variances, and osteopenia or osteoporosis ; problems related to the use of surgical technique by an inexperienced operator, such as inadequate planning, overworking of the implant drilling, mishandling, and incorrect manipulation, have also been suggested as possible factors5,6. according to theisen.,14 during the tightening of the healing screw, the implant was displaced inferiorly into the preparation, and attempts to remove the screw and retrieve the implant body resulted in the further inferior displacement of the implant body. thus, we assumed that the nerve had been damaged when the implant fell down into the mandible. although dental implant surgery is considered a simple and predictable procedure, unforeseen complications and rare accidents could occur especially in the posterior segments with lower trabecular bone density than in anterior segments. as previously reported by theisen.,14 when the location of the inferior alveolar canal could not be delineated on a panoramic radiograph, large medullary components could be detected on the ct section. thus, additional radiographic evaluations such as preoperative ct may be necessary for patients whose molar teeth were extracted much earlier in their life, particularly among postmenopausal women. we recommend securing the healing screw in the implant body prior to the placement of the implants. even if implants fall down into fobmd, removal of the implants was performed carefully without any damage to nerve and vessel bundles, and replacement of implants was done as a common method of immediate placement after teeth extraction. nonetheless, additional studies with more cases and long - term follow - up should be conducted.	focal osteoporotic bone marrow defect (fobmd) is a radiolucent area corresponding to the presence of hematopoietic tissue rarely found in the jaws. fobmd is most commonly located in the mandibular edentulous posterior area of a middle - aged female. from november 2011 to november 2012, we experienced three cases involving removal of implants that had accidentally fallen into the fobmd area. all patients happened to be female, with a mean age of 54 years (range : 51 - 60 years). one case involved hypoesthesia of the lower lip and chin, while two cases healed without any complication. displacement of an implant into the fobmd area is an unusual event, which occurs rarely during placement of a dental fixture. the purpose of this study was to report on three cases of fobmd and to provide a review of related literature.
with a prevalence of 1 in 5,000, fragile x syndrome is the most common form of hereditary mental retardation. patients suffer from mild to severe mental retardation, mild facial dismorphologies and macroorchidism (enlarged testicles). in addition, behavioral problems like autism and hyperactivity may be associated with the syndrome. in 1991, defects in the fmr1 gene (fragile x mental retardation gene 1) were identified as the cause of the syndrome. an expansion of a cgg repeat in the 5 ' untranslated region (utr) of the fmr1 gene causes transcriptional silencing, resulting in absence of the fragile x protein, fmrp. it was soon discovered that fmrp can bind rna and plays a role in the transport of mrnas from the nucleus to ribosomes. several recent studies have provided new insights into the motifs responsible for rna binding and revealed a list of mrna targets bound by fmrp. the fmr1 gene is ubiquitously expressed, with particularly high expression in neurons but barely detectable expression in glia. the fmr1 gene product can be up to 631 amino acids long and has five functional domains : two types of rna - binding structures, namely two kh domains (kh denotes heterogeneous nuclear ribonucleoprotein k homology) and an rgg box (containing repeats of an arg - gly - gly motif - rgg, in the single - letter amino - acid code) ; a nuclear localization signal (nls) ; a nuclear export signal (nes) ; and two coiled - coil domains. fmrp is predominantly cytoplasmic but shuttles between the cytoplasm and nucleus, a process mediated by the nuclear import and nuclear export signals. fmrp can bind 4% of all brain mrnas, including its own mrna, presumably with the aid of its rna - binding domains. in the nucleus, the fmrp protein forms messenger ribonucleoprotein (mrnp) precursor particles along with other proteins, including nucleolin (which is known to shuttle between the nucleus and cytoplasm) and the fragile x - related proteins fxr1p and fxr2p. the fxr1 and fxr2 genes are autosomal paralogs of fmr1, and both gene products have high overall sequence similarity to fmrp, especially within the functional domains. the fmrp - mrnp particles are transported out of the nucleus into the cytoplasm and to actively translating ribosomes. it has been hypothesized that, in neurons, fmrp transports specific mrnas along the dendrites towards actively translating ribosomes near the synapses. which neuronal rna targets are bound and why absence of the fmrp protein causes mental retardation remained unknown until recently, however. initially, the kh domains were thought to be crucial for the function of fmrp, because an exceptional patient had been described with a point mutation (ile304 asn) in the second kh domain. this patient had a more severe phenotype than any other patient lacking fmrp, including profound mental retardation and excessive macroorchidism. experiments in vitro demonstrated that the fmrp kh domains interact with high affinity with various mrnas, including those of fxr1 and fxr2. fmrp is able to suppress translation of mrnas in vitro, presumably through its kh domains. mutation of fmrp at position 304 abrogates its association with ribosomes and the mutated protein does not suppress translation. it came as a surprise, therefore, that a series of recent papers revealed that the rgg box, not the kh domains, is responsible for the binding of a specific subset of mrnas to fmrp. rgg boxes are usually found within proteins in combination with other rna - binding domains. darnell. identified which rnas were preferentially bound by fmrp, by screening a pool of 96-mer rnas containing 52 bases of random sequence. after multiple rounds of selection a limited set of rnas was identified with a common motif of [dwggn(0 - 2)]4 (where d is any nucleotide except c, w is u or a, and n is any nucleotide). at the same time, schaeffer. determined the minimal site within the fmr1 mrna necessary for fmrp binding. they concluded that a 100-nucleotide fragment in the 3 ' coding region of the fmr1 gene is responsible for the specific binding of fmrp with its own mrna, in contrast to earlier experiments in which fmrp was reported to bind to the 3 ' utr of the fmr1 mrna. both the random rnas with the repeated dwgg motif that are preferentially bound by fmrp and the 100 nucleotides at the 3 ' end of the fmr1 gene necessary for fmrp binding have the ability to form planar structures, so - called g quartets. g quartets are hydrogen - bonded structures formed from four guanosine residues in a square - planar array stabilized preferentially by k (figure 1). g - quartet structures have been observed in telomeric sequences and stabilize chromosome ends ; they dimerize the rna genome of the human immunodeficiency virus (hiv) and are also involved in the site - specific recombination required for immunoglobulin maturation. the presence of a stable g - quartet structure near the 3 ' end of the coding sequence of fmr1 was investigated by reverse transcriptase (rt)-coupled pcr. adding k to the rt buffer caused an abrupt pause in reverse transcription before and within the purine - rich region and this block was resolved when na was added. binding of fmrp to a g - quartet - containing sequence was strongly reduced when na and li cations were used compared with k. surprisingly, the mrna - binding site of fmrp that binds the g quartet overlaps with the rgg box, which is responsible for the binding of specific mrnas. when translated, therefore, the product of the g - quartet sequence, which is the rgg box of fmrp, is able to bind to its own mrna. g - quartet - containing rnas were also shown to be targets of fmrp in vivo. using microarrays, brown. identified from total murine brain mrnas associated with mrnps that interact with actively translating ribosomes. fmrp - rnp particles were isolated from wild - type and knockout mouse brains and hybridized to microarrays containing 25,181 rnas with sequences taken from the unigene database of the national center for biotechnology information and 19,021 expressed sequence tags (ests) from the tigr gene indices database. after rigorous selection, brown. obtained 432 different mrnas specifically associated with the mouse fmrp - rnp complexes. subsequently, comparing polyribosome fractions from normal human lymphoblastoid cells with those from cells isolated from fragile x patients on a microarray containing 35,000 human genes or ests, they identified 251 human mrnas that show abnormal polyribosome profiles in fragile x patients. the set of genes whose mrnas were found in the mrnp complexes in mrnas from total mouse brain were compared with the set of human genes that gave an abnormal polyribosomal profile in the absence of the fragile x protein, using human orthologs of the mouse genes and the murine orthologs of the human genes, respectively. of the genes found to be associated with the murine fmrp - rnp complexes, thus, fmrp within cells binds preferentially to mrnas that contain g - quartet structures. identifying the mrna targets of fmrp in the cell may help us understand the clinical consequences of the absence of fmrp. only around 0.01% of the sequences present in the unigene database are predicted to have a g - quartet structure ; thus only a minor fraction of all proteins may be transported through the cell by fmrp. many of the predicted g - quartet - containing genes identified have a role in synaptogenesis, a process that is impaired in fragile x syndrome. the rna that was the most enriched in the fmrp - rnp particles isolated from normal mouse brain, when compared with particles from fragile x mouse brain, codes for a guanine - nucleotide exchange factor related to the yeast secretion protein sec-7 and is predicted to have a g - quartet structure. several other members of gtpase pathways, including genes encoding oligophrenin-1 (a rho - gtpase - activating protein), gdi (a guanine nucleotide dissociation inhibitor) and the guanine - nucleotide exchange factor arhger6 have been found mutated in patients with non - specific mental retardation. examples of genes that were identified in the abnormal polyribosomal fractions in fragile x patients and that were present in the murine fmrp - rnp complexes are those encoding munc13, a phorbol ester receptor involved in the maturation of golgi vesicles and vesicle transport in neurons, and sapap4, which is involved in neuronal cell signaling. from the list of 14 mrnas containing a g quartet that bind fmrp, as identified by darnell., including those encoding nap22, which is present in axon terminals and dendritic spines and plays a role in maturation or maintenance of synapses, and the microtubule - associated protein map1b, which is involved in transport within neurons and is the human ortholog of the drosophila futsch gene. drosophila has only one homolog (dfxr) of the fragile x gene family (consisting of fmr1, fxr1 and fxr2). dfxr acts as a translational represser of futsch, thereby regulating the synaptic microtubule cytoskeleton. flies with dfxr mutations display, among other symptoms, enlarged synaptic terminals, whereas double mutants deficient in both dfxr and futsch have a normal phenotype, demonstrating the importance of the dfxr - futsch interaction in flies. it remains to be resolved whether each of the identified fmr1 target genes is responsible for a small effect, or whether a few genes will turn out to be responsible for the majority of clinical symptoms of fragile x patients. the identification of the rgg box as the site of rna binding and the role of secondary rna structures could perhaps explain some of the differences in function between fmr1 and its autosomal paralogs fxr1 and fxr2. we compared the rgg boxes of fmrp, fxr1p and fxr2p, and found that, although overall the sequences are reasonably well conserved, fmrp has six repeated blocks of the consensus motif rgg, whereas fxr1p and fxr2p each have only a single rgg motif (figure 2a). fxr1p and fxr2p may have one additional rgg motif each when not aligned with fmrp (boxed sequences in figure 2a) and fxr1p has two more motifs downstream of the rgg - box consensus sequence. although the rgg - box sequence is poorly defined and the number of rgg - repeats necessary for rna - protein interaction is not known, it seems unlikely that the rgg boxes of fmrp, fxr1p and fxr2p have identical rna - binding capacities. we also compared the g - quartet sequences of fmr1, fxr1 and fxr2 mrnas (figure 2b, 2c). of the 31 guanines conserved between fmr1 genes in human, mouse and rat rna, only 20 were detectable in fxr1 and 15 in fxr2. in fxr2 in particular, however, extra guanines at other positions may compensate for the absence of these consensus guanines. it therefore remains to be verified whether fxr1 and fxr2 mrnas are able to form a g - quartet structure similar to that of fmr1 mrna. given the importance for the function of fmrp of interactions between the rgg box and the g - quartet structure, the differences in rgg - box sequence between fmrp, fxr1p and fxr2p could perhaps explain why fxr1p and fxr2p, which, unlike fmrp, are present in the mrnps of fragile x patients, can not fully compensate for the absence of the fragile x protein fmrp. further characterization of the mrna targets of the fragile x protein and possibly the identification of the mrnas bound by fxr1p and fxr2p will aid our understanding of the complex mechanisms underlying fragile x syndrome. financial support for the fragile x research in antwerp was obtained through grants of the belgian national fund for scientific research - flanders (fwo), an interuniversity attaction pole (iuap - v) and the fragile x research foundation (fraxa). the sequence of sc1 rna is used to illustrate the three - dimensional structure of a gquartet ; sc1 is one of a series of six random rnas specifically bound by fmrp. the g - quartet structure is stabilized by cations such as k. comparison of consensus sequences in the human members of the fragile x gene family. (a) protein sequence alignment of the rgg boxes of fmrp, fxr1p and fxr2p. the consensus sequence is indicated in dark blue and differences are highlighted in light blue. we used the rgg - box consensus motif determined by burd and dreyfuss and the alignment of the three proteins generated by zhang.. (b, c) transcript sequence comparison of the purine - rich region between fmr1 and the autosomal paralogs fxr1 and fxr2. guanines conserved between human, mouse and rat fmr1 genes are indicated in dark blue ; some of these are not conserved in fxr1 and fxr2 (highlighted in light blue).	ten years after the identification of the gene responsible for fragile x syndrome, recent studies have revealed a list of mrnas bound by the fragile x gene product and have identified specific sequences required for the interaction between the fragile x protein and its targets. these results are a breakthrough in understanding why absence of the fragile x protein leads to mental retardation.
a 55-year - old female patient presented with a four - month history of a continuous burning sensation of the oral mucosa. the patient had witnessed an accident and suffered from major depression 3 years prior to visiting our clinic. the patient had been on the following medication for 3 years : monoprolol 40 mg, benzhexol (parkin) 2 mg, sodium valproate (valprol) 50 mg, haloperidol (trancodol) 5 mg and fluoxetin 100 mg. in the course of managing her depression, the dosage of the drug fluoxetin was briefly increased one year prior to her visit from 100 to 200 mg per day for a month. during this period, the patient experienced a mild burning sensation in her mouth, which returned to normal when the dosage was decreased. the dosage of the same drug was again increased from 100 to 200 mg six months prior to her visit, and the patient had been experiencing symptoms of burning mouth for the last four months. on examination, the patient was apparently healthy, conscious, co - operative, well - oriented, well - built and nourished. intraoral examination revealed that the oral mucosa appeared normal and healthy ; there were no mucosal lesions to explain the pain. the intensity of the burning sensation was rated on a visual analogue scale, with a score of 8. the patient was referred to her psychiatrist with a request to alter or reduce the dosage of the drug fluoxetin. the drug was discontinued, and her depression was managed instead with the drug colostrinin (cognate). following this change, the patient 's glossodynia disappeared completely within one month. based on her history, clinical features and response after drug cessation, a final diagnosis of selective serotonin reuptake inhibiter (ssri) antidepressant - induced bms burning mouth syndrome is defined by the international association for the study of pain as burning pain in the tongue or other oral mucous membrane associated with normal signs and laboratory findings, lasting for at least 4 to 6 months. one of the most commonly affected sites is the tip and anterior two thirds of the tongue, which are the areas of greatest movement in the oral cavity. the prevalence of burning mouth symptoms reported by international studies ranges from 0.6% to 15%. patients are around 60 years of age, and females are more commonly affected than males. lamey and lewis have suggested classifying bms into three patterns : types 1, 2, and 3. type 1 includes symptom - free waking, with sensations developing in the morning and progressively rising to a severe level by evening. type 2 involves continuous symptoms throughout the day ; whereas type 3 features intermittent symptom - free periods throughout the day. nonpsychologic causative factors, such as nutritional deficiencies, have been linked to type 1, chronic anxiety to type 2, and food allergies to type 3. another approach in classifying bms is to divide patients into either primary or secondary groups. while primary bms is idiopathic, secondary bms may be caused by local factors or systemic conditions. local factors associated with bms include mucosal diseases, fungal infections, bacterial invasion, allergies, temporomandibular joint dysfunctions, and salivary gland abnormalities. deficiency diseases, hormonal and immunologic disturbances, and pharmacotherapeutic side effects are included in the systemic conditions. secondary bms requires appropriate diagnosis and treatment of the underlying condition to manage symptoms. in primary bms, set out the guidelines for the management of primary bms based on a systematic review of randomized controlled trials, and recommended the following drugs : ssri, clonazepam, alphalipolic acid, amisulpride and cognitive behavioral therapy. these include the following : efavirenz, clonazepam, hormonal replacement therapies, fluoxetine, sertraline and a broad range of antihypertensive agents such as captopril, enalapril, and lisinopril. thirty - three percent of drug - induced bms have been seen to be dose - dependent phenomena, because the burning sensation appears after elevating the drug dose in search of increased therapeutic efficacy. levenson reported a case of a patient with major depression who developed bms after an increase in the dosage of ssri (fluoxetine, sertraline) and whose burning sensation was completely relieved after discontinuation of the medication. the pathogenesis of bms has been described in terms of local factors or conditions that alter the peripheral nerves, resulting in a decreased threshold of burning sensation and peripheral sensitization. if the peripheral sensitization continues for a long period of time, it adversely affects the central nervous system and results in neuroplasticity and central sensitization. the dose of her medication was increased to improve the efficacy of treatment for her depression. however, this resulted in the symptoms of a burning mouth. cessation of the drug is the best way to manage such patients after obtaining physician consultation for the medical management of depression.	burning mouth syndrome (bms) is defined as a chronic orofacial pain syndrome, without evidence of mucosal lesions and other clinical signs of disease or laboratory abnormalities. patients with bms complain of burning pain in the mouth, xerostomia and taste disturbances. it is more common among women and the median age of occurrence is about 60 years. bms may be primary or secondary to other diseases. the mainstay in the treatment of bms includes antidepressants, benzodiazepines, and anticonvulsants. a few cases of bms caused due to medication have been reported. the causative drugs include angiotensin - converting enzyme inhibitors, anticoagulants, antipsychotics, antiretrovirals, and benzodiazepines. this is a case report of a patient on antidepressants who developed symptoms of bms thereby causing a dilemma in management.
the international diabetes federation states that there are currently 316 million people with impaired glucose tolerance (igt), which will increase to 471 million people by 2035 (1). there is considerable debate as to whether these subjects should be considered to have a medical problem (2). however, in subjects with igt, the risk of developing type 2 diabetes ranges from 3.6 to 8.7% per year (3). furthermore, igt is also independently associated with the traditional microvascular complications of diabetes, including retinopathy, microalbuminuria, and neuropathy (4). there appears to be a good rationale for identifying subjects with igt, but there are limited data identifying subjects with igt who may be at greatest risk for developing diabetes and its complications. in relation to neuropathy, the specific focus of this study, the uk prospective diabetes study, showed that at the time of diagnosis of type 2 diabetes, 57% of patients already had neuropathy (5), and longitudinal data from the rochester cohort have shown that duration and severity of exposure to hyperglycemia are related to the severity of neuropathy (6). in a recent study of patients with 2 years of type 2 diabetes, there was also evidence of a significant neuropathy (7). however, there is debate as to whether igt is associated with neuropathy, with some studies showing evidence of neuropathy (812), while others do not (1315). we recently showed that a significant small - fiber neuropathy occurred in 40.5% of 37 subjects with igt (16). interestingly, a recent study evaluating electrochemical sweat conductance, a proxy for small - fiber neuropathy, has shown that healthy subjects with an abnormal response have a significantly increased odds ratio for the development of igt over 2 years (17). of relevance, lifestyle modification has been shown to improve intraepidermal nerve fiber density (ienfd) (11) and to improve after chemical axotomy (18). we previously showed an improvement in corneal nerve morphology after an improvement in glycemic control, lipids, and blood pressure (19) after simultaneous pancreas and kidney transplantation (20) and more recently in patients on continuous subcutaneous insulin infusion (21), suggesting a dynamic regenerative capacity of the small fibers in relation to metabolic change. we have undertaken a longitudinal study in subjects with igt to assess whether baseline and follow - up measures of neuropathy, particularly small - fiber neuropathy, relate to changes in glucose tolerance over 3 years. we assessed 30 subjects with igt based on an oral glucose tolerance test (ogtt) (2-h glucose 7.811.1 mmol) at central manchester and manchester children s university hospital and 17 health control subjects. exclusion criteria were any history of neuropathy due to a nondiabetic cause and any history of ocular pathology or systemic disease with corneal involvement. this study was approved by the central manchester research and ethics committee, and written informed consent was obtained from all subjects prior to participation. all study participants underwent assessment at baseline and 12, 24, and 36 months. participants underwent assessment of bmi, blood pressure, ogtt, hba1c, lipid profile (total cholesterol, ldl, hdl, and triglycerides), albumin - to - creatinine excretion ratio, and estimated glomerular filtration rate (egfr). symptoms of diabetic peripheral neuropathy were assessed using the neuropathy symptom profile (nsp). neurological deficits were evaluated using the simplified neuropathy disability score (nds), which is comprised of vibration perception, pinprick, temperature sensation, and presence or absence of ankle reflexes. vibration perception threshold (vpt) was tested using a neurothesiometer (horwell ; scientific laboratory supplies, wilfrod, nottingham, u.k.). cold (ct) and warm (wt) thresholds were established on the dorsolateral aspect of the left foot (s1) using the tsa - ii neurosensory analyzer (medoc, ramat - yishai, israel). electro - diagnostic studies were undertaken using a dantec keypoint system (dantec dynamics, bristol, u.k.) equipped with a disa temperature regulator to keep limb temperature constantly between 32 and 35c. sural sensory nerve amplitude (snap), sural sensory nerve conduction velocity (sncv), and peroneal motor nerve conduction velocity (pmncv) and amplitude (pmna) were assessed by a consultant neurophysiologist. a 3-mm punch skin biopsy was taken from the dorsum of the foot, 2 cm above the second metatarsal head under local anesthesia (1% lidocaine). sections (50 m) were stained using anti - human pgp 9.5 antibody (abcam, cambridge, u.k.), and nerve fibers were demonstrated using sg chromogen (vector laboratories, peterborough, u.k.). ienfd was quantified in accordance with established criteria and expressed as no. per millimeter (22). twenty z - stack images per case were taken using a zeiss axioimager m2 microscope, and mean dendritic length (mdl) (length of ienf from piercing the dermo - epidermal junction to its terminal in the epidermis) was manually traced and quantified using the imagepro 6.2 program (mediacybernetics, marlow, u.k.). patients underwent examination with the corneal confocal microscopy (ccm) (heidelberg retinal tomograph iii rostock cornea module ; heidelberg engineering, heidelberg, germany) as per our previously established protocol (23). six nonoverlapping images / patient from the center of the cornea were selected and quantified in a masked fashion. three corneal nerve parameters were quantified : corneal nerve fiber density (cnfd), the total number of major nerves per square millimeter of corneal tissue ; corneal nerve branch density (cnbd), the number of branches emanating from all major nerve trunks per square millimeter of corneal tissue ; and corneal nerve fiber length (cnfl), the total length of all nerve fibers and branches (millimeter per square millimeter) within the area of corneal tissue. analysis of the images was done using purposefully designed automated software called accmetrics (24). analysis was carried out on spss for mac (version 19.0 ; ibm corporation, armonk, ny). the data were tested for normality by using the shapiro wilk normality test and by visualizing the histogram and normal q - q plot. to assess within- and between - group differences, we used one - way anova (nonparametric kruskal - wallis). a significant p value was considered to be < 0.05 (post hoc tukey). we assessed 30 subjects with igt based on an oral glucose tolerance test (ogtt) (2-h glucose 7.811.1 mmol) at central manchester and manchester children s university hospital and 17 health control subjects. exclusion criteria were any history of neuropathy due to a nondiabetic cause and any history of ocular pathology or systemic disease with corneal involvement. this study was approved by the central manchester research and ethics committee, and written informed consent was obtained from all subjects prior to participation. all study participants underwent assessment at baseline and 12, 24, and 36 months. participants underwent assessment of bmi, blood pressure, ogtt, hba1c, lipid profile (total cholesterol, ldl, hdl, and triglycerides), albumin - to - creatinine excretion ratio, and estimated glomerular filtration rate (egfr). symptoms of diabetic peripheral neuropathy were assessed using the neuropathy symptom profile (nsp). neurological deficits were evaluated using the simplified neuropathy disability score (nds), which is comprised of vibration perception, pinprick, temperature sensation, and presence or absence of ankle reflexes. vibration perception threshold (vpt) was tested using a neurothesiometer (horwell ; scientific laboratory supplies, wilfrod, nottingham, u.k.). cold (ct) and warm (wt) thresholds were established on the dorsolateral aspect of the left foot (s1) using the tsa - ii neurosensory analyzer (medoc, ramat - yishai, israel). electro - diagnostic studies were undertaken using a dantec keypoint system (dantec dynamics, bristol, u.k.) equipped with a disa temperature regulator to keep limb temperature constantly between 32 and 35c. sural sensory nerve amplitude (snap), sural sensory nerve conduction velocity (sncv), and peroneal motor nerve conduction velocity (pmncv) and amplitude (pmna) were assessed by a consultant neurophysiologist. a 3-mm punch skin biopsy was taken from the dorsum of the foot, 2 cm above the second metatarsal head under local anesthesia (1% lidocaine). sections (50 m) were stained using anti - human pgp 9.5 antibody (abcam, cambridge, u.k.), and nerve fibers were demonstrated using sg chromogen (vector laboratories, peterborough, u.k.). twenty z - stack images per case were taken using a zeiss axioimager m2 microscope, and mean dendritic length (mdl) (length of ienf from piercing the dermo - epidermal junction to its terminal in the epidermis) was manually traced and quantified using the imagepro 6.2 program (mediacybernetics, marlow, u.k.). patients underwent examination with the corneal confocal microscopy (ccm) (heidelberg retinal tomograph iii rostock cornea module ; heidelberg engineering, heidelberg, germany) as per our previously established protocol (23). six nonoverlapping images / patient from the center of the cornea were selected and quantified in a masked fashion. three corneal nerve parameters were quantified : corneal nerve fiber density (cnfd), the total number of major nerves per square millimeter of corneal tissue ; corneal nerve branch density (cnbd), the number of branches emanating from all major nerve trunks per square millimeter of corneal tissue ; and corneal nerve fiber length (cnfl), the total length of all nerve fibers and branches (millimeter per square millimeter) within the area of corneal tissue. analysis of the images was done using purposefully designed automated software called accmetrics (24). analysis was carried out on spss for mac (version 19.0 ; ibm corporation, armonk, ny). the data were tested for normality by using the shapiro wilk normality test and by visualizing the histogram and normal q - q plot. to assess within- and between - group differences, we used one - way anova (nonparametric kruskal - wallis). a significant p value was considered to be < 0.05 (post hoc tukey). the control and igt subjects were age matched (62.3 1.8 vs. 60 2.1 years, p = 0.2). subjects with igt had a significantly higher hba1c (42.7 0.9 vs. 38.3 0.7 mmol / mol, p < 0.0001) and bmi (32.0 1.0 vs. 27.6 0.9 kg / m, p = 0.01) and lower hdl (1.2 0.1 vs. 1.7 0.1 mmol / l, p = 0.03) but comparable total cholesterol, triglycerides, egfr, and blood pressure compared with control subjects. clinical and metabolic parameters and neuropathy assessment in control subjects and subjects with igt at baseline and follow - up data are means sem. all symbols represent statistically significant differences. p value igt baseline vs. control ; igt baseline vs. 36 months. the igt group had a significantly higher nsp (3.4 0.7 vs. 0.3 0.1, p < 0.0001), nds (2.9 0.5 vs. 1.1 0.3, p = 0.03), and vpt (16.2 2.1 vs. 8.4 1.5, p = 0.02) compared with the control group. there was no significant difference in sncv and pmncv and amplitude between subjects with igt and the control subjects. there was no difference in ienfd ; however, mdl was significantly lower in the igt group compared with control subjects (25.1 1.6 vs. 63.0 4.2 m, p < 0.0001). cnfd (24.4 1.3 vs. 30.7 1.5 no./mm, p < 0.0001) and cnfl (15.3 0.6 vs. 20.4 3.14 mm / mm, p = 0.004) were significantly lower, but there was no difference in cnbd between subjects with igt and control subjects. there was no correlation between hdl and ccm measures at baseline (cnfl r = 0.2, p = 0.2 ; cnbd r = 0.2, p = 0.1 ; and cnfd r = 0.2, p = 0.3). control subjects showed no significant change in metabolic parameters or neuropathy measures over 3 years (repeat skin biopsy not performed in control subjects). in subjects with igt, bmi, hba1c, lipids, and blood pressure remained stable, and there was a small but significant reduction in egfr (79.1 3.0 vs. 74.3 4.3 ml / min/1.73 m, p = 0.03) over 3 years (table 1). there was no significant change in nsp, nds, vpt, or thermal thresholds. there was a significant reduction in sncv (49.7 1.4 vs. 46.6 1.3 m / s, p = 0.007) and ienfd (6.4 0.8 vs. 3.2 0.8 no./mm, p = 0.02), but no change in mdl or ccm measures, from baseline to 36 months. all subjects with igt underwent an annual ogtt over 36 months, 10 developed type 2 diabetes, 15 remained with igt, and 5 regressed to normal glucose tolerance (ngt) (table 2). neuropathy assessments at baseline and 36 months in subjects who reverted to ngt, remained with igt, or developed type 2 diabetes at 36 months data are means sem. p < 0.0001, baseline vs. control or baseline vs. 36 months. corneal confocal images from control subject at baseline (a), control subject at follow - up (b), igt subject who developed type 2 diabetes at baseline (c), igt subject who developed type 2 diabetes at follow - up (d), igt subject who remained igt at baseline (e), igt subject who remained igt at follow - up (f), igt subject who reverted to ngt at baseline (g), and igt subject who reverted to ngt at follow - up (h). red arrow, corneal nerve fiber ; yellow arrow, corneal nerve branch. in the 10 subjects who developed type 2 diabetes, their baseline cnfd (20.0 2.2 vs.30.7 1.5 no./mm, p = 0.003), cnbd (25.6 5.2 vs. 37.0 2.7 no./mm, p = 0.04), and cnfl (13.7 1.2 vs. 20.4 3.2 mm / mm, p = 0.04) were significantly lower compared with control subjects. over 36 months, there was a significant increase in hba1c (42.4 1.0 vs. 50.3 1.4 mmol / mol, p = 0.02) and a significant decrease in cnfl (13.7 1.2 vs. 11.8 1.0 mm / mm, p = 0.006), mdl (21.9 2.1 vs. 16.5 0.32 m, p = 0.02), and ienfd (6.5 1.2 vs. 3.9 0.9 no./mm, p = 0.002), with no significant change in any other measure of neuropathy (fig. 2). of the igt subjects who had a significant (a cnfd value < 2 sd below the mean for control subjects) reduction in cnfd at baseline, 87.5% developed type 2 diabetes and 12.5% remained igt or reverted to ngt (p = 0.007). in subjects who had a significant (a cnfl value < 2 sd below the mean for control subjects) reduction in cnfl change in corneal nerve fiber morphological parameters in subjects at baseline (black) and 36 months (red). in the 15 igt subjects who remained igt, their baseline cnfd (28.6 1.5 vs. 30.7 1.5 no./mm, p = 0.33), cnbd (38.8 3.7 vs. 37.0 2.7 no./mm, p = 0.54), and cnfl (16.8 0.8 vs. 20.4 3.2 mm / mm, p = 0.75) were comparable with control subjects. there was a significant reduction in ienfd (6.7 1.1 vs. 2.8 0.3 no./mm, p = 0.02) with no change in any measure of neuropathy over 36 months. in the 5 subjects who became ngt, baseline cnfd (25.4 1.9 vs. 30.7 1.5 no./mm, p = 0.06), cnbd (29.3 5.6 vs. 37.0 2.7 no./mm, p = 0.07), and cnfl (15.7 1.3 vs. 20.4 3.2 mm / mm, p = 0.24) did not differ from control subjects. however, there was a significant increase in cnfd (25.4 1.9 vs. 29.8 1.5 no./mm p = 0.05), cnbd (29.3 5.6 vs. 44.9 6.2 no./mm, p = 0.04), and cnfl (15.7 1.3 vs. 17.2 0.9 mm / mm, p = 0.05). there was a significant decrease in ienfd (6.5 1.1 vs. 3.0 0.4 no./mm, p = 0.02) with no significant change in any other measure of neuropathy over 36 months (table 2 and fig. the control and igt subjects were age matched (62.3 1.8 vs. 60 2.1 years, p = 0.2). subjects with igt had a significantly higher hba1c (42.7 0.9 vs. 38.3 0.7 mmol / mol, p < 0.0001) and bmi (32.0 1.0 vs. 27.6 0.9 kg / m, p = 0.01) and lower hdl (1.2 0.1 vs. 1.7 0.1 mmol / l, p = 0.03) but comparable total cholesterol, triglycerides, egfr, and blood pressure compared with control subjects. clinical and metabolic parameters and neuropathy assessment in control subjects and subjects with igt at baseline and follow - up data are means sem. all symbols represent statistically significant differences. p value igt baseline vs. control ; igt baseline vs. 36 months. the igt group had a significantly higher nsp (3.4 0.7 vs. 0.3 0.1, p < 0.0001), nds (2.9 0.5 vs. 1.1 0.3, p = 0.03), and vpt (16.2 2.1 vs. 8.4 1.5, p = 0.02) compared with the control group. there was no significant difference in sncv and pmncv and amplitude between subjects with igt and the control subjects. there was no difference in ienfd ; however, mdl was significantly lower in the igt group compared with control subjects (25.1 1.6 vs. 63.0 4.2 m, p < 0.0001). cnfd (24.4 1.3 vs. 30.7 1.5 no./mm, p < 0.0001) and cnfl (15.3 0.6 vs. 20.4 3.14 mm / mm, p = 0.004) were significantly lower, but there was no difference in cnbd between subjects with igt and control subjects. there was no correlation between hdl and ccm measures at baseline (cnfl r = 0.2, p = 0.2 ; cnbd r = 0.2, p = 0.1 ; and cnfd r = 0.2, p = 0.3). control subjects showed no significant change in metabolic parameters or neuropathy measures over 3 years (repeat skin biopsy not performed in control subjects). in subjects with igt, bmi, hba1c, lipids, and blood pressure remained stable, and there was a small but significant reduction in egfr (79.1 3.0 vs. 74.3 4.3 ml / min/1.73 m, p = 0.03) over 3 years (table 1). there was no significant change in nsp, nds, vpt, or thermal thresholds. there was a significant reduction in sncv (49.7 1.4 vs. 46.6 1.3 m / s, p = 0.007) and ienfd (6.4 0.8 vs. 3.2 0.8 no./mm, p = 0.02), but no change in mdl or ccm measures, from baseline to 36 months. all subjects with igt underwent an annual ogtt over 36 months, 10 developed type 2 diabetes, 15 remained with igt, and 5 regressed to normal glucose tolerance (ngt) (table 2). neuropathy assessments at baseline and 36 months in subjects who reverted to ngt, remained with igt, or developed type 2 diabetes at 36 months data are means sem. p < 0.0001, baseline vs. control or baseline vs. 36 months. corneal confocal images from control subject at baseline (a), control subject at follow - up (b), igt subject who developed type 2 diabetes at baseline (c), igt subject who developed type 2 diabetes at follow - up (d), igt subject who remained igt at baseline (e), igt subject who remained igt at follow - up (f), igt subject who reverted to ngt at baseline (g), and igt subject who reverted to ngt at follow - up (h). red arrow, corneal nerve fiber ; yellow arrow, corneal nerve branch. in the 10 subjects who developed type 2 diabetes, their baseline cnfd (20.0 2.2 vs.30.7 1.5 no./mm, p = 0.003), cnbd (25.6 5.2 vs. 37.0 2.7 no./mm, p = 0.04), and cnfl (13.7 1.2 vs. 20.4 3.2 mm / mm, p = 0.04) were significantly lower compared with control subjects. over 36 months, there was a significant increase in hba1c (42.4 1.0 vs. 50.3 1.4 mmol / mol, p = 0.02) and a significant decrease in cnfl (13.7 1.2 vs. 11.8 1.0 mm / mm, p = 0.006), mdl (21.9 2.1 vs. 16.5 0.32 m, p = 0.02), and ienfd (6.5 1.2 vs. 3.9 0.9 no./mm, p = 0.002), with no significant change in any other measure of neuropathy (fig. 2). of the igt subjects who had a significant (a cnfd value < 2 sd below the mean for control subjects) reduction in cnfd at baseline, 87.5% developed type 2 diabetes and 12.5% remained igt or reverted to ngt (p = 0.007). in subjects who had a significant (a cnfl value < 2 sd below the mean for control subjects) reduction in cnfl change in corneal nerve fiber morphological parameters in subjects at baseline (black) and 36 months (red). in the 15 igt subjects who remained igt, their baseline cnfd (28.6 1.5 vs. 30.7 1.5 no./mm, p = 0.33), cnbd (38.8 3.7 vs. 37.0 2.7 no./mm, p = 0.54), and cnfl (16.8 0.8 vs. 20.4 3.2 mm / mm, p = 0.75) were comparable with control subjects. there was a significant reduction in ienfd (6.7 1.1 vs. 2.8 0.3 no./mm, p = 0.02) with no change in any measure of neuropathy over 36 months. in the 5 subjects who became ngt, baseline cnfd (25.4 1.9 vs. 30.7 1.5 no./mm, p = 0.06), cnbd (29.3 5.6 vs. 37.0 2.7 no./mm, p = 0.07), and cnfl (15.7 1.3 vs. 20.4 3.2 mm / mm, p = 0.24) did not differ from control subjects. however, there was a significant increase in cnfd (25.4 1.9 vs. 29.8 1.5 no./mm p = 0.05), cnbd (29.3 5.6 vs. 44.9 6.2 no./mm, p = 0.04), and cnfl (15.7 1.3 vs. 17.2 0.9 mm / mm, p = 0.05). there was a significant decrease in ienfd (6.5 1.1 vs. 3.0 0.4 no./mm, p = 0.02) with no significant change in any other measure of neuropathy over 36 months (table 2 and fig. hughes. (25) found that in 50 consecutive subjects with pn and 50 consecutive control subjects, there was no significant difference in the prevalence of igt, but in the pn group serum triglycerides were significantly higher. fujimoto. (26) showed that subjects with igt had comparable nerve conduction studies but had a greater prevalence of retinopathy and nephropathy compared with control subjects. more recently, dyck. (27) showed that the frequency of pn was comparable in healthy subjects (1.7%) and subjects with impaired glycemia (2.0%) and was only increased in those with type 2 diabetes (7.8%). in a cohort of 393 subjects, ziegler. (28) found that there was an increased prevalence of polyneuropathy in those with igt (13%) compared with those with impaired fasting glycemia (11.3%) and control subjects (7.4%), although this was not significant. these findings may be attributed to the fact that neuropathy was diagnosed by assessing predominantly large fibers (13,29). indeed, there are accruing data to suggest that there is an increased prevalence of painful symptoms (3032) and evidence of a small - fiber neuropathy in subjects with igt (10,11,16,32). thus, small - fiber neuropathy may be the earliest change in the spectrum of pn, with injury beginning in the small myelinated a and unmyelinated c fibers, which over time progresses to affect larger nerves (33). while ienfd is accepted as the gold standard for quantifying ienf pathology, interestingly, pittenger. (34) showed that mdl was reduced before ienfd in subjects with metabolic syndrome and may therefore be an early marker of sensory neuropathy. our data support these findings, as mdl was significantly reduced, while ienfd was comparable in the igt cohort compared with control subjects at baseline. furthermore, mdl appears to be more responsive to changes in glucose tolerance status with a further worsening in only those igt subjects who developed type 2 diabetes, while ienfd showed a reduction in all three groups. in relation to causal factors, pittenger. (34) also reported a correlation between pn and hdl cholesterol. in the current study, we show that hdl cholesterol was lower in the igt group compared with the control subjects ; however, this was not associated with lower ccm measures. in an 18 week open - label trial, boyd at al. (35) showed that treatment with topiramate resulted in a significant improvement in mdl at the forearm and proximal leg and an increase in ienfd at the proximal leg. (11) have shown that a 1-year diet and lifestyle intervention program leads to an increase in ienfd. however, the much larger da qing study showed that lifestyle intervention over 6 years reduced the incidence of severe retinopathy but had no impact on neuropathy, although the end point was monofilament insensitivity (36). more recently, a 6-month twice weekly individualized exercise program significantly improved the rate of cutaneous nerve regeneration in a capsaicin nerve ablation model (18). our recent study in patients with type 1 diabetes undergoing simultaneous pancreas kidney transplantation showed that ccm can detect small - fiber regeneration as early as 6 months postsurgery (37). we have also shown that improvement in glycemia as well as blood pressure and lipids leads to corneal nerve regeneration (19). this leads to the notion that if there is an improvement in glycemia, then it may improve neuropathy. in the current study, we show that subjects with igt have evidence of small - fiber neuropathy, as evidenced by a greater prevalence of painful symptoms and abnormalities in ccm as well as a reduction in mdl in keeping with our recent study (16). however, we now show that patients who progress to type 2 diabetes have worse baseline corneal nerve morphology and mdl at a time when they are diagnosed with igt. this is in keeping with a recent study showing that subjects with ngt, but with abnormal electrochemical sweat conductance, have a significantly increased odds ratio for the development of igt (17). furthermore, subjects who progressed to type 2 diabetes also showed a further significant reduction in cnfl and mdl. in subjects who remained with igt, there was no baseline loss or any change over time. in subjects who reverted to ngt, the baseline ccm values did not differ significantly from control subjects, and indeed there was a significant increase in all ccm parameters. while this is a small study, the detailed quantification, particularly of the small fibers, provides insights into the dynamic relationship between small - fiber damage and repair in relation to overall glucose tolerance status. we confirm the data from our previous study showing that small - fiber neuropathy, detected using ccm, is prevalent in subjects with igt (16). more importantly, both ccm and mdl appear to be early and dynamic markers of small - fiber neuropathy, which may allow risk stratification of subjects with igt who are likely to progress to type 2 diabetes.	objectiveimpaired glucose tolerance (igt) through to type 2 diabetes is thought to confer a continuum of risk for neuropathy. identification of subjects at high risk of developing type 2 diabetes and, hence, worsening neuropathy would allow identification and risk stratification for more aggressive management.research design and methodsthirty subjects with igt and 17 age - matched control subjects underwent an oral glucose tolerance test, assessment of neuropathic symptoms and deficits, quantitative sensory testing, neurophysiology, skin biopsy, and corneal confocal microscopy (ccm) to quantify corneal nerve fiber density (cnfd), branch density (cnbd), and fiber length (cnfl) at baseline and annually for 3 years.resultsten subjects who developed type 2 diabetes had a significantly lower cnfd (p = 0.003), cnbd (p = 0.04), and cnfl (p = 0.04) compared with control subjects at baseline and a further reduction in cnfl (p = 0.006), intraepidermal nerve fiber density (ienfd) (p = 0.02), and mean dendritic length (mdl) (p = 0.02) over 3 years. fifteen subjects who remained igt and 5 subjects who returned to normal glucose tolerance had no significant baseline abnormality on ccm or ienfd but had a lower mdl (p < 0.0001) compared with control subjects. the igt subjects showed a significant decrease in ienfd (p = 0.02) but no change in mdl or ccm over 3 years. those who returned to ngt showed an increase in cnfd (p = 0.05), cnbd (p = 0.04), and cnfl (p = 0.05), but a decrease in ienfd (p = 0.02), over 3 years.conclusionsccm and skin biopsy detect a small - fiber neuropathy in subjects with igt who develop type 2 diabetes and also show a dynamic worsening or improvement in corneal and intraepidermal nerve morphology in relation to change in glucose tolerance status.
human performance is affected by both the genetic makeup of the individual and the environmental factors. current research and interest in sports genetics focus on genetic variants that may make a significant contribution to the individual s performance. it is known that personal traits such as endurance, strength, power, muscular coordination, and psychological willingness and motivation, all have a genetic background.1 the most frequently investigated genetic polymorphisms in terms of athletic performance or predisposition to athletic capacity are angiotensin-1 converting enzyme (ace) gene and -actinin-3 (actn3) gene.2 variants in these genes have been reported to be associated with elite athletic performance and with quantitative physical performance traits in the general population.35 actn3 was the first structural gene specific to skeletal muscle that has been associated with athletic performance.6 the actinins, major and important z line functional and structural proteins in sarcomers, are members of the actin - binding protein family.7 actn3 codes for actn3 in humans, and its expression is restricted to fast, type 2 fibers.8 north reported that a deficiency of -actinin protein because of a premature stop codon in actn3 alters the designation for the amino acid arginine to a stop codon at position 577 (r577x ; dbsnp rs1815739) in exon 16 and may contribute to the possibility of differential fitness. the actn3 577r allele and 577rr genotype are associated with top - level, power - orientated athletic performance in a wide array of ethnic groups.10 the actn3 r577x polymorphism has been reported to be associated with elite athletic status,6 endurance athletes,11 and many other groups.1216 ace is located on chromosome 17q23 and comprises 26 exons and 25 introns. it contains a polymorphism due to an insertion (i) or a deletion (d) of a 287 bp alu sequence in intron 16, resulting in the three genotypes of insertion / insertion (ii), insertion / deletion (i d), and deletion / deletion (dd).17 the i / d polymorphism is associated with circulating and tissue ace levels. individuals homozygous for the d allele had higher tissue and plasma ace concentrations than heterozygotes and ii homozygotes.18 many of the case control studies reported that success in speed - strength disciplines, such as short - distance running, long jump, high jump, and short - distance swimmers, is associated with ace dd genotype.19,20 on the other hand, individuals with the ii genotype have a lower ace serum concentration and have more success in endurance - related disciplines such as medium- and long - distance running, race walking, and rowing.21,22 two different studies involving spanish and lithuanian football players showed that players had a significantly higher percentage of the ace i d genotype when compared to the nonathletic population.23,24 previous studies, including related genetic polymorphisms and football players, are very limited, especially in turkish subjects. the aim of this study was to determine the genotype and allele distribution of actn3 and ace in turkish male football players and assess the impact on predisposition to football. all players enrolled for the study had turkish ancestry and were members of a turkish football team. the study was conducted in accordance with the principles of the declaration of helsinki ii. skdar university ethical committee approved the study protocol, and written informed consent was obtained from each player once prospective participants understood and accepted the aim and protocol of the study. dna isolation was carried out by using high pure polymerase chain reaction template preparation kit (roche diagnostics) using peripheral blood. the region of interest, actn3, was amplified using the following primers : forward 5-ctg ttg cct gtg gta agt ggg-3 and reverse 5-tgg tca cag tat gca gga ggg-3, as described previously.5 polymerase chain reaction (pcr) was performed by initial denaturation at 95c for five minutes, followed by 35 cycles of denaturation at 95c for 30 seconds, annealing and extension at 72c for one minute, and a final extension for seven minutes at 72c. the 290-bp amplicons were digested by ddei (new england biolabs) as recommended by the manufacturer. digested fragments were separated on 10.0% polyacrylamide gel electrophoresis and visualized under uv light by ethidium bromide staining. the wild - type allele, 577r, showed fragments of 205 and 85 bp, whereas the variant allele, 577x, showed fragments of 108, 97, and 85 bp (fig. the final volume of the pcr mixture was 50 l and contained 50100 ng genomic dna, 1 mm of each primer, 50 mm kcl, 1 mm deoxynucleotide triphosphate (dntp), 1.5 mm mgcl2, 10 mm tris hcl, an initial denaturation at 94c for five minutes was followed by annealing at 58c for one minute and extension at 72c for two minutes. amplification was finalized with 30 cycles : denaturation at 94c for one minute, annealing at 58c for one minute, and extension at 72c for two minutes, followed by a final elongation at 94c for one minute, annealing at 58c for one minute, and extension at 72c for seven minutes. amplicons were separated by electrophoresis on a 2% agarose gel and visualized under uv light after ethidium bromide staining. electrophoresis gave rise to three possible patterns : a 490-bp band (ii genotype), a 190-bp band (dd genotype), or both 490- and 190-bp bands (i / d genotype) (fig. amplicons had 190 bp in the presence of the d allele and 490-bp fragment in the presence of the i allele. all players enrolled for the study had turkish ancestry and were members of a turkish football team. the study was conducted in accordance with the principles of the declaration of helsinki ii. skdar university ethical committee approved the study protocol, and written informed consent was obtained from each player once prospective participants understood and accepted the aim and protocol of the study. dna isolation was carried out by using high pure polymerase chain reaction template preparation kit (roche diagnostics) using peripheral blood. the region of interest, actn3, was amplified using the following primers : forward 5-ctg ttg cct gtg gta agt ggg-3 and reverse 5-tgg tca cag tat gca gga ggg-3, as described previously.5 polymerase chain reaction (pcr) was performed by initial denaturation at 95c for five minutes, followed by 35 cycles of denaturation at 95c for 30 seconds, annealing and extension at 72c for one minute, and a final extension for seven minutes at 72c. the 290-bp amplicons were digested by ddei (new england biolabs) as recommended by the manufacturer. digested fragments were separated on 10.0% polyacrylamide gel electrophoresis and visualized under uv light by ethidium bromide staining. the wild - type allele, 577r, showed fragments of 205 and 85 bp, whereas the variant allele, 577x, showed fragments of 108, 97, and 85 bp (fig. the final volume of the pcr mixture was 50 l and contained 50100 ng genomic dna, 1 mm of each primer, 50 mm kcl, 1 mm deoxynucleotide triphosphate (dntp), 1.5 mm mgcl2, 10 mm tris an initial denaturation at 94c for five minutes was followed by annealing at 58c for one minute and extension at 72c for two minutes. amplification was finalized with 30 cycles : denaturation at 94c for one minute, annealing at 58c for one minute, and extension at 72c for two minutes, followed by a final elongation at 94c for one minute, annealing at 58c for one minute, and extension at 72c for seven minutes. amplicons were separated by electrophoresis on a 2% agarose gel and visualized under uv light after ethidium bromide staining. electrophoresis gave rise to three possible patterns : a 490-bp band (ii genotype), a 190-bp band (dd genotype), or both 490- and 190-bp bands (i / d genotype) (fig. amplicons had 190 bp in the presence of the d allele and 490-bp fragment in the presence of the i allele. the percentage of the ace genotype in the examined players was 16, 44, and 40 for ii, i d, and dd genotypes, respectively. for the actn3 genotype, the respective frequencies were 20, 36, and 44 for xx, rx, and rr. an allelic count gave rise to 19 (38%) i and 31 (62%) d alleles for ace and 31 (62%) r and 19 (38%) x alleles for actn3. table 1 lists the genotype and allelic frequencies of ace and actn3. according to the genotypes, nine different combinations were found, five of the players had dd + rr, the same number of the players had i d + rr, four players had i d + rx, three players had dd + rx, two players had dd + xx, the same number of the players had i d + xx, two players had ii + rx, one player had ii + xx, and one player had ii + rr (data not shown in table). recent studies have indicated that several genes are involved in determining the performance of the players, both physiologically and psychologically.25 genetic models could be developed and used to find the optimal genetic endowment of a player to help scientists establish which genetic polymorphisms are advantageous for proper performance in different sports types. therefore, the creation of genomic databases will be very useful for sport scientists. in this study, we solely examined 25 senior football players in terms of ace and actn3 polymorphisms and aimed to associate these polymorphisms with predisposition to football. in our study cohort, the r allele, considered to be the wild - type allele of the gene and associated with the rapid contraction of the sarcomers, was also high when compared to x allele. egorova reported similar results in russian football players ; they examined 240 football players, and 46.25% had the rr genotype. santiago analyzed 60 brazilian football players, and in their cohort, 48.3% of the players had rr genotype. pimenta aimed to compare acute inflammatory responses, muscle damage, and hormonal variations with eccentric training in soccer athletes and examined 37 professional soccer players of which 40.5% were rr. to the best of our knowledge, this is the first report in turkish male professional soccer players, and our results were in agreement with previous studies, indicating r allele and rr genotype superiority for soccer players. in our cohort, 44% of the players had i d genotype, 40% of the players had dd genotypes for ace, and 84% of the players had at least one d allele. d allele is responsible for high ace concentration and is associated with success in speed - strength disciplines.17 gineviciene examined 199 lithuanian football players and reported similar results to ours. they showed that i d was the highest genotype (46.7%), and together with dd genotype, the percentage of the players having at least one d allele was 76.3%. juffer analyzed 54 male professional soccer players, and i d was the most detected genotype. unlike our results, egorova examined 213 russian football players and found the frequency of i d and dd genotypes as 28.6% and 50.7%, respectively, giving both genotypes an overall frequency of 79.3%, which is similar to our results. the cumulative effect of genotypes in human metabolism had always received great attention. in an attempt to find the optimal genotypes, several researchers have examined candidate genes separately, in different populations, to create a pool of genomic data. genotype combinations are very useful for identifying a certain metabolism, or a part of a metabolism, if they are part of the same reaction. for example, actn3 and ace have crucial roles for their metabolisms, but they do not have roles in the same biologic pathway. but it is important to understand performance - enhancing polymorphisms to create an optimal genomic score for being an elite athlete. in our cohort, five of the players had the dd + rr genotype combinations and the same number had i d + rr genotypes. the d and r alleles were found together in these genotypes, the most important similarity between these genotypes. on the other hand, one player had the ii + xx genotype, thought to be associated with endurance capacity. we hope this preliminary study will guide new studies, with extended numbers of players in different kinds of sports to evaluate the effect of the combination of these polymorphisms more accurately. in this study, we examined the distribution of ace and actn3 polymorphisms in male turkish professional soccer players for the first time. actn3 rr genotype and r allele and ace i d genotype and d allele dominated our cohort. therefore, we did not compare the genomic results with sedentary people ; rather, we examined the combined effect of these polymorphisms. according to our previous and current results, we hypothesize that elite soccer players tend to have a power / strength - oriented genotype. these polymorphisms, alone or in combination with the additional polymorphisms, should be taken into account when deciding a genomic score profile for success in sports.	angiotensin-1 converting enzyme (ace) gene and -actinin-3 (actn3) gene polymorphisms are considered to be the most important candidate genes for genetic predisposition to human athletic performance. in the present study, we aimed to analyze the distribution of ace and actn3 polymorphisms for the first time in male turkish soccer players. in this prospective study, our cohort consisted of 25 professional players, all with turkish ancestry. polymerase chain reaction (pcr)-restriction length polymorphism was used for the characterization of the genotype of actn3 and single pcr for ace. for ace genotype, 16%, 44%, and 40% of the players had insertion / insertion (ii), insertion / deletion (i d), and deletion / deletion (dd) genotypes, respectively, whereas 20% had xx, 36% had rx, and 44% had rr genotypes for actn3. when we examined the allelic percentages, for ace, d allele was recorded as 62 and i as 38, and for actn3, r allele was 62 and x was 38. our results were in agreement with the previous reports, indicating the presence of actn3 d and ace x allele in soccer players. we suggest that ace and actn3 genotypes are important biomarkers for genetic counseling for the individuals who are prone to be successful soccer players.
over the last decade, progress observed in sub - saharan african countries in increasing pmtct service coverage and in reducing new hiv infections among children is promising. for example, in the 21 global plan priority countries in sub - saharan africa, coverage of antiretroviral medicines for pregnant women living with hiv to prevent mother - to - child transmission has nearly doubled in 2012 as compared to the 2009 level, increasing from 34% in 2009 to 65% in 2012. this increased coverage has contributed to reducing new hiv infections among children by 38% in 2012 as compared with 2009 levels. in this regard, botswana, ethiopia, malawi, namibia and zambia have demonstrated a 50% or more decline on new hiv infections among children. on the other hand, angola, chad, cote divoire, democratic republic of congo, lesotho and nigeria showed slow declines in averting new hiv infections among children. specifically, nigeria, which accounts more than one - quarter of new hiv infections among children in the region, pmtct coverage was very low (17% in 2012) and consequently, the decline in hiv infection aversion among children has been less effective. such slow progress in high burden countries such as nigeria also affects achievement of regional targets. the main purpose of this paper is to share the evidence on a major factor that may impede scale - up of pmtct service coverage and thereby advocate for integration of pmtct and maternal and child health services, fundamental for achieving the global plan and global fast - track commitments of zero transmission and to improve maternal and child health in general. the paper specifically analyses disparities across income in the use of antenatal care services in sub - saharan african countries. it also analyses the extent to which use of antenatal care services differentiated by women s wealth status explains variations in pmtct service coverage in the region. all sub - saharan african countries that conducted demographic and health survey (dhs) between 2008 and 2015 have been selected for analysis. if countries conducted two or more dhs surveys, the most recent survey has been considered. the dhs are nationally - representative household surveys (with large sample sizes ranging between 5000 and 30,000) that provide a wide range of data in the areas of population, health, and nutrition. the dhs surveys are normally implemented approximately every five years. from the dhs survey results, unicef pooled data on anc coverage and released the results in february 2016. for this analysis, anc use refers to least four antenatal care visits to a health facility or health personnel during the last pregnancy. on the other hand, the most recent pmtct coverage data have been pooled from the unaids dataset released in 2016. using spss, linear regression analysis were conducted to assess the disparity in the use of anc that are associated with income ; and to understand how much the disparity in anc use could explain pmtct service coverage. wealth quintiles, as measured by dhs, refer to household income classified into five categories using principal component analysis. thirty - one sub - saharan african countries with complete data on anc use by wealth quintile and pmtct service coverage. have been identified. the complete dataset extracted from unicef and unaids 2016 online datasets are presented in supplementary table s1. the anc use (at least four visits) in the lowest and highest wealth quintiles, in their respect, average 42% and 69%. this implies that in the 31 sub - saharan african countries, women in the highest wealth quintile are about 3 times more likely to use anc services (at least four visits) as compared to those in the lowest wealth quartile (95%ci : 1.7 - 5.7, p<0.000). the pmtct service service coverage in these countries ranges between 3% in madagascar and 95% in mozambique, namibia, swaziland, togo, and uganda ; while the average pmtct coverage is about 71% (table 1). a regression analysis shows that the disparity in anc use associated with income (i.e., between women in the highest and lowest wealth quintiles) is a statistically significant predictor of pmtct service coverage (p<0.05). also, the analysis shows that disparity in anc use associated with income can explain nearly a quarter of the variation on pmtct service coverage in sub - saharan african countries (p<0.05). the analysis and graph bellow show that the higher the disparity in anc use associated with income, the lower the pmtct service coverage (figure 1 ; see also supplementary table s2 for more details on the output of the regression analysis). sub - saharan africa accounts the highest burden of hiv / aids world wide. in this regard, by the end of 2012, about 69% of people living with hiv globally were in this region, where nearly 1 in every 20 adults (4.9%) were living with hiv. similarly, over 90% of the children who acquired hiv infection in 2011 lived in sub - saharan africa. however, there are also encouraging results that are being observed, specifically over the last decade. in this regard, for example, new hiv infection in the region has declined by one - quarter ; i.e., from 2.4 million in 2001 to 1.8 million in 2011. also, in the global plan s 21 priority countries in sub - saharan africa, the number of children newly infected fell by 38% ; i.e., from 340,000 new infections in 2009 to 210,000 in 2012. but, achieving the global target of eliminating new hiv infections among children and keeping their mothers alive requires taking the full advantages of the advancements in the biomedical field on pmtct. advancements in biomedical interventions have created huge opportunities by reducing the risk of mother - to - child transmission (mtct) of hiv from 35% to 2%. however, translating such advancements into practice is faced with programmatic barriers specifically in sub - saharan africa ; one of such challenges being lack of integration of pmtct with other maternal and child health services. the world health organization strategic directions for scaling - up quality and effective pmtct calls for integration of hiv prevention, care and treatment services with maternal, new - born and child health and reproductive health programs as one of the critical approaches for reaching zero new infections among children. in most of the sub - saharan african countries, anc use (at least four visits) therefore, reaching women who are already using anc service is a low hanging fruit for expanding pmtct services. while maternal health and pmtct service coverage are generally low and at varying levels in sub - saharan africa, access to services by the poorest women is worse, which has major impact on national and regional level achievements. in this regard, for example, the relatively wealthiest women are three times more likely to use anc services as compared to the poorest women in the region (p<0.05) (table 1). also, the higher the disparity across income in using anc services, the lower the pmtct service coverage at the national level (p<0.05). the impact of health inequality in the region is beyond pmtct and anc. in this regard, in sub - saharan african countries, while there is no association between national poverty rate and hiv prevalence, a systematic review of studies shows existence of clear and significant association between income inequality (measured with gini coefficient) and hiv prevalence across countries ; i.e., countries with greater income inequality have higher hiv prevalence. the specific factors as to why women in the lower wealth quintile have lower access to health services might require further in - depth research. however, available studies on the general population might also enlighten some of the potential factors that need to be considered when designing maternal and pmtct programs. it is also important to note that the anc use disparity across income only explains about a quarter of the variation on pmtct service coverage (supplementary table s2). in this respect, there remain many other factors that determine access / utilization of pmtct and other maternal health services. for example, a study in holeta town of ethiopia shows that anc attendance is significantly associated with demographic, socio - economic and health related factors including age at last birth, literacy status of women, average monthly family income, media exposure, attitude towards pregnancy, knowledge on danger signs of pregnancy and presence of husband approval on anc. studied women s practice of disclosing their hiv status to their partners in four sub - saharan africa countries - burkina faso, kenya, malawi and uganda. disclosure has important effect for hiv positive women s desires to access pmtct services. according to hardon., rates of disclosure to partners in the four countries varied between 32.7% and 92.7% ; the lowest being in malawi. a similar study in tanzania showed that six in ten hiv positive pregnant women had not disclosed their results of the hiv test to their partners. an important reason for not disclosing was women s economic dependency on their partners and hence fear of being expelled from house. such findings suggest the importance of also targeting male patterns in pmtct and maternal health programs. a cross - sectional study conducted in southeast nigeria shows limitations of women s knowledge / awareness about available maternal and child health services at the different service delivery points in their areas, which potentially leads to lack of trust and wastage of their time and other resources by unknowingly traveling to facilities where services are not available. in these regards, pmtct and maternal health program planning processes need to further look into the factors, validate those that deem most important and earmark resources on how to break the barriers for women (especially to those in the poorest wealth category) for accessing the services. ameur. emphasized on the importance of eliminating user fees for maternal health services, specifically at the time of delivery, in order to address issue of maternal health inequity. in this regards, while the study by ameur. focused on burkina faso, they also cited the positive outcomes in ghana after the country introduced exception policy making delivery care free. in this respect, 18 months after user fees were abolished in ghana, the greatest increase in health facility use was by the poorest segment of the population. analysis of data from 269 sites in 20 pmtct programs and 15 sub - saharan countries from 2002 to 2005 showed two important factors that can increase pmtct coverage higher knowledge about pmtct and higher government investment on health. in order to address the maternal health disparities across income through integrated service delivery, other potential areas to be considered are : introducing routine hiv testing for women and their partners (e.g., through provider initiated testing and counselling - pict) ; supporting alternate models of care delivery (such as shifting basic tasks from physicians to nurses) ; and providing incentives for service providers as well as clients (e.g., incentive for health workers for agreeing to relocate to rural areas if necessary ; or outreach pregnant women in neighboring villages ; and reimbursing pregnant women for the cost of transportation to the clinic). the goal of the global plan was to move towards eliminating new hiv infections among children by 2015 and keeping their mothers alive. the plan focused on reaching pregnant women living with hiv and their children, from the time of pregnancy until the mother stops breastfeeding. the global fast - track commitments to end aids by 2030 also aims to eliminate hiv infections among children by 2020. in this regards, one of the principles for reaching these goals is leveraging synergies, linkages and integration for improved sustainability, i.e., national plans leverage opportunities to strengthen synergies with existing programmes for hiv, maternal health, new - borns and child health, family planning, orphans and vulnerable children, and treatment literacy. pmtct is more than the simple administration of arvs ; and the full set of its intervention components should be implemented as an integral component of essential maternal, neonatal and child health services. on the other hand, funding is critical, there is never enough money for global health ; never enough for any one intervention ; and this creates a double rationale for support. while the hiv / aids and maternal health challenges in sub - saharan africa are immense, the gains (especially over that last decade) are also promising. in these regards, achieving the global plan target and global fast - track commitments of eliminating new hiv infections among children and keeping their mothers alive would definitely require strategic approaches by building on existing health - care delivery systems / structures and specifically integrating the service deliveries with other maternal and child health services ; and with deliberate effort to reach women who are most disadvantaged.	about 69% of people living with hiv globally and over 90% of the children who acquired hiv infection are in sub - saharan africa. despite this, promising results have been observed, especially over the last decade for example, a 25% decline in new hiv infections as compared to 2001 and a 38% decline in the number of children newly infected by hiv in 2012 as compared to 2009. however, the global plan and the global fast - track commitments of eliminating new hiv infections among children require addressing impediments to service expansion. in this regard, this paper attempts to draw attention to the extent to which disparities across income in using antenatal care (anc) services may constrain the prevention of mother - to - child transmission (pmtct) service expansion in sub - saharan africa. the analysis is based on anc service coverage data from demographic and health surveys conducted between 2008 and 2015 in 31 sub - saharan african countries ; and pmtct coverage data from unaids datasets released in 2016. our analysis found that women in the highest wealth quintile are about three times more likely to frequently use anc services (at least four visits) as compared to those in the lowest wealth quintile (95%ci : 1.7 - 5.7, p<0.0001). a regression analysis shows that one - quarter of the pmtct service coverage can be explained by the disparity in anc use associated with income ; and the higher the disparity in anc use, the lower the pmtct service (p<0.05). the findings suggest that achieving the ambitious plan of zero new hiv infections among children while keeping their mothers alive will require on - going pmtct / anc service integration and ensuring that programs reach women who are most in need ; specifically those in the poorest income categories.
various essential biological tasks that involve motion and mechanical work are performed by molecular motor proteins. the activity of these motors is modulated dynamically through complex biological pathways in response to environmental stimuli. allosteric regulation plays a key role in biological systems in order to switch between active and inactive states. since allosteric effects involve conformational changes (cf. dynamic motion), the operating principles of allosteric systems are very important in the design of artificial molecular motors and machines. furthermore, allosteric control has been applied to various supramolecular systems to inhibit or activate, for example, catalysis and substrate binding. different successful approaches have been taken to develop synthetic motors and machinery that mimic their biological counterparts. in this respect, the use of light as the energy source has proven to be particularly attractive for nanotechnology applications. however, activity regulation of synthetic nanomotors by multiple external stimuli, as is observed in biological systems, still remains a formidable challenge. overcrowded alkene - based rotary motors, developed by our group, are among the most promising candidates to perform work at the nanoscale. they have been applied to control a wide variety of processes, such as self - assembly, substrate binding, wettability, and gel formation. in these motors, repetitive unidirectional rotation is accomplished through sequential photochemical and thermal isomerization steps (scheme 1a). that is, light excitation can induce a double bond isomerization giving a higher energy (unstable) isomer. this isomer then thermally relaxes to the energetically most favored (stable) isomer via a helix inversion process. in the latter thermal helix inversion (thi) step, the aromatic unit in the upper half of the molecule flips along the lower half. when these photochemical and thermal isomerization steps are repeated, one - half of the motor completes a full 360 rotation with respect to the other half. the thi process is the rate - determining step in the overall rotary cycle and, hence, is what determines the speed of rotation at infinite photon flux. please note that the two line drawings for stable l1, as well as those for unstable l1, represent identical molecular structures with different viewpoints. the development of strategies that allow for regulation of the rotational speed of molecular motors is crucial for bringing nanoscale machinery to a higher level of sophistication and complexity. within our group, considerable effort has gone into adjusting the frequency of rotation via structural modifications, which required tedious synthetic work. in addition, we have reported on the supramolecular locking of rotation using an acid base - responsive self - complexing pseudorotaxane, as well as reversing the direction of rotation by base - catalyzed epimerization. up until now, however, we have not been able to control the speed of rotation in a dynamic way. recently, the molecular motor l1 was developed (see scheme 1a), which incorporates a 4,5-diazafluorenyl lower half that can bind metal ions. our initial studies focused on ruthenium bipyridine complex formation with the goal of shifting the excitation wavelength to the visible region, but interestingly, also an effect on the energy barrier for thi was observed. reversible metal complexation has been applied in the past to control free rotation around a single bond using a molecular brake approach as described by the group of kelly. we envisioned that metal complexation to l1 could also offer an extremely powerful method to postmodify the rotational behavior of light - driven molecular motors in a reversible manner (scheme 1b). herein, we describe the synthesis, characterization, and isomerization behavior of different metal dichloride complexes of l1, that is, zn(cl)2(l1), pd(cl)2(l1), and pt(cl)2(l1). predictions on the relative rates of rotation can be made using dft optimized molecular structures, which reveal a correlation between the energy barrier for thi and certain structural parameters. we demonstrate that the steric crowding in the fjord region, and hence the rotational speed, can be controlled through metal ion binding. thus, the metal ion can act as a kind of allosteric effector, which induces a structural change. importantly, the metal complexes can be prepared in situ and metal decomplexation occurs upon addition of a competing ligand. this opens up new opportunities to dynamically modulate the properties of light - driven molecular motors and represents a significant step forward toward higher levels of control in nanomechanical devices. dft calculations have proven to provide accurate predictions for the structural parameters and the barrier for thi of sterically overcrowded alkene - based molecular motors. it was previously found that the tpsstpss functional along with the 6 - 31g+(d, p):lanl2dz mixed basis set gives the best approximation of the thermodynamic properties of transition metal complexes. therefore, this method was chosen together with an iefpcm, ch2cl2 solvation model to calculate the structures and relative free energies (g) of the stable and unstable isomers, in addition to the transition states (ts) for thi. the coordination of zinc, palladium, and platinum to 4,5-diazafluorenyl ligands has been studied before and is known to afford stable metal ligand complexes. for that reason furthermore, the resulting metal ligand complexes are diamagnetic so that h nmr can be used as a convenient tool to study the photochemical and thermal isomerization processes (vide infra). in the earlier reported x - ray crystal structures of palladium and platinum 4,5-diazafluoren-9-one dichloride complexes, a square planar coordination geometry was observed, whereas the zinc dichloride complex of a structurally related 4,5-diazafluorene derivative revealed a tetrahedral coordination environment. the input geometries for dft energy minimization were adapted as such, and the obtained molecular structures and relative g values are presented in figure 1. plots of the relative gibbs free energies (20 c) between the stable, unstable, and transition state (ts) geometries of (a) zn(cl)2(l1), (b) pd(cl)2(l1), and (c) pt(cl)2(l1) obtained by dft using the tpsstpss/6 - 31g+(d, p):lanl2dz level of theory. for similar to what has been found for uncomplexed l1, the metal motor complexes exist in a twisted conformation, which is known to be preferred over anti folding when the lower half of these structures is sufficiently rigid. furthermore, inherent to this type of molecular motors, the methyl group is oriented pseudoaxially in the thermodynamically most stable form and equatorially in the higher energy unstable isomer. the metal - to - nitrogen bond lengths and diazafluorenyl bite angles in the dft optimized complexes of stable l1 closely resemble those found in the crystal structures of related metal n bond lengths in the calculated zinc, palladium, and platinum complexes of the stable ligand are about 2.19, 2.09, and 2.07, respectively (cf. 2.13, 2.08, and 2.06 in the reported solid state structures of 4,5-diazafluorene complexes). in addition, the observed diazafluorenyl n m n bite angles range from 83.2 for zinc and 84.0 for palladium to 83.6 for platinum (cf. 84.8, 83.7, and 83.6 in the solid state structures). the ts geometries were searched via a dihedral scan over the central olefinic bond (see the supporting information for details). for the palladium and platinum complexes, two different ts structures were found and energy minimized, whereas for the zinc analogue only one ts could be optimized (see figure 1). the first and lowest energy transition state (ts1) has a planar diazafluorenyl moiety and the c(2)h and c(2)ch3 protons are located on the same side of the lower half (see scheme 1 for the atom numbering). the second transition state (ts2), found for pd(cl)2(l1) and pt(cl)2(l1), is slightly higher in energy (34 kj mol) and is structurally similar to that calculated earlier for the uncomplexed ligand l1. it has a slightly bent diazafluorenyl lower half with c(2)h and c(2)ch3 positioned on opposite sides of this unit. the most important structural parameters of all the transition states obtained are summarized in table 1. see scheme 1 for the atom numbering. from these structural parameters, it can be derived that metal complexation leads to a contraction of the lower diazafluorenyl half of the molecular motor, which is reflected in the shortened n the angle enlargement implies a reduction of the steric crowding in the fjord region. it has been well - established by our group that a decrease of the steric hindrance in this region lowers the barrier to thi and hence, enhances the speed of rotation of the motor. based on these structural features, it is anticipated that the rotary speed of metal - complexed l1 will increase in the order zn(cl)2(l1) < the gibbs free energy barriers [g(20 c) ] for thi, as calculated by dft (see figure 1), decrease from zinc to palladium and platinum, which is also expected based on the structural features. the molecular motor l1 was obtained by following a recently reported procedure, and it was used as a racemate. subsequent stirring with zncl2 in etoh at room temperature afforded zn(cl)2(l1) as a precipitate, which was collected by filtration. likewise, the addition of l1 to a solution of na2[pdcl4 ] in ch3oh / ch2cl2 at room temperature led to precipitation of pd(cl)2(l1), whereas the corresponding platinum complex pt(cl)2(l1) was synthesized by heating a solution of l1 with k2[ptcl4 ] in h2o / mecn under reflux. the photochemical and thermal isomerization behavior of the metal - complexed motors was studied in degassed ch2cl2 by low temperature uv vis and h nmr spectroscopy. in the uv vis spectrum (figure 2), the absorption maxima of zn(cl)2(l1) and pd(cl)2(l1) are situated around = 405 nm, whereas pt(cl)2(l1) has a maximum absorption at = 422 nm. these maxima are all bathochromically shifted relative to the parent uncomplexed l1 (max = 390 nm). irradiation of the uv vis samples with 365 nm light at 20 c resulted in a further bathochromic shift of the major absorption bands to = 426 nm for zn(cl)2(l1) and pd(cl)2(l1) and to = 431 nm for pt(cl)2(l1) (see figure 2). uv vis absorption spectra of (a) zn(cl)2(l1), (b) pd(cl)2(l1), and (c) pt(cl)2(l1) in degassed ch2cl2 (2 10 m) before (solid line) and after (dashed line) irradiation with 365 nm light at 20 c. irradiation was continued until no further changes were observed, meaning that the photostationary state (pss) had been reached. during the course of irradiation, clear isosbestic points were observed indicating that the conversion from stable to unstable isomer is a unimolecular process. upon warming of the uv vis samples to room temperature, the original absorption spectra were obtained in all cases, which is consistent with thermal isomerization from the unstable back to the thermodynamically most stable form. likewise, h nmr analysis of solutions in cd2cl2, irradiated with 365 nm light at 75 c, showed a new set of peaks that can be ascribed to the unstable isomer (see figures s16s18 in the supporting information). by integration of either the c(2)ch3 or c(3)ch signals, which are shifted downfield for the photogenerated isomer, the unstable / stable isomer ratios at the pss were determined (table 2). pleasingly, comparison of these pss365 ratios with that of the uncomplexed l1 reveals that they do not substantially decrease upon metal complexation. when the h nmr samples were allowed to warm to room temperature, full conversion to the stable isomer furthermore, upon the repetition of this irradiation / warming cycle, no degradation products could be detected (figures s16s18 in the supporting information). half - life, t1/2 = ln(2)/k. maximum rotation rate, = 1/2k. the quantum yields for the photoisomerization process were determined by irradiation of concentrated solutions of the stable isomers in ch2cl2. the absorption increase at = 450 was monitored over time by uv vis spectroscopy and was used to calculate the rates of formation of the photogenerated, unstable isomers (see figures s12s15 in the supporting information). comparison of these rates to the one measured for the formation of fe ions from ferrioxalate under identical conditions afforded the photochemical quantum yields, which are given in table 2. these quantum yields range between 3.9% and 6.4% and point out that metal binding has only limited impact on the photoconversion efficiency. furthermore, the smallest value found for pd(cl)2(l1) is in line with the lower pss365 ratio, whereas the decreased pss365 ratio for pt(cl)2(l1) may be ascribed to a higher quantum yield for the reverse reaction (i.e., photoconversion from the unstable to the stable isomer). the rates of the thermal isomerization step were determined over a range of temperatures (30 to 0 c, see figures s8s11 in the supporting information for details) for each of the metal motor complexes by following the decrease in absorption at = 450 nm in the uv vis spectrum. application of the eyring equation afforded the thermodynamic parameters (see figures s8s10 in the supporting information) of which the gibbs free energies of activation (g) are given in table 2. the corresponding rate constants (k) were obtained by extrapolation to 20 c and used to calculate the half - lives (t1/2) at this temperature. since the thi step is the rate - determining step in the rotary cycle, the rate constant can also be used to define a maximum rotation rate () at 20 c given infinite photon flux (table 2). from this data, it is evident that regulation of the rotational speed is possible through metal complexation to l1. the experimentally obtained g values for thi compare well with the dft calculated ones (see figure 1). n distances, the c(8)c(8)c(9) angles (, see table 1), and the experimentally determined g and k values correlate with each other. in agreement with the predictions based on the dft optimized molecular structures (vide supra), the speed of rotation increases in the order : l1 < zn(cl)2(l1) < region of l1 and can be used as a convenient tool to regulate the rotational speed. previous approaches to achieve such changes in the speed of rotation required a new motor design and synthesis. the metal ligand coordination strategy presented here offers significant benefits over existing methods. when the metal complexes were dissolved in a coordinating solvent (dmso - d6) or when a strongly coordinating ligand was added (pyridine - d5), the h nmr spectrum of uncomplexed l1 was recovered (see figures s19 and s20 in the supporting information). this observation triggered us to investigate the possibility to switch between a metal associated and dissociated state via the addition of chemical stimuli. in that way, the rotary speed of the molecular motor could be reversibly tuned in situ, which is important toward the development of more complex molecular motor and machine - like systems. the in situ metal complex formation and metal decomplexation were studied by h nmr and uv vis spectroscopy. zinc and palladium were used because the corresponding metal motor complexes are formed at room temperature. addition of zncl2 or na2[pdcl4 ] to ligand l1 dissolved in cd2cl2/cd3od afforded h nmr spectra consistent with those of the previously isolated zn(cl)2(l1) and pd(cl)2(l1) complexes (figure 3). subsequent addition of pyridine - d5 induced metal decomplexation as evidenced by the regeneration of the h nmr spectrum of l1 (see figures s21 and s22 in the supporting information). (a) h nmr spectrum of l1 (2 10 m solution in cd2cl2/cd3od) and h nmr spectrum after (b) the addition of either zncl2 to the solution of l1, which generates zn(cl)2(l1), or (c) the addition of na2[pdcl4 ] to l1, which generates pd(cl)2(l1). during these h nmr experiments significant color changes were observed upon addition of the metal salts, which prompted us to additionally monitor this in situ reversible complexation by uv vis spectroscopy. adding either zncl2 or na2[pdcl4 ] to a solution of l1 in ch2cl2/ch3oh resulted in the bathochromic shifting of the absorption maxima, which is consistent with formation of the metal complex (figure 4). again, metal dissociation was successfully achieved through the addition of pyridine as is clear from the hypsochromic shift back to the original absorption maximum. in the case of zinc, these metal complexation and decomplexation steps were repeated multiple times (see figure s23 in the supporting information). hence, these combined h nmr and uv vis experiments reveal that dynamic regulation of the rotation rate is feasible via addition of chemical stimuli. uv vis spectrum of l1 (2 10 m in ch2cl2/ch3oh, 40:1, solid line) upon addition of either (a) zncl2 or (b) na2[pdcl4 ] (dashed line) followed by the addition of pyridine (dotted line). in conclusion, we have presented the synthesis and characterization of zinc, palladium, and platinum dichloride complexes of molecular motor l1. the photochemical and thermal isomerization behavior of these metal motor complexes has been studied in detail. interestingly, the speed of rotation of molecular motor l1 increases through metal complexation in the order zn(cl)2(l1) < pd(cl)2(l1) < pt(cl)2(l1), which is in agreement with predictions based on dft geometry optimization. binding of metal ions to l1 leads to a contraction of the 4,5-diazafluorenyl lower half and the simultaneous reduction of the steric crowding in the fjord region. furthermore, the metal motor complexes can be generated in situ, and the addition of a competitive ligand causes full metal decomplexation. consequently, the rotational behavior of these motors can be tuned reversibly in a highly practical and versatile manner with a level of control that is unprecedented. therefore, this work represents a critical step forward to integrated chemical regulation in molecular machines that are operated by light. other applications are foreseen in photoswitchable transition metal catalysis, surface anchoring, and molecular sensing.	the rotational speed of an overcrowded alkene - based molecular rotary motor, having an integrated 4,5-diazafluorenyl coordination motif, can be regulated allosterically via the binding of metal ions. dft calculations have been used to predict the relative speed of rotation of three different (i.e., zinc, palladium, and platinum) metal dichloride complexes. the photochemical and thermal isomerization behavior of these complexes has been studied in detail using uv vis and 1h nmr spectroscopy. our results confirm that metal coordination induces a contraction of the diazafluorenyl lower half, resulting in a reduction of the steric hindrance in the fjord region of the molecule, which causes an increase of the rotational speed. importantly, metal complexation can be accomplished in situ and is found to be reversible upon the addition of a competing ligand. consequently, the rotational behavior of these molecular motors can be dynamically controlled with chemical additives.
a 55-year - old man traveled to mainland china from march 5 to march 15. by march 18 the patient was given a course of antimicrobial drugs, and the fever resolved. by march 23, at this point, sars was suspected, and the patient was moved into isolation in an intensive care unit the same day and had to be intubated for 5 days. results from the polymerase chain reaction (pcr) test indicated the presence of the sars - associated coronavirus. the patient had no previous history of serious respiratory illness or any other serious coexisting conditions, and the source of the exposure could not be determined. the 50-year - old wife of patient 1 was exposed to sars upon her husband 's return on march 15. on march 23, a cough developed, and she had shortness of breath and chills. because sars was suspected, the patient was placed on antiviral treatment, and sars was confirmed by pcr testing. patient 2 was placed in isolation on a medical ward on march 25 and remained there until april 11. the 15-year - old daughter of patients 1 and 2 also became exposed to sars when her father returned on march 15., she complained of stomach pain and, upon examination, had a temperature of 37.7c. the 23-year - old daughter of patients 1 and 2, who lived away the family home, became exposed to sars through contact with both patients 1 and 2 upon a brief visit (lasting < 1 hour) on march 19. by march 27 a pcr test was not completed because her fever resolved, and no other symptoms developed. a 55-year - old man traveled to mainland china from march 5 to march 15. by march 18 the patient was given a course of antimicrobial drugs, and the fever resolved. by march 23, at this point, sars was suspected, and the patient was moved into isolation in an intensive care unit the same day and had to be intubated for 5 days. results from the polymerase chain reaction (pcr) test indicated the presence of the sars - associated coronavirus. the patient had no previous history of serious respiratory illness or any other serious coexisting conditions, and the source of the exposure could not be determined. the 50-year - old wife of patient 1 was exposed to sars upon her husband 's return on march 15. on march 23, a cough developed, and she had shortness of breath and chills. because sars was suspected, the patient was placed on antiviral treatment, and sars was confirmed by pcr testing. patient 2 was placed in isolation on a medical ward on march 25 and remained there until april 11. the 15-year - old daughter of patients 1 and 2 also became exposed to sars when her father returned on march 15., she complained of stomach pain and, upon examination, had a temperature of 37.7c. the 23-year - old daughter of patients 1 and 2, who lived away the family home, became exposed to sars through contact with both patients 1 and 2 upon a brief visit (lasting < 1 hour) on march 19. by march 27 a pcr test was not completed because her fever resolved, and no other symptoms developed.	with cases of severe acute respiratory syndrome (sars) occurring across geographic regions, data collection on the effectiveness of intervention strategies should be standardized to facilitate analysis. we propose a minimum dataset to capture data needed to examine the basic reproduction rate, case status and criteria, symptoms, and outcomes of sars.
the histological features include the presence of newly formed blood vessels and the identifying feature of multinucleated giant cells. however, to date, there has been no molecular method of distinguishing the variants. surgical curettage showed high recurrence rate in case of aggressive lesions, so resection with and without continuity defects was reported to prevent recurrence. intra - lesional injections were considered among the possible means of treatment which may be either intralesional steroid, intralesional calcitonin, intralesional interferon, intralesional bisphosphonates and denosumab (a monoclonal antibody that binds rankl and directly inhibits osteoclastogenesis). the concept of using medical alternatives or adjuncts to surgery was to evade the need for resection and reconstruction in this young group of patients. we present a case report in which an aggressive variant of central giant cell granuloma was managed using a combined protocol. the patient presented with cushonoid facial features during the phase of intralesional corticosteroid injection. in response, the surgical team shifted to completing the treatment by surgical curettage. a nine years old female patient presented with swelling in the left side of the mandible related to the ipsilateral lower first molar. the chief complaint was pain and localized swelling, the initial diagnosis made by the pediatric department was periapical abscess. a pretreatment radiograph (fig. the opt showed well defined radiolucency related to the mandibular left first molar measuring 3.5 cm in antero the case was then referred to the oral and maxillofacial department clinic. upon history taking, the patient reported a history of malignancy in large intestine, which had been managed, by chemotherapy. intraorally, a swelling related to a badly broken down lower left 1st molar which proved to have pulpal exposure was detected (fig. a multi slice ct scan over the facial bones with contrast was ordered for further assessment of the lesion s nature. the multi slice ct showed wide bucco - lingual extension with perforations through both the buccal and lingual bone plates (figs. 3, 4, 5). the assessment of the collected data at this point led to a differential diagnosis list. histological analysis depicted multinucleated giant cell most probably of fibroblast origin and extravasated erythrocytes and indicated that the lesion is giant cell lesion (fig. laboratory investigations were ordered, which included pth, blood calcium and phosphorus levels and all were within the normal levels, furthermore, renal function tests were also normal ruling out the possibility of primary or secondary hyperparathyroidism. intra - lesional steroid injections of 5 ml per injection of (10 mg / ml triamcinolone (kenacort - a) with lidocaine 2%) was administered with a disposable syringe for 3 months twice weekly. the follow up monthly opt starting showed gradual increase in the opacity of the radiolucent zone denoting bone apposition (fig. e). during the follow up period, the patient presented with an increase in weight. this was associated with features coinciding with moon face appearance and hirsutism associated with cushing s disease (fig. this alerted the surgical team to a possibility of cushing disease secondary to the local administration of corticosteroids. furthermore, laboratory investigations were ordered revealing that na / k level was normal and that serum acth was normal excluding full blown cushing disease. at this point, the operating team attributed the appearance to systemic effects secondary to escape of the intralesional steroids to the circulation. the radioleucent lesion decreased in size gradually over the follow up period. at the end of follow up period, accidental intraoperative bleeding took place due to injury of lingual tissues through perforated lingual plate and was stopped by cauterization and packing. the patient lost about 800 cc of blood and her hb dropped from 12.1 to 9.1 and arterial blood gas before recovery was 7.27 leading to mild acidosis, the anesthetic team managed the acidosis by oxygen administration postoperatively and sodium bicarbonate iv injection and fluid loss by crystalloids. the post operative biopsy was histologically assessed revealing fibrous tissue free from giant cell and showed with multiple bony trabeculae specks indicating healing process and bony nature of these specks were confirmed by immunohistochemistry using osteopontine (fig. however, the patient suffered from pain and infection related to lower left second molar attributed to loss of vitality, the causative tooth was extracted later due to endodontic difficulty. many studies considered the aggressive form of cgcg as true giant cell tumor of jaw based on resemblance in nature and histological features with giant cell tumor of long bones. findings of another study conducted by auclair. suggest that the gct and the cgcg represent a spectrum of a single disease process modified by the age of the patient and the site of occurrence. however, the term cgcg is used more in oral and maxillofacial field than the term giant cell tumor of bone. steroid injections were reported to decrease the inflammatory response within the lesion (triamcinolone for 6 weeks). the operating team believed the cushonoid presentation could be attributed to the escape of the intralesional steroids toward the highly vascularized lingual tissues. hence, although direct intravascular injection did not take place, however, the steroids found their way into the circulation through the perforated lingual cortex. in summary, this case report raises a warning sign when using intralesional corticosteroids in treatment of central giant cell granulomas. although this treatment line has been proved effective, however, caution should be taken to evade developing the complication for which intralesional injections were preferred to systemic injections. there is no conflict of interest between our case and any organization or any funding organization. yasser el hadidi : clinical instructor of oral and maxillofacial surgery, faculty of dentistry, ain shams university responsible for the case starting from presentation till follow up. amr amin ghanem : lecturer and consultant in department of oral and maxillofacial surgery ain shams university responsible for the treatment plan. iman helmy : professor of oral pathology performed the histopathological assessment pre and post treatment.	highlightscentral giant cell granuloma is considered one of the giant cell lesion affecting the oral cavity with variable histological forms ; may be aggressive or non-aggressive.many studies consider giant cell granuloma the same entity as giant cell tumor of long bones.different treatment modalities arise due to its different histological and clinical behavior. treatment may be medical or surgical.intralesional injection is one of the medical lines used in management of cgcg ; with high success and no side effects.in our clinical case, cushing developed in 9 years old patient forced our team to shift toward surgical line and stop intralesional steroid injections.
positron emission tomography (pet) is a procedure for evaluating biochemical and physiological processes by use of radiopharmaceuticals labeled with positron - emitting radionuclides. f - fluoro-2-deoxyglucose (f - fdg) pet scanning has been widely utilized for the diagnosis of various tumors ; for the staging and restaging of various malignancies, including head and neck, esophageal, lung, breast, colorectal, and gynecological cancers, as well as melanoma and lymphoma ; and for assessing brain function and heart muscle metabolism. however, the positive predictive value of f - fdg pet is low for cancer, including for prostate cancer. fdg uptake in the prostate is nonspecific for cancer and may result from an inflammatory condition such as prostatitis. moreover, the maximum standardized uptake value (suv) of fdg is not essential for the differential diagnosis of prostatic lesions ; nevertheless, several studies have reported a high correlation between fdg uptake and prostate cancer. for example, fdg uptake without coincidental calcification indicates the possibility of prostate cancer, suggesting the need for additional diagnostic methods, including digital rectal examinations (dres) and measurements of serum concentrations of prostate - specific antigen (psa). incidental fdg uptake in the prostate is often experienced in clinical practice, but suv alone can not determine whether increased uptake indicates a malignancy or a benign state. detection of second primary cancers, particularly early cancers that require radical treatment, is important because these cancers can significantly influence patient management. we therefore investigated the prevalence and clinical significance of incidental prostate fdg uptake and evaluated its impact on patient management. the medical records of all patients who underwent fdg pet at our hospital between october 2004 and march 2014 were reviewed. the study was performed with the approval and oversight of the institutional review board, which waived the requirement for informed consent because of the retrospective design of this study. during the study period, of the 47,109 males, 1,335 (2.83%) demonstrated incidental fdg uptake in the prostate. of the 144 patients who underwent prostate biopsy, the 45 who had undergone fdg pet for staging or restaging of prostate cancer were excluded. finally, 99 patients who underwent prostate biopsy following fdg uptake in the prostate were included in this study (fig. patients underwent fdg pet on one of the three scanners operated in our hospital (discovery st [ge healthcare, wauwatosa, wi, usa ] and biograph 16 and biograph 40 [siemens medical solutions, malvern, pa, usa ]). after their venous blood glucose concentration was confirmed as being < 150 mg / dl, the patients were intravenously injected with f - fdg (5.2 mbq / kg body weight), with pet / computed tomography (ct) scanning started 50 minutes later. images were reconstructed by using a three - dimensional ordered subset expectation maximization algorithm, and ct attenuation maps were used for attenuation correction. pet, ct, and fused pet / ct images were available for review and were displayed in axial, coronal, and sagittal planes on a viewer system. the suv was calculated according to the standard formula, with the use of lean body mass as the body weight. fdg uptake in the prostate was visually defined as positive or negative, with physiological uptake in the prostate urethra on coronal, sagittal, and axial views considered negative. patterns of fdg uptake (focal or diffuse) were evaluated on axial views (fig. baseline demographic and clinical characteristics, including patient age, serum total psa concentration, dre findings, and transrectal ultrasound (trus)-derived information (prostate volume) were obtained by reviewing medical records. psa concentrations were measured by using a psa - riact assay system (cis bio international, gif - sur - yvette, france). all patients underwent trus - guided laterally directed systematic 12-core biopsies of the prostate the primary study end point was the histological presence of adenocarcinoma of the prostate in the biopsy specimen. all samples were graded by genitourinary pathologists at our institute, with grading based on the gleason scoring system. the outcomes in patients with prostate cancer, including treatment methods, survival, and causes of death, were also assessed. patients were divided into three groups according to their prostate biopsy results, as follows : those with normal prostate, prostate cancer, and prostate invasion, except for prostate cancer. clinicopathological factors were compared in the benign lesion and prostate cancer groups by using pearson chisquare test for categorical variables and student t - test for continuous variables. factors associated with a diagnosis of prostate cancer were evaluated by using logistic regression analysis, with variables of p<0.1 on univariate analysis included in the multivariable analysis. correlations between clinical outcomes and the assessed variables are expressed as odds ratios (ors) and 95% confidence intervals (cis). the group of patients with prostate invasion, except for prostate cancer, was analyzed separately. all statistical analyses were performed by using the pasw statistics ver. 18.0 (spss inc., chicago, il, usa). during the study period, 92,203 patients (47,109 males and 45,094 females) underwent fdg pet. of the 47,109 males, 1,335 (2.83%) demonstrated incidental fdg uptake in the prostate. of the 144 patients who underwent prostate biopsy, the 45 who had undergone fdg pet for staging or restaging of prostate cancer were excluded. finally, 99 patients who underwent prostate biopsy following fdg uptake in the prostate were included in this study (fig. patients underwent fdg pet on one of the three scanners operated in our hospital (discovery st [ge healthcare, wauwatosa, wi, usa ] and biograph 16 and biograph 40 [siemens medical solutions, malvern, pa, usa ]). all patients fasted for at least 6 hours before the examination. after their venous blood glucose concentration was confirmed as being < 150 mg / dl, the patients were intravenously injected with f - fdg (5.2 mbq / kg body weight), with pet / computed tomography (ct) scanning started 50 minutes later. images were reconstructed by using a three - dimensional ordered subset expectation maximization algorithm, and ct attenuation maps were used for attenuation correction. pet, ct, and fused pet / ct images were available for review and were displayed in axial, coronal, and sagittal planes on a viewer system. the suv was calculated according to the standard formula, with the use of lean body mass as the body weight. fdg uptake in the prostate was visually defined as positive or negative, with physiological uptake in the prostate urethra on coronal, sagittal, and axial views considered negative. patterns of fdg uptake (focal or diffuse) were evaluated on axial views (fig. baseline demographic and clinical characteristics, including patient age, serum total psa concentration, dre findings, and transrectal ultrasound (trus)-derived information (prostate volume) were obtained by reviewing medical records. psa concentrations were measured by using a psa - riact assay system (cis bio international, gif - sur - yvette, france). the primary study end point was the histological presence of adenocarcinoma of the prostate in the biopsy specimen. all samples were graded by genitourinary pathologists at our institute, with grading based on the gleason scoring system. the outcomes in patients with prostate cancer, including treatment methods, survival, and causes of death, were also assessed. patients were divided into three groups according to their prostate biopsy results, as follows : those with normal prostate, prostate cancer, and prostate invasion, except for prostate cancer. clinicopathological factors were compared in the benign lesion and prostate cancer groups by using pearson chisquare test for categorical variables and student t - test for continuous variables. factors associated with a diagnosis of prostate cancer were evaluated by using logistic regression analysis, with variables of p<0.1 on univariate analysis included in the multivariable analysis. correlations between clinical outcomes and the assessed variables are expressed as odds ratios (ors) and 95% confidence intervals (cis). the group of patients with prostate invasion, except for prostate cancer, was analyzed separately. table 1 shows the clinical characteristics of the 52 patients with benign lesions and the 41 patients (44.1%) with prostate cancer. the mean age of all 93 patients was 65.8 years, with the mean ages of the benign lesion and prostate cancer groups being 62.8 and 69.6 years, respectively (p=0.001). patients with prostate cancer were more likely to have higher serum psa concentrations (p=0.001) and focal fdg uptake (p=0.036). only 1 of the 26 patients (3.8%) with serum psa<2.5 ng / ml had prostate cancer, compared with 40 of the 67 patients (59.7%) with serum psa2.5 ng / ml. the trus - determined prostate volume in all 93 patients was 38.8 ml and was similar in the benign lesion and prostate cancer groups (39.7 ml vs. 37.7 ml, p=0.672). in addition, mean maximum suv was similar in the two groups (p=0.116). univariate analysis showed that age (or, 1.08 ; 95% ci, 1.02 - 1.13 ; p=0.003), serum psa concentration (or. 1.31 ; 95% ci, 1.14 - 1.50 ; p=0.001), and the presence of focal lesions (or, 3.80 ; 95% ci, 1.10 - 14.50 ; p=0.043) were significant predictors of a final diagnosis of prostate cancer. multivariable analysis showed that the presence of focal lesions was an independent predictor of a diagnosis of prostate cancer (or, 5.50 ; 95% ci, 1.09 - 27.64 ; p=0.038), as were age (or, 1.06 ; 95% ci, 1.01 - 1.11 ; p=0.031) and serum psa concentration (or, 1.28 ; 95% ci, 1.10 - 1.47 ; p=0.001) (table 2). table 3 shows the clinical characteristics of the six patients with prostate invasion, except for prostate cancer. these six patients had a mean age of 59.0 years, a mean serum psa concentration of 3.2 ng / ml, and a mean prostate volume of 49.3 ml. histologically, two of these patients (33.3%) had bacillus calmette - guerin - granuloma, two (33.3%) had lymphoma, one (16.7%) had gastrointestinal stromal tumor, and one (16.7%) had colon cancer. table 4 shows the patterns of treatment in the 41 patients with prostate cancer, most of whom had organconfined tumors. of these 41 patients, 12 (29.3%) underwent radical prostatectomy and 25 (60.9%) received hormone therapy. of the 11 patients who died, 9 did so from primary cancer progression, with only 1 patient dying from prostate cancer. because f - fdg pet is becoming more widely used in cancer diagnosis, staging, and restaging, as well as in monitoring response to treatment, more incidental lesions, including prostate lesions, are being detected. the rise in incidental findings has created a problem for clinicians and often leads to unnecessary diagnosis and treatment, consequently increasing patient concern. thus, determining the clinical significance of fdg uptake in the prostate is extremely important. our results showed that prostate cancer was strongly associated with serum psa concentration, with tumors detected in 40 of 67 patients (59.7%) with serum psa2.5 ng / ml but in only 1 of 26 patients (3.8%) with serum psa<2.5 ng / ml. this difference is much greater than that observed in community - based populations. in general, prostate cancers are detected in approximately 2% of men with psa of 2 - 4 ng / ml and in 11% of those with psa of 4 - 10 ng / ml. our multivariate analysis also showed that the serum psa concentration was an independent predictor of a diagnosis of prostate cancer (or, 1.28 ; 95% ci, 1.10 - 1.47 ; p=0.001). these findings suggest that patients with fdg uptake in the prostate should undergo secondary evaluation, including measurement of serum psa, and that those with high serum psa should be evaluated by prostate biopsy. fdg pet is relatively insensitive in the detection of primary tumors, with sensitivities ranging from 19% to 64%. fdg uptake in the prostate is not specific to cancer, but is also positive in patients with benign prostatic hypertrophy (bph) and inflammatory conditions such as prostatitis. fdg uptake tends to be higher in poorly differentiated prostate tumors and in those with higher serum psa concentrations than in tumors with lower psa levels, a more localized clinical stage, and lower gleason scores, perhaps because the level of glucose transporter-1 expression rises with increasing malignancy grade. therefore, fdg pet may not be useful in the diagnosis or staging of clinically organ - confined disease, because the levels of fdg accumulation in normal prostate tissue, bph, and prostate cancer may overlap. however, focal fdg uptake is important because most of our patients with prostate cancer had focal fdg uptake, and other studies have reported that focal uptake in the peripheral zone correlates with prostate cancer. bph is characterized by nodular hyperplasia of the fibromuscular tissue and epithelium within the transition zone and periurethral area. by contrast, most prostate cancers arise from the peripheral zone. in some solitary tumors, maximum suv closely correlates with pathologic grade, which suggests that this parameter could be used for diagnosis and for assessing prognosis. in our study and others, however, there was no correlation between the maximum suv of uptake in the prostate and prostate cancer. although the degree of fdg uptake by primary prostate tumors was greater in patients with higher psa than in those with lower psa levels, psa levels can also increase in benign conditions such as bph. an ideal candidate for radical prostatectomy is free of comorbidities that may make the operation unacceptably risky. surgery is not indicated for patients with a large number of comorbidities or a short life expectancy, however, and these patients should receive hormone therapy or radiation therapy, which may effectively control tumor progression. of our 41 patients with prostate cancer, 37 (90.2%) received treatment, with only 1 patient (2.4%) dying of prostate cancer ; thus, a diagnosis of prostate cancer in patients with incidental prostate fdg uptake could have affected their prognosis. this study had several limitations, including its retrospective design, the relatively small patient cohort, and the significant differences in several clinical variables between the groups of patients with normal prostate and prostate cancer. moreover, our study was limited by selection bias, because prostate cancer was not histologically confirmed in all patients. although the most accurate tool for diagnosing prostate cancer is biopsy, some patients showing incidental focal fdg uptake on pet did not undergo prostate biopsy ; thus, the sensitivity of fdg pet could not be determined in this patient cohort. nonetheless, the present study is the first to analyze the clinical significance of incidental prostate fdg uptake and to evaluate the prevalence of prostate cancer according to psa and clinical outcomes. this study showed that patients with fdg uptake and elevated serum psa should be evaluated by prostate biopsy, especially if their scans show focal lesions. the prevalence of incidental fdg uptake in the prostate was not high ; however, patients with elevated serum psa had a high incidence of prostate cancer. therefore, patients showing fdg uptake in the prostate should undergo further evaluation, including measurements of serum psa concentration, with those having a high serum psa evaluated by prostate biopsy. the results of this study may be helpful in planning the management of patients with incidental fdg uptake in the prostate. because the study cohort was relatively small, however, further studies are needed to determine the clinical validity of this management strategy.	purposeto investigate the prevalence and clinical significance of incidental prostate fluoro-2-deoxyglucose (fdg) uptake and to evaluate its impact on patient management.materials and methodsof 47,109 men who underwent fdg positron emission tomography between 2004 and 2014, 1,335 (2.83%) demonstrated incidental fdg uptake in the prostate, with 99 of the latter undergoing prostate biopsy. the primary end point was the histological presence of prostate adenocarcinoma in the biopsy specimen. outcomes, including treatment methods, survival, and causes of death, were also assessed. factors associated with the diagnosis of prostate cancer were evaluated by using logistic regression analysis.resultspatients with prostate cancer were more likely to have higher serum prostate - specific antigen (psa) (p=0.001) and focal fdg uptake (p=0.036) than were those without. prostate cancer occurred in 1 of 26 patients (3.8%) with serum psa<2.5 ng / ml, compared with 40 of 67 patients (59.7%) with serum psa2.5 ng / ml. multivariable analysis showed that focal lesions (odds ratio [or ], 5.50 ; p=0.038), age (or, 1.06 ; p=0.031), and serum psa (or, 1.28 ; p=0.001) were independent predictors of prostate cancer diagnosis. most patients with prostate cancer had organ - confined tumors. of these, 12 (29.3%) underwent radical prostatectomy and 25 (60.9%) received hormone therapy. of the 11 patients who died, 9 died of primary cancer progression, with only 1 patient dying from prostate cancer.conclusionsthe prevalence of incidental fdg uptake in the prostate was not high, although patients with elevated serum psa had a higher incidence of prostate cancer. patients with fdg uptake in the prostate should be secondarily evaluated by measuring serum psa, with those having high serum psa undergoing prostate biopsy.
although diagnosis of moderate to severe cases of glaucoma is relatively straightforward, with diagnoses confirmed based on the presence of typical visual field (vf) defects on standard automated perimetry (sap) and corresponding signs of glaucomatous onh damage, the disease typically remains asymptomatic in the early stages. standard automated perimetry has been widely used for diagnosis, staging, and monitoring of glaucoma, but is only likely to detect functional deficits after at least 20%40% of rgcs have been lost. furthermore, visual field testing is often variable, and diagnosis may require repeated testing. identification of early glaucomatous structural damage, such as structural remodeling of the onh and inner retinal layers, is essential for early diagnosis, management, and prevention of vision loss. clinical assessment using multiple parameters, including peripapillary rnfl, onh, and macular parameters, has proven useful, not only for management and diagnosing glaucoma at various levels of severity, but for evaluating risk in glaucoma suspects. although the use of multiple parameters could increase false - positive results, structural damage may be present in one parameter and not the other, and thus it is helpful to have information from the macula, onh, and rnfl in glaucoma diagnosis. current sd - oct rnfl thickness parameters alone, however, have good diagnostic accuracy and help clinicians in determining severity stages and differentiating normal from glaucomatous eyes in the early stages. retinal nerve fiber layer parameters most extensively researched include : global average circumpapillary rnfl thickness (average of thickness measurements in the circumpapillary circle centered on the onh), thickness deviation map, and thickness parameters measured by quadrants or clock - hour sectors. in general, average circumpapillary rnfl thickness and inferior sector rnfl thicknesses are the oct parameters with the best diagnostic accuracy, with superior quadrant thickness values following in terms of sensitivity. this agrees with prior studies showing superior and inferior areas of the optic nerve most commonly affected glaucoma (fig. the diagnostic accuracy of ss - oct circumpapillary rnfl parameters are similar to those of sd - oct. for detection of glaucomatous damage, the sd - oct rnfl parameters have sensitivities ranging from 60% to 98% and specificities ranging from 80% to 95%. diagnostic performance decreases, however, for detection of early disease to 48% to 77% at the same specificity range for patients with minimal visual field losses. recent evidence shows, however, that even before the appearance of any vf defects on sap, rnfl average thickness parameters could detect glaucomatous damage : at 95% specificity, up to 35% of eyes had abnormal thickness values 4 years prior to detectable vf loss and 19% had abnormal values 8 years prior. the reproducibility of current sd - oct rnfl thickness parameters is excellent, with global average rnfl thickness generally being the most reproducible. retinal nerve fiber layer analysis from spectralis - oct (heidelberg engineering, heidelberg, germany) demonstrating glaucomatous damage. the rnfl evaluation in the right eye (od) shows abnormalities in the superior and inferior quadrants. evaluation of the macular region is also important in glaucoma diagnosis ; and oct has become an attractive means for identifying glaucomatous macular damage. glaucomatous damage of the macula is difficult to detect and generally overlooked or underestimated by clinicians when using the most common automated perimetry testing : 24 - 2 (6 grid) vf test. macular damage occurs early in the disease process and can take the form of arcuate defects, diffuse, widespread damage, and/or local damage, or some combination of these. in eyes without other macular pathology, there is less variability and a lower likelihood of the presence of anomalous structural characteristics in the macula compared with the optic disc and peripapillary region. assessment of the macula may also avoid some limitations of circumpapillary measurements, such as interference from retinal and optic nerve head vasculature, peripapillary atrophy, and variable placement of the measurement circle around the disc. structures that thin and diminish in glaucoma comprise a large proportion of total macular thickness ; these include rnfl and rgcs, but also inner plexiform layer. the rgc layer is thickest in the perimacular region, and its thinning is likely responsible for the decreased total macular thickness observed in glaucomatous eyes. preservation of central vision until late in the disease may suggest macular assessment is not useful for glaucoma detection. the macular rgc layer, however, is up to seven cells thick and contains more than 50% of the eye 's rgcs, and structural changes in the macula can thus easily precede detectable vf losses. additionally, changes in this layer are more likely be the result of pathologic change rather than normal variation, and thus measurements of this layer could potentially be more sensitive than rnfl thickness parameters. segmentation of the ganglion cell layer alone, however, remains very difficult due to low reflectivity. advances in oct have allowed for better quantitative evaluation of macular rgc damage and have enabled more detailed segmentation of the macular inner retinal layers and the entire macular thickness. although some studies show that macular rnfl (mrnfl) thickness in sd - oct is less accurate than circumpapillary rnfl in glaucoma diagnosis or detecting preperimetric glaucoma, novel segmentation algorithms have increased the diagnostic utility of macular evaluation. parameters, such as mrnfl, ganglion cell layer with inner plexiform layer (cgipl), and the ganglion cell complex (gcc), which includes mrnfl, ganglion cell layer, and inner plexiform layer, can distinguish glaucomatous eyes from those of healthy subjects and can differentiate between early, moderate, and advanced glaucoma. the gcc and gcipl parameters from both ss - oct and sd - oct carry a diagnostic performance at least equal to that of circumpapillary rnfl parameters ; all three parameters, including mrnfl, now have comparable diagnostic performance in detection of preperimetric glaucoma. once vf losses are apparent, there is a significant association of vf defect patterns with gcipl defect patterns. the most commonly observed gcipl and inner macular layer defect pattern in glaucoma subjects is thinning in the inferior perifoveal region, seen clinically as superior vf defects (figs. 2, 3). ganglion cell analysis from cirrus - oct (carl - zeiss meditec, dublin, ca, usa), which includes the combination of ganglion cell and inner plexiform layers (gcipl), shows thinning in the inferior and inferotemporal perifoveal regions of both eyes. macular parameters are of increasing importance in the management of glaucoma, especially given improvements in oct technology. recent software updates have reduced gcc segmentation errors in patients with macular degeneration, which should allow these patients to be evaluated for glaucoma with more confidence. posterior pole asymmetry analysis (ppaa) combines mapping of the posterior pole retinal thickness with asymmetry analysis between eyes and between hemispheres of each eye. posterior pole asymmetry analysis, although not presently having a built - in normative database, is highly reproducible and matches circumpapillary rnfl measurements in terms of diagnostic accuracy of early glaucoma. spectral - domain oct may also be superior to ss - oct in detecting gcipl thinning in the outer temporal zone, where the glaucomatous damage commonly occurs. however, sd - oct and ss - oct have similar glaucoma diagnosis abilities based on macular inner layer thickness analysis. ultimately, clinical assessment of the macular region is helpful in glaucoma diagnosis and evaluation and clinicians should incorporate macular scans into clinical protocols. patients with abnormal or borderline macular structural parameters likely require close follow - up and initiation of treatment to avoid vision loss. in addition to glaucoma, other macular diseases are also common in the aging population. these conditions may affect oct macular thickness measurements and render them useless for the evaluation of glaucoma, including diabetic retinopathy, macular edema, macular degeneration, and epiretinal membranes. advances in oct technology have also allowed for higher - resolution imaging of the onh and quantification of onh parameters, but the clinical value of oct onh parameters remains controversial. compared with previous oct technology, sd - oct relies less on data interpolation, and this has resulted in far better delineation of onh structures than could be achieved with td - oct. studies have found that onh parameters have an excellent ability to discriminate between normal eyes and eyes with even mild glaucoma. parameters such as rim area, vertical rim thickness, and vertical cup to disc ratio were found to have the greatest diagnostic ability and were as good as rnfl thickness parameters in diagnosing glaucoma. in some cases, onh parameters were found to be better at initial glaucoma detection and discriminating glaucoma and glaucoma suspect subjects from normal subjects. however, a number of studies show onh parameters are inferior to standard circumpapillary measurements for glaucoma detection. one study, although limited by using vfs as a reference standard, found that rnfl and macular parameters were significantly better for glaucoma diagnosis than onh parameters, especially for early - stage glaucoma. differences in these results are at least partially a consequence of the reference standard used to select cases and controls given that a reference standard must be employed to select cases and controls for any diagnostic accuracy study. there is a greater chance, for example, that patients with clearly abnormal optic disc features will be classified as cases if the reference criteria include optic disc appearance. this type of reference standard introduces a bias towards favoring accuracy of topographic onh parameters. furthermore, differences in commercially available oct devices could at least partially explain the differences between studies, such as differences in the acquisition speed, scanning rate, spatial resolution, layer detection algorithms, and analytical software. each of the different devices, therefore, may report different rnfl thickness values and onh measurements. additional reasons could also contribute to variable results, including differences in the number of subjects, differences in ethnicity, or differences in the representation of the different stages of glaucoma. consideration of a combination of circumpapillary and onh parameters is likely the best approach for glaucoma detection. given recent advances in sd - oct technology, evaluating the onh with oct is useful in the diagnosis and management of glaucoma. segmentation of the onh was greatly improved with new software, as was implementation of referencing the fovea 's position and bruch 's membrane as anatomic landmarks, which allowed for better measurement of the onh rim and rnfl thickness. bruch 's membrane opening minimal rim width (bmo - mrw), a relatively new anatomical parameter describing the neuroretinal rim, consists of the minimum distance between the bmo and the internal limiting membrane. bruch 's membrane opening minimal rim width has a high association with glaucomatous functional changes on sap and, compared with previous bmo methods, has a better ability to detect early glaucoma. it has an advantage over other sd - oct methods of neuroretinal thickness measurement by considering the variable orientation of rim tissue in the onh. rim area, however, appears to be a more useful onh parameter for detecting early glaucoma. ultimately, assessment of circumpapillary rnfl, macular, and onh parameters is useful for quantifying risk, diagnosis, and management of glaucoma at different levels of severity. detection of disease progression remains challenging in glaucoma due to the variable and slowly progressive nature of the disease, measurement variability of sap and of imaging devices, and the lack of a commonly acceptable reference standard. some eyes show structural changes in the onh or rnfl before any indication of glaucomatous damage can be detected with sap. because sd - oct is relatively new technology, only a few reports exist using sd - oct rnfl parameters for detecting glaucoma progression. most progression studies use td - oct due to the longer follow - up period. assessment of multiple oct parameters from the macula, onh, and rnfl is important, not only in diagnosis, but to detect disease progression and longitudinal change. retinal nerve fiber layer evaluation is less sensitive than vf when tracking progression in advanced cases due to a floor effect that occurs when the residual rgc layer has nearly diminished. although average rnfl thickness may be the main parameter to consider when evaluating for structural progression in advanced glaucoma patients, rnfl thicknesses in the inferior quadrant and inferotemporal sector may be the most predictive of progression (fig. retinal nerve fiber layer thinning in the superior quadrant has also been associated with subsequent vf losses in td - oct. spectral - domain oct instruments can not be used interchangeably, however, especially for glaucoma progression assessment, due to variability in circumpapillary rnfl thickness calculations. interestingly, other studies found that average macular thickness is more sensitive than circumpapillary rnfl for detection of disease progression. additionally, analysis of the total retinal thickness (gcipl, along with outer plexiform layer to rpe) may be more sensitive in detecting progression than circumpapillary rnfl. guided progression analysis from cirrus - oct (carl - zeiss meditec) demonstrating early glaucomatous progression. compared with baseline examinations from december 2007, the patient has statistically significant focal rnfl loss in the inferior quadrant in the left eye. more commonly, the corresponding rnfl thickness map to exam 6 may show a red wedge - shaped defect in the inferior quadrant of the onh. approaches that combine structure and function improve diagnosis both in cross - sectional and longitudinal investigations and algorithms that combine structural and functional measurements will likely improve the detection of glaucoma progression. prior studies suggest that using a combination of perimetry and circumpapillary rnfl values is the best approach when monitoring for progression, especially given that sd - oct rnfl values have a strong relationship to functional deficits. in td - oct, structural progression was associated with functional progression in preperimetric, glaucoma suspect, and glaucomatous eyes and eyes with significant sap progression have higher rates of rnfl thickness loss compared with nonprogressing eyes. clinically, it can be difficult to determine whether rnfl losses that precede sap changes reflect true progression. important from a clinical perspective, the 24 - 2 vf test, although the gold standard in sap for glaucoma evaluation, is not an ideal strategy for detecting glaucomatous damage of the macula ; the 10 - 2 vf will often detect damage missed with the 24 - 2 pattern. as oct evolves, it will continue to provide more accurate detection of progression and enhance our understanding of the structural pathogenesis of glaucoma, including the role of the lc in glaucoma progression. as the presumed site of axonal injury in glaucoma, the lc may play a role in neuronal death seen in glaucoma. lamina cribrosa microstructure likely provides the mechanical support to optic nerve fibers within the deep optic disc region. quantitative measurements of lc microarchitecture, such as pore diameter, pore area, and lc beam thickness, were found to have good reproducibility in a multimodal sd - oct with adaptive optics technology and offer the potential to serve as biomarkers for glaucoma progression. using a prototype ss - oct with 100,000 a - scan / s scanning speed and 5-m axial resolution, wang. demonstrated good reproducibility of lc parameters, including pore diameter sd, pore aspect ratio ; beam thickness, pore area, beam thickness sd, and beam thickness to pore diameter ratio. lamina cribrosa microarchitecture changes have been observed with ss - oct in glaucomatous eyes, and lc pore shape and size also have been correlated with the severity and progression of glaucoma. additionally, the lc was found to be displaced both anteriorly and posteriorly in glaucomatous eyes compared with age - matched healthy eyes, and thinner lc was associated with glaucoma progression. overall, the structural thinning and displacement of the lc likely cause lc pores to deform, impeding axoplasmic flow within the optic nerve fibers and disrupting transport of factors crucial for the survival of rgcs. in addition, there may be biological changes that occur due to deformations of the lamina in the axons or microglia that result in axonal stress or rgc impairment or death. although the lc 's role in glaucoma progression is yet to be fully determined, ss - oct and sd - oct have undoubtedly improved current understanding of the lc and its microarchitecture. optical coherence tomography has impacted the ways that patients are diagnosed and followed clinically and remains a dynamic and evolving imaging modality. optical coherence tomography technology and software algorithms are improving and newer technologies are continually under development, increasing oct 's clinical utility. clinicians should be aware of oct 's limitations and should be aware of possible scan artifacts. clinical decisions should never be driven by oct results alone, but should also be based on a complete ophthalmic examination and vf assessment.	optical coherence tomography (oct) has established itself as the dominant imaging modality in the management of glaucoma and retinal diseases, providing high - resolution visualization of ocular microstructures and objective quantification of tissue thickness and change. this article reviews the history of oct imaging with a specific focus on glaucoma. we examine the clinical utility of oct with respect to diagnosis and progression monitoring, with additional emphasis on advances in oct technology that continue to facilitate glaucoma research and inform clinical management strategies.
the control of dental biofilm is one of the cornerstones of preventive dentistry and can be achieved by mechanical means, use of chemical agents, or a combination of the two.1 mouth - washes are used as adjuvant agents in daily oral hygiene routine, aiding in the chemical control of dental biofilm. in brazil, most mouthwashes are freely available at pharmacies, drugstores, supermarkets, and other commercial establishments and usually do not require a prescription from a dentist, making these products readily available to children and adults. the indiscriminate use of mouthwashes by the general population has generated concern because the presence of acid components in their formulations could make the products potentially erosive to hard dental tissue over time.2 dental erosion is the progressive and irreversible loss of tooth enamel as a result of chemical processes not involving bacterial action.3 previous studies have demonstrated that several mouthwashes available in the brazilian2,4 and uk5 markets present low endogenous ph. a ph equal to or less than 5.5 is traditionally considered critical for enamel dissolution, although mineral loss may begin at higher ph.6 the purpose of this study was to evaluate the endogenous ph, titratable acidity, and total soluble solid content (bx) of mouthwashes available in the brazilian market. ten commercial brands of mouthwashes comprising various active ingredients were selected for this study (table 1). the products were evaluated in a randomized experiment, with 3 repetitions for each sample, with values averaged to provide a single value per sample. data were collected by a single calibrated examiner (kappa=0.83) and recorded in study - specific charts. the endogenous ph of each mouthwash was measured immediately after package was opened at room temperature (20c) using a ph meter (tec-2 ; tecnal, sion paulo, sp, brazil) accurate to 0.1 mm. titratable acidity was measured according to the method adopted by the association of official analytical chemists, that is, the amount of 0.1 n potassium hydroxide (koh) solution needed for the product to reach ph equal to or greater than neutral ph. an abbe refractometer (pzo - rl1, warszawa, poland) was used to measure bx. mean values and standard deviations were analyzed statistically using spss statistical software (spss inc., distribution of the mouthwashes according to mean values and standard deviations is presented in table 2. ph values ranged from 3.56 (peroxyl) to 7.43 (cepacol), and three mouthwashes (clinerize, listerine cool citrus, and peroxyl) had ph less than the critical value of 5.5, thus classified as potentially erosive. oral b and clinerize demonstrated the lowest (4.7%) and the highest (23.70%) bx, respectively. mouthwashes have been used for centuries for medicinal and cosmetic purposes, but it is only in recent years that the rationale for use of the active ingredients of these products has been subject to scientific research and clinical trials.7 based on studies published in the brazilian2,4 and international5,810 dental literature, the present investigation evaluated three important physicochemical properties of mouthwashes commercially available in brazil : ph, titratable acidity, and bx. although a ph value equal to or less than 5.5 is considered critical for enamel dissolution, mineral loss may begin even at higher ph;6 therefore, the prolonged use of oral rinses with ph below this value may be potentially harmful to dental tissue. in the present study, three mouthwashes were classified as potentially erosive (ph<5.5), corroborating the findings of previous investigations.2,4,5,8,10 the low ph of oral care products increases the chemical stability of some fluoride compounds and favors the incorporation of fluoride ions into the lattice of hydroxyapatite and the precipitation of calcium fluoride onto the tooth surface.11 based on this statement, product labels were examined to identify mouthwashes containing fluoride. among the three mouthwashes with ph less than 5.5, only oral - b has fluoride (0.05% naf) in its formulation. the label of the other two mouthwashes with ph below the critical value for enamel dissolution (listerine cool citrus and periogard) did not list fluoride in their ingredients. lack of fluoride and low ph may make these products harmful to dental tissues if not used carefully. although mouthrinses have been formulated as pre- and post - brushing products for routine use, findings of a previous in situ study evaluating the erosive effects of some mouthrinses on enamel have suggested that low ph mouthrinses should not be considered for long - term or continuous use and never as pre - brushing rinses;8 however, it must be emphasized that erosive potential of a substance can not be attributed exclusively to ph.4 other important physicochemical properties, such titratable acidity, bx, and viscosity, should be also be considered. in this study, titratable acidity determined the amount of acid present and the volume of koh necessary to buffer the test solution, a characteristic directly related to the buffering capacity of the saliva. substances with low titratable acidity are readily neutralized by oral fluids, while those with high titratable acidity cause a prolonged drop in ph and greater demineralization of dental tissues.12 in the present study, prevident 220 exhibited high titratable acidity even with ph close to 6. a possible explanation for this result is that some ingredients present in its composition did not react with the base used to neutralize the mouthwash (0.1n koh). four of the mouthwashes exhibited bx greater than 20%, that is, 20 g of solids dissolved in 100 g of mouthwash. among the tested mouthwashes, clinerize presented the highest bx. lack of similar studies evaluating bx of oral rinse products hinders comparison of the present results to data published in the literature. brix refractometry is a physical method for measuring the amount of soluble solids (sugar, salts, proteins, acids, etc) present in an aqueous solution.13 the majority of medicinal formulations, if not all, have some side effects, whether local or systemic. in each case risk clearly will be influenced by the likely incidence and severity of side effects. in the case of dental erosion, the regimen and duration of use of a potentially erosive agent will be critical to the outcome. mouthwashes in general have similar regimens of use, namely 1020 ml rinsed twice a day for 3060 seconds. it is recommended that low ph oral rinse products be used as short to medium term adjuncts to oral hygiene and never as prebrushing rinses.8 the findings of this investigation can not be directly extrapolated to the clinical situation ; however, results indicate that some of the mouthwashes evaluated exhibited low endogenous ph, even below the critical value for enamel dissolution (5.5), high titratable acidity, and high total soluble solid content, which may make these products potentially erosive to dental tissue if not properly used.	objectives : to evaluate in vitro the endogenous ph, titratable acidity and total soluble solid content (tssc) of mouthwashes available in the brazilian market.methods:the study sample was composed of 10 commercial brands of mouthwashes based on different active ingredients : cepacol, clinerize, equate, listerine cool citrus, oral - b, periogard, peroxyl, plax overnight, prevident 220 and sanifill. the experiments were performed in triplicate. the endogenous ph was evaluated by potentiometry, titratable acidity was evaluated by the addition of 0.1n koh increments to the mouthwashes, and tssc readings were performed by brix refractometry using the abb refractometer.results:ph values ranged from 3.56 (peroxyl) to 7.43 (cepacol) and three mouthwashes presented phs below 5.5. the titratable acidity values ranged from 0.007 (periograd) to 0.530 (prevident). oral b and clinerize presented the lowest (4.7%) and the highest (23.70%) tssc, respectively.conclusions:some of the mouthwashes evaluated in this study presented low endogenous ph, even below the critical value for enamel dissolution (ph<5.5), high titratable acidity and high tssc, and may be potentially erosive to the dental tissues if not properly used.
metabolic syndrome affects a significant proportion of the population and is becoming increasingly more prevalent in adolescents [14 ]. the syndrome embodies many components including central obesity, insulin resistance, dyslipidemia, and hypertension [3, 5 ]. in addition, the syndrome features a chronic low grade inflammatory state, vascular endothelial dysfunction, and a prothrombotic environment. long standing metabolic syndrome predisposes an individual to type 2 diabetes (t2d), atherosclerosis, microvasculature disease (of retina), stroke, renal injury, and neuropathy. due to the complicated mechanisms involved in the syndrome and its sequelae, current clinical standard of care embodies polypharmacological therapeutics aimed at controlling atherogenic dyslipidemia, hyperglycemia, and hypertension as well as intervening in secondary conditions such as renal dysfunction, stroke, and microvascular disease related to retinopathy. development of new chemical entities with the potential to control more than one risk factor is hampered by the paucity of highly translational animal models. the most frequently used rat and mouse models for obesity, metabolic syndrome, and t2d have defects in the leptin pathway. the zucker diabetic fatty (zdf) has been a gold standard for this disease complex and as such has many of the characteristics of the human condition but it becomes obese due a leptin receptor defect and becomes diabetic before the animals are mature. to circumvent these zdf complications, the zdsd / pco (zdsd) rat has been developed to be a more translational model of obesity, metabolic syndrome, and diabetes and to aid the study of these conditions and the development of drugs that could assist in control and treatment. the model was developed by crossing a homozygous lean zucker diabetic fatty (zdf) male rat with a substrain of the crl : cd (sd) rats that had been selectively bred for high fat diet induced obesity [6, 7 ]. the standard crl : cd (sd) rat is a substrain of sd rats that is significantly heavier and more obese than other lines of sd rats ; a percentage of these rats is very susceptible to develop obesity, fed high fat diets [6, 7 ]. the original design was to combine the defect in -cell gene transcription that is found in lean and obese zdf rats with the obesity of the crl : cd (sd) model to produce an obese diabetic model that preserves the critical leptin pathway. the animals were fed regular rodent chow (purina 5008) during the 12 years of the model development process. the offspring from the initial crosses were screened and selected for obesity, the propensity to become diabetic and the expression of the other characteristics of metabolic syndrome. this model has been selectively inbred for > 30 generations. in the current study, zdsd rats were followed for 6 months ; clinically relevant metabolic endpoints were included throughout the study to extensively characterize the phenotype and development of metabolic disease. the authors believe that the zdsd rat displays a phenotype with close similarity to metabolic syndrome / t2d in humans further representing a one rat, many models tool that will enable the study of early metabolic dysregulation, overt diabetes, and late stage complications of t2d in one rat. it is the sum of the presence of all the characteristics of the human prediabetic state, t2d, and the growing number of associated comorbidities that creates the belief that the zdsd rat is highly translational to the human condition. it is our belief that this model will reduce development and clinical costs and facilitate discovery of new agents with potential to impact multiple components of the disease. this study was designed to follow a cohort of male zdsd rats from early in life and as they progress through obesity / metabolic syndrome into full t2d with beta cell failure. the study objectives included comparison of the model 's disease progression to the development of diabetes in the human condition. this paper will be followed by a publication that will use these pieces of data to compare this progression in the zdsd rat to what occurs in the human condition using a model similar to a published paper. twenty - four male zdsd rats (zdsd / pco) of comparable body weight were sourced at 6 weeks of age for the study (preclinomics inc., indianapolis, in, usa). animals were housed 2 per cage, fed purina 5008 chow, and given water ad libitum for the duration of the study unless otherwise noted. the definition of the permanent onset of diabetes in this model is quite simple, two subsequent weekly morning glucose readings of over 250 mg / dl. when this occurs, the animals will consistently continue to remain hyperglycemic and continue to get more overtly diabetic. for the purposes of this publication, three metabolic states are defined and used to describe the progressive disease states of the model that are typically used in describing the human conditions : (1) metabolic syndrome / prediabetic hyperglycemia : fed / fasted glucose levels above 125 mg / dl, 50% increase in glucose auc in the ogtt and a 25% increase in hba1c ; (2) diabetic : fed glucose levels above 250 mg / dl, > 100% increase in hba1c and a > 200% increase in auc in the ogtt ; and (3) overt diabetes : equaling or exceeding values in (2) above decreases in insulin levels and weight loss. depending on the methodology used for glucose measurements in an experiment body weight was recorded and whole blood was collected every 2 weeks at 6 a.m. for purposes of obtaining fed analyte values. the whole blood (nonfasted samples) was collected by tail clip at 6 a.m. between 731 weeks of age for assessment of fed glucose levels using statstrips (statstrip xpress glucometer, nova biomedical, waltham, ma, usa). animals were then fasted for 6 hours and a sample of lithium heparin anticoagulated blood (100150 l) was taken from a second tail clip. hba1c was measured in 20 l of whole blood prior to processing to plasma (au480 ; beckman coulter, brea, ca, usa). glucose, triglycerides, and cholesterol were assayed on fresh plasma samples using a clinical chemistry analyzer (au480 ; beckman coulter, brea, ca, usa). oral glucose tolerance tests (ogtts) were performed monthly after a 6-hour fast using a glucose load (2 g / kg, p.o.) and sampling from the tail at 0 (prior to glucose administration), 15, 30, 60, and 120 minutes postglucose load. the 7-week ogtt was done using blood glucose (statstrip) methodology ; plasma insulin levels were assayed at each time point of the ogtt. for subsequent ogtts, glucose was determined from fresh plasma on the au480 and insulin levels were determined from frozen plasma using a rat / mouse meso scale discovery kit (k152bzc, rockville, md, usa). to insure that fasting did not negatively affect the phenotype, the fasts were limited to 6 hours (6:00 a.m.12:00 p.m.) every two weeks. the effect of age on measured parameters (body weight, triglycerides, cholesterol, hba1c, nefa, glucose, glucose auc, insulin, and insulin auc) was determined using one - way anova with repeated measures (p < 0.05) and when necessary tukey 's multirange t - test was performed (p < 0.05). the effect of age on the 5-point oral glucose tolerance tests (ogtt) was determined using two - way anova with repeated measures (p < 0.05). a mahalanobis distance plot and t statistic were used to identify outliers based on average fed glucose levels over the course of the study. a technical issue regarding the collection and processing of fasted blood samples was noted at the 17-week time point. this necessitated elimination of these samples (glucose, triglycerides, cholesterol, and insulin) from analysis. fasting plasma glucose and insulin levels were used to calculate the homeostasis model assessment of -cell function (homa-) and insulin sensitivity index (isi). homa- was calculated as [20 fasting insulin (u / ml)/fasting glucose (mmol / l) 3.5 ]. insulin sensitivity was calculated from the ogtt using matsuda index (1,000,000/square root fasting glucose fasting insulin glucose auc insulin auc). homeostatic assessment of insulin resistance (homa ir) was calculated using fasting glucose (mmol / l) fasting insulin (u / ml)/22.5 [10, 11 ]. male zdsd rats weighed on average 221.9 2.0 g at 7 weeks of age as study observations began. body weights reached a plateau and peaked at 23 weeks of age (564.4 8.2 g). thereafter there was a significant (p < 0.0001) decline (8.2% from the peak) in body weight over the next 8 weeks (figure 1). blood glucose (bg) values in the fed state were obtained from whole blood using statstrips. bg in male zdsd rats progressed significantly (p < 0.0001) from prediabetic hyperglycemia to diabetic levels with age (table 1). most animals (56.5%) were hyperglycemic as early as 7 weeks of age with a mean fed bg of 127.4 2.3 mg / dl. bg levels rose steadily, reaching 227.3 21.7 and 299.91 26.4 mg / dl at 19 and 21 weeks, respectively, such that most animals (56.5%) could be classified as diabetic by 21 weeks. bg levels continued to increase such that 100% of animals were diabetic by 27 weeks of age (478.4 15.6 mg / dl). bg levels rose to 527.8 11.0 mg / dl at 31 weeks of age. similarly, mild hyperglycemia in the fasted state was also observed in animals as young as 7 weeks of age (125.7 2.1 mg / dl). fasted plasma glucose levels remained relatively stable up to 19 weeks of age. then significant (p < 0.0036) increases were noted (151.65 15.4 versus 236.8 27.6 mg / dl at 19 and 21 weeks, resp.). fasted plasma glucose values progressed steadily to 498.1 10.0 mg / dl in 31-week - old animals (figure 2). since fed blood glucose (bg) and fasted plasma glucose were taken using different methods, statistical comparison was not appropriate. comparison of these two methods of glucose determination demonstrated that the bg levels run about 88% of the plasma glucose levels. this would effectively make the difference between fed bg and fasted plasma glucose significantly greater than what is shown in figures 2 and 11. all animals that were analyzed in this study were overtly diabetic at the end of the study (as defined in methods above). chronic progressive hyperglycemia with an average fed bg level of 281.5 11.1 mg / dl was confirmed as evidenced by a significant (p < 0.0001) increase in glycated hemoglobin (hba1c) levels taken throughout the study period. hba1c values in 7-week - old crl : cd (sd) control rats ranged from 3.35 to 3.60 and remained relatively stable throughout their life span (data not shown). hba1c levels in 7-week - old male zdsd (3.45 0.03) rats were the same as those of the control animals (3.44 0.03%). despite relatively steady morning glucose levels between 7 and 15 weeks of age (figure 2), the hba1c levels increased by 42% from baseline values during this period (3.45 0.03 versus 4.92 0.05%) suggesting that there likely were unseen hyperglycemic excursions during this period (see discussion on pm glucose levels). thereafter, hba1c levels rose rapidly, reaching 10.88 0.23% (figure 3). oral glucose tolerance tests (ogtts) were performed every four weeks during the experiment. for clarity purposes, significant abnormalities in glucose disposal were observed in aging zdsd rats during performance of oral glucose tolerance tests. glucose values taken just prior to glucose load (time zero) increased significantly (p < 0.0001) over time (124.9 2.1, 149.4 2.4, 314.7 34.0, and 498.1 10.0 mg / dl for 7, 15, 23, and 31 weeks, resp.). similarly, the peak glucose excursion and the glucose levels 2 hours after administration of glucose load rose dramatically as animals aged, creating a significant (p < 0.0001) age - dependent increase in the area under the curve (10 for glucose disposal, 18.4 0.2, 33.5 0.4, 58.5 4.2, and 72.1 2.4 for 7, 15, 23, and 31 weeks, resp.) in contrast to glucose values obtained during ogtt, fasted insulin values obtained just prior to glucose load increased significantly (p < 0.0001) in animals 7 to 19 weeks of age, after which a precipitous drop in insulin level was noted. pg / ml) and then fell significantly (p < 0.0001) from the peak to 189.4 27.4 pg / ml in 31-week - old animals (figure 5). in 7-week - old animals, the insulin response to glucose loading was rapid, occurring at 15 minutes, and represents a dramatic 4.3-fold increase over values prior to glucose loading (448.8 37.4 versus 1935.3 106.4 further aging resulted in reduction of the magnitude of the insulin response in all age groups (1.7-, 1.35-, and 1.9-fold for 15, 23, and 31-week - old animals, resp.). the curve for the insulin response to glucose load revealed an increasingly flat profile as animals aged, such that insulin responses in 31-week - old animals were minimal throughout the 2-hour period. the area under the curve (10) for the insulin response increased significantly (p < 0.0001) and peaked at 19 weeks followed by a significant drop from peak in 23- (p < 0.0016) and 31- (p < 0.0001) week - old animals (118.4 5.3, 225.3 17.5, 146.5 21.7, and 32.1 5.2 at 7, 19, 23, and 31 weeks, resp.) (figures 6(a) and 6(b)). fasting plasma glucose and insulin levels obtained from ogtt 's were used to calculate the homeostasis model assessment of -cell secretory capacity. the values followed the same time course as did the fasted insulin levels presented in figure 3, that is, a progressive increase 719 weeks of age, followed by a precipitous decline to values indicating very little insulin secretory capacity at 31 weeks. homa- values were 3.92 0.31 at 7 weeks of age, peaked at 19 weeks (12.6 1.22), and fell abruptly (p < 0.0001) to 0.44 0.1 at 31 weeks, indicating only minimal secretion at 31 weeks (figure 7). insulin sensitivity in zdsd animals reflects the progressive hyperglycemia and augmented insulin secretion before beta cell loss as animals age. isi in 7-week - old animals (99.3 5.8) decreased quickly to 61.9 7.5 at 11 weeks (p < 0.0002) and plateaued at 19 weeks (33.01 4.7). this decline represents a significant 67% decrease in sensitivity within the 719 week old animals (p < 0.0001) (figure 8). fasted triglyceride levels increased significantly (p < 0.0001) compared to that of 7-week - old rats and peaked at 19 weeks of age (107.5 3.7 and 292.5 17.8 mg / dl, resp.). a slight but not significant decrease (p < 0.1170) to 251.1 16.7 mg / dl was apparent at 21 weeks ; however, levels remained relatively stable for the remainder of the study. average triglyceride levels were 230.3 8.8 mg / dl over the last 12 weeks (figure 9). fasted cholesterol levels increased significantly (p < 0.0001) over the course of data collection, ranging from 95.4 1.1 to 127.9 2.4 mg / dl at 7 and 31 weeks, respectively (figure 10). over the last decade, there has been a global increase in obesity and type 2 diabetes (t2d). in 2011 ninety percent of individuals with diabetes were reported as having t2d, largely the result of obesity and lack of physical activity (who, 1999, 2003). t2d is thought to occur, in large part, as a consequence of the development of metabolic syndrome which presents as a cluster of conditions defined by obesity, insulin resistance, hypertension, hyperlipidemia, and hyperglycemia. in humans, the natural course of t2d progresses from insulin resistance to compensatory hyperinsulinemia resulting in beta cell failure and overt diabetes. development can be initiated in humans by genetic and/or environmental factors and is thus considered a multifactorial disease. genetic polymorphisms linked to t2d have been identified in at least 3 dozen genes, most of which influence both hepatic and peripheral insulin resistance, and adipogenesis as well as beta cell mass and function ; however, no mutations in leptin signaling have been identified in human t2d [18, 19 ]. since obesity is a major environmental factor predisposing humans to t2d, it becomes necessary for a translational animal model to become obese by a polygenetic mechanism. the zdsd rat was developed to address the disparity in disease development from the human condition that is apparent in commonly used rodent models. previous models (zucker fatty rat, zdf rat, ob / ob mice, and db / db mice) have proven useful in drug development and in research in the areas of obesity, metabolic syndrome, and diabetes. however, their mutations compromise the leptin pathway which is essential for the normal function of cns obesity and feeding behavior. since leptin pathway mutations are a very rare condition in the human population, the continued exclusive use of these models will impede the studies of cns mechanisms that are central to combating the obesity conditions in the human population. the zdsd rat model is similar to an animal model that was developed at the university of california at davis [23, 24 ], in as much as it involved crossing homozygous lean zdf rats with obese crl : cd (sd) rats. although direct comparisons have not been made, the model and the disease are similar to the zdsd and thus it is probable that it will perform similarly in many experiments. the zdf rat is the most commonly used leptin deficient rat model of metabolic syndrome, obesity, and diabetes. hyperglycemia with hyperinsulinemia and dyslipidemia are apparent as early as 8 weeks of age, before the animal reaches maturity, and are sustained throughout life [25, 26 ]. the time course for progression to beta cell failure is rapid in this model, leading to the absence of the extended metabolic syndrome (prediabetic) condition common to human t2d. hypertension is a major contributor to the end - organ damage seen with long standing diabetes and as such is included in the cluster related to metabolic syndrome. reports detailing blood pressure in zdf rats vary greatly ; when assessed by radio - telemetry, blood pressure is normal, but hypertension has been reported when pressure is assessed by tail cuff. although marked renal damage with albuminuria is also apparent with long standing diabetes, this model also has significant hydronephrosis which compromises its value for application. while this rat exhibits some components of metabolic syndrome, the rats progress rapidly to overt diabetes. the largest divergence from the human t2d for this model resides in the lack of a significant prediabetic period and the development of overt diabetes in the adolescent state. in contrast to the zdf male, the male zdsd rat maintains a moderately long period (approximately eight weeks) of prediabetic metabolic syndrome with obesity, followed by progression to overt diabetes and its complications. the female zdsd rat does not become diabetic on ordinary rat chow such as 5008, but under the right conditions, feeding the females diabetogenic diet (5sca or rd12468) can trip them into overt diabetes. when compared to the rodent models extensively used over the past two decades, the zdsd rat appears to be a better translational model of obesity, metabolic syndrome, and diabetes as well as its secondary complications associated with t2d. the zdsd rat 's polygenic phenotype, as it relates to metabolic syndrome, includes visceral obesity, insulin resistance, hyperglycemia, dyslipidemia, and mild hypertension. in addition, zdsd rats spontaneously develop diabetes independently of special diets or monogenic mutations making this a much more translational model. moreover, the development of diabetes can be synchronized with the feeding of diabetogenic diets (purina 5sca or research diets 12468) beginning at 16 weeks of age ; this has been described in previously published papers [29, 3133 ]. as demonstrated here, the zdsd males become spontaneously hyperglycemic when maintained on purina 5008 and express a heterogeneous onset of overt diabetes which mimics that seen in t2d humans. when either spontaneous or synchronized diabetes is inadequately treated, this model develops the translational complications of a t2d profile with its sequelae. the resultant naturally occurring phenotype, in the presence of a functional leptin pathway, translates to a rodent model more characteristic of the disease conditions as expressed in the human population. the characteristics of the zdsd rat, as it goes through the prediabetic condition (metabolic syndrome) into overt diabetes, are described in this paper. from 7 to 19 weeks of age the zdsd male rat demonstrated obesity, impaired glucose tolerance, dyslipidemia, and prediabetic hypertension. time dependent elevation in hba1c, which occurs during the metabolic syndrome / prediabetic phase, was also noted. although the morning fed glucose levels are quite stable in animals 715 weeks of age, they are higher than those in control rats ; this study did not include blood glucose from control animals. more recent data using continuous blood glucose monitoring and statstrip reading in the am and pm have reliably demonstrated that afternoon glucose levels are significantly higher than the morning levels in this model. they are not strictly nocturnal feeders and tend to eat a significant amount during the day. it should also be noted that the late afternoon glucose elevations might be related to the human dawn effect since late afternoon just before lights go off is equivalent to the rat 's morning since they are nocturnal animals. as metabolic syndrome developed in the zdsd rat, insulin levels increased in a compensatory fashion to combat increasing insulin resistance. shortly after the peak in insulin levels at 19 weeks of age, insulin values fell precipitously as beta cells began to fail. progressive decreases in beta cell secretory capacity were demonstrated by the dramatic decrease in insulin and in homa- calculated during this time. as the zdsd model became more diabetic, further abnormalities in glucose disposal became apparent as the magnitude and duration of the glucose excursion following glucose load area under the glucose curve in the ogtt increased while area under the insulin curve was drastically diminished. in fact, in 31-week - old animals, the insulin response to glucose load was barely visible. the pattern of changes in glucose homeostasis in zdsd is very much like that in humans as the disease progressed. initially, obesity leads to insulin resistance due to many factors, including contributions from adipokines and cytokines from visceral fat. glucose levels are temporarily maintained at normal level through hypersecretion of insulin from beta cells. insulin resistance continues to progress until beta cells begin to cease functioning, insulin levels drop, and overt diabetes ensues. cholesterol levels increased as animals age ; interestingly, the rate of increase changed upward as zdsd rats became overtly diabetic. furthermore, although this study does not demonstrate the long - term complications of diabetes, we believe that this model will also develop complications that are seen in the human population with long - lasting, inadequately controlled diabetes. indeed, in other studies, the zdsd rat has been shown to develop diabetic osteoporosis, diabetic nephropathy, diabetic connective tissue changes [29, 32 ], and diabetic neuropathy and hypertension prior to development of diabetes. a review of the literature on the development of t2d in the human population demonstrated a high degree of translation with the zdsd model. the review and analysis by many authors have led them to an understanding of the time course of events that occur in humans in the prediabetic and diabetic conditions [15, 3739 ]. figure 11(a) captures this in a single graphic that represents the progression of the natural history of type 2 diabetes in the human population. this figure produced by kendall and bergenstal represents the changes that occur during the prediabetic stages through the overt diabetic stages of diabetes. similar to figures 11(a) and 11(b) graphs from this paper have combined to compare the pattern which is observed in zdsd rats where insulin resistance is represented by the progressive decrease in glucose disposal (increase glucose auc) and insulin response is represented by the insulin response to a glucose load during an ogtt. insulin auc in the prediabetic state is exaggerated while a steady decline in insulin response is noted during the diabetic state. the current study demonstrates similarities in fasting and fed glucose levels (figure 2), insulin levels (figure 5), the insulin auc (figure 6), and the homa- (figure 7) in the progression of the disease from the prediabetic state to the overt diabetic state. this comparison appears to support the case for translatability of the progression of t2d in zdsd model to the human disease. the zdsd rat model exhibits all stages and multiple components of metabolic syndrome / t2d and the complications of these conditions. although further research is clearly needed, this study demonstrates that the zdsd model may represent a truly translational model for the investigation of causative mechanisms, targeted interventions, and treatments for this multifaceted disease. the authors believe that, in addition to the scientific merits of the zdsd, the use of the zdsd in the one rat, many models paradigm presents an opportunity for investigators to significantly reduce costs while evaluating compound effects on the multiple components of t2d and its sequelae.	metabolic syndrome and t2d produce significant health and economic issues. many available animal models have monogenic leptin pathway mutations that are absent in the human population. development of the zdsd rat model was undertaken to produce a model that expresses polygenic obesity and diabetes with an intact leptin pathway. a lean zdf rat with the propensity for beta - cell failure was crossed with a polygenetically obese crl : cd (sd) rat. offspring were selectively inbred for obesity and diabetes for > 30 generations. in the current study, zdsd rats were followed for 6 months ; routine clinical metabolic endpoints were included throughout the study. in the prediabetic metabolic syndrome phase, zdsd rats exhibited obesity with increased body fat, hyperglycemia, insulin resistance, dyslipidemia, glucose intolerance, and elevated hba1c. as disease progressed to overt diabetes, zdsd rats demonstrated elevated glucose levels, abnormal oral glucose tolerance, increases in hba1c levels, reductions in body weight, increased insulin resistance with decreasing insulin levels, and dyslipidemia. the zdsd rat develops prediabetic metabolic syndrome and t2d in a manner that mirrors the development of metabolic syndrome and t2d in humans. zdsd rats will provide a novel, translational animal model for the study of human metabolic diseases and for the development of new therapies.
the 2013 aha / acc guideline on lifestyle management to reduce cardiovascular risk recommends a healthy dietary pattern with 5% to 6% of calories from sfa for lowering ldlc, a primary target for cvd risk reduction. both mufa and pufa the food sources of mufa in the mediterranean diet provide many micronutrients and bioactives that also may confer cardioprotective benefits. the predimed (prevencin con dieta mediterrnea) trial reported that a mediterranean diet supplemented with mufarich foods that included either extravirgin olive oil or mixed nuts (walnuts, almonds, hazelnuts) reduced the incidence of major cvd events by 30% after approximately 5 years in men and women (50 to 80 years of age) at high risk for cvd. avocados are another nutrientdense source of mufa, rich in vitamins, minerals, fiber, phytosterols and polyphenols that have not been studied extensively. the hass variety, which is mainly consumed in the united states, is relatively high in mufa and other fatsoluble vitamins. one hass avocado (136 g, without skin and the seed) contains 13 g of oleic acid, which is similar to the amount of oleic acid in 1.5 oz (42 g) almonds or 2 tablespoons (23 g) of olive oil. based on their fatty acid and nutrient profile, evidence about the beneficial effects of avocados on the lipid / lipoprotein profile is based on relatively few diet studies. studies with avocados (0.5 to 2 avocados per day) have reported similar or greater tg, ldlc, and tclowering effects while maintaining hdlc compared with lowfat, cholesterollowering diets. there are several limitations, however, of previous studies that include the presence of diabetes, cvd, and other illnesses in some participants, weightloss during the feeding period, lack of control of the background diet, and variations in the diet interventions, including the length of the feeding period and diet design, as well as a small sample size. furthermore, in these studies, avocados were a source of mufa that was substituted for other macronutrients to evaluate only the effect of fat content on lipids and lipoproteins. the present study evaluated the effects of avocados on traditional and novel lipid risk factors by replacing sfa with carbohydrate (cho) or mufa from avocados or high oleic acid oils. this diet design also enabled us to evaluate the effects of a lower fat and 2 moderate fat diets, the latter of which differed in nutrients and bioactive components provided by avocados. therefore, we were able to further evaluate the nutrients and bioactive compounds in avocados beyond mufa on established and emerging cardiometabolic risk factors. a randomized, 3period crossover study design was implemented. a 2week runin average american diet (aad : 34% fat, 51% cho, 16% protein [pro ]) was fed to participants before they were randomly assigned to a treatment sequence of 3 diet periods (5 weeks each) with a 2 week compliance break between diet periods. the study design is presented in figure 1. the nutrient profile and major food groups for the lf, mf, and av diets are shown in table 1. the lf diet was designed by replacing 6 to 7% of energy from sfa with cho (from grains that were incorporated in the diet in place of sfa) in the aad. likewise, the av and mf diets were designed by replacing 6 to 7% of energy from sfa with mufa using either one hass avocado (136 g fruit pulp, 13 g mufa) per day (for the av diet) or high oleic acid oils (eg, sunflower oil and canola oil, for the mf diet) as the main sources of mufa. to match the macronutrients and fatty acids in the mf and av diets, and to adjust for the different calorie levels, high oleic acid oils, lowfat cheese, and nuts were used in both diets. about 90% of foods in the 2 diets were identical. thus, the major difference between the nutrient profiles of the av and mf diets were due to the bioactives from one avocado beyond its fatty acid profile. nutrient profile and food groups of study diets aad indicates average american diet ; av, avocado diet ; lf, lowerfat diet ; mf, moderatefat diet ; mufa, monounsaturated fatty acids ; pufa, polyunsaturated fatty acids ; sfa, saturated fatty acids. based on 2100 kcals / day. aad indicates average american diet ; av, avocado diet ; lf, lowerfat diet ; mf, moderatefat diet. menus (6day rotating) were developed using food processor sql software (esha research, salem, or) for 7 calorie levels (1800 to 3600 kcals) to meet participants energy requirements. the harrisbenedict equation with a physical activity factor was used to estimate each participant 's basal metabolic rate (bmr) and daily energy requirements. during the 2week runin the 3 experimental diets met current foodbased dietary recommendations except for dairy products in lower calorie levels (1600 to 2100 kcals), which provided around 2 servings per day. participants were weighed daily (monday through friday) to assess diet compliance and ensure that body weight was maintained. participants were asked to maintain their habitual level of physical activity throughout the study. at the end of each diet period, serum and edta plasma were collected and stored at 80c until the end of the study. one day lf, mf and av diet menus av indicates avocado diet ; lf, lowerfat diet ; mf, moderatefat diet. one of the 6 rotating menus used in the study ; all menus have 7 calorie levels (1800 to 3600 kcal). the institutional review board at the pennsylvania state university healthy, overweight men and women (21 to 70 years old, bmi 25 to 35 kg / m) with ldlc in the 25th to 90th percentile (nhanes ; 105 to 194 mg / dl for males ; 98 to 190 mg / dl for females) and normal blood pressure (140/90 mm hg) or well controlled with blood pressurelowering medication were recruited. all participants were nonsmokers, free of cvd, diabetes, liver, or renal disease, and not taking lipidlowering medications or supplements. nine participants were excluded during the 2week runin diet due to a schedule conflict, personal health issue, or inability to comply. five participants dropped out during the study but completed as least one diet treatment (figure 2). study participants were predominantly caucasian and nonhispanic (n=41) with 3 asians and 1 african american. baseline characteristics of study participants (n=45, 27 males and 18 females) are shown in table 3. participants atherosclerotic cardiovascular disease (ascvd) risk and metabolic syndrome (mets) criteria at baseline are shown in table 4. baseline characteristics of study participants (n=45) bmi indicates body mass index ; dbp, diastolic blood pressure ; hdlc, highdensity lipoprotein cholesterol ; hscrp, high sensitivity creactive protein ; ldlc, lowdensity lipoprotein cholesterol ; sbp, systolic blood pressure ; tc, total cholesterol. baseline values were obtained before randomization, at the end of 2 weeks runin aad. participants ascvd risk and mets criteria at baseline ascvd risk estimate is calculated using the web based risk estimator tools. avcsd indicates atherosclerotic cardiovascular disease ; mets, metabolic syndrome. the ascvd 10year risk is only calculated for the 40 to 79 year range. endpoint visits took place in the clinical research center (crc) at pennsylvania state university. serum lipid, lipoprotein subclasses cholesterol, and apolipoproteins were assayed using vertical auto profile (vap ii) (atherotech diagnostics lab, birmingham, al), which directly measures cholesterol in all lipoprotein classes. plasma lipoprotein particle number and size were assessed by a proton magnetic resonance spectroscopy assay (nmr lipoprofile iii ; liposcience, raleigh, nc), which measures the particle concentrations of lipoprotein subclasses and average particle size of lipoproteins. serum high sensitivity creactive protein (hscrp) was determined by a nephelometric method utilizing latex particles coated with monoclonal antibodies ; the analytical sensitivity was 0.2 mg / dl (quest diagnostics ; assay cv < plasma fasting insulin was measured by immunoassay ; fasting glucose was measured by spectrophotometry (quest diagnostics ; assay cv was < 8% for both). blood pressure was measured 3 times by a standard mercury stadiometer (w. a. baum co, copiague, ny) after sitting for 5 minutes (before the blood draw) and the average of the second and third measurements was used for the final value. statistical analyses were performed with sas (version 9.2 ; sas institute inc) and minitab software (version 16 ; minitab inc). the mixed models procedure (proc mixed) was used comparing the effects of 3 diets on the change value (from baseline) of all outcome variables. potential carryover effects were assessed by including diet sequence, period and dietperiod interaction as a fixed effect in the model ; age, bmi, sex, dietsex interaction were included as covariates. the shapirowilk test was used to assess normality of residuals in the mixed model. fisher 's ztransformation was used to compare the correlations between lipid biomarkers in different diets. ascvd 10year and lifetime risk was calculated using the online ascvd risk estimator tool (http://tools.cardiosource.org/ascvd-risk-estimator/) for every participant at baseline and at the end of each diet period. the change in risk score for each diet was estimated as the median with 95% ci by the wilcoxon signed rank test. the ascvd risk estimate is based on each participant 's age, gender, race, smoking status, diabetes status, hypertension treatment, blood pressure, total cholesterol, and hdlc. due to the large variation in previous avocado studies, we determined sample size based on a pistachio study that used a similar diet design (similar difference in macronutrients between the lowfat diet and the moderatefat, pistachio diet). using the sas procedure procpower with pairedmeans statement, a final sample size of 37 was needed to provide 95% power to detect a 10% decrease in ldlc by the avocado diet versus the lowerfat diet with a 2tailed =0.05. considering a 10% to 20% dropout rate a randomized, 3period crossover study design was implemented. a 2week runin average american diet (aad : 34% fat, 51% cho, 16% protein [pro ]) was fed to participants before they were randomly assigned to a treatment sequence of 3 diet periods (5 weeks each) with a 2 week compliance break between diet periods. the study design is presented in figure 1. the nutrient profile and major food groups for the lf, mf, and av diets are shown in table 1. the lf diet was designed by replacing 6 to 7% of energy from sfa with cho (from grains that were incorporated in the diet in place of sfa) in the aad. likewise, the av and mf diets were designed by replacing 6 to 7% of energy from sfa with mufa using either one hass avocado (136 g fruit pulp, 13 g mufa) per day (for the av diet) or high oleic acid oils (eg, sunflower oil and canola oil, for the mf diet) as the main sources of mufa. to match the macronutrients and fatty acids in the mf and av diets, and to adjust for the different calorie levels, high oleic acid oils, lowfat cheese, and nuts were used in both diets. about 90% of foods in the 2 diets were identical. thus, the major difference between the nutrient profiles of the av and mf diets were due to the bioactives from one avocado beyond its fatty acid profile. nutrient profile and food groups of study diets aad indicates average american diet ; av, avocado diet ; lf, lowerfat diet ; mf, moderatefat diet ; mufa, monounsaturated fatty acids ; pufa, polyunsaturated fatty acids ; sfa, saturated fatty acids. based on 2100 kcals / day. aad indicates average american diet ; av, avocado diet ; lf, lowerfat diet ; mf, moderatefat diet. menus (6day rotating) were developed using food processor sql software (esha research, salem, or) for 7 calorie levels (1800 to 3600 kcals) to meet participants energy requirements. the harrisbenedict equation with a physical activity factor was used to estimate each participant 's basal metabolic rate (bmr) and daily energy requirements. during the 2week runin the 3 experimental diets met current foodbased dietary recommendations except for dairy products in lower calorie levels (1600 to 2100 kcals), which provided around 2 servings per day. participants were weighed daily (monday through friday) to assess diet compliance and ensure that body weight was maintained. participants were asked to maintain their habitual level of physical activity throughout the study. at the end of each diet period, serum and edta plasma were collected and stored at 80c until the end of the study. one day lf, mf and av diet menus av indicates avocado diet ; lf, lowerfat diet ; mf, moderatefat diet. one of the 6 rotating menus used in the study ; all menus have 7 calorie levels (1800 to 3600 kcal). the institutional review board at the pennsylvania state university healthy, overweight men and women (21 to 70 years old, bmi 25 to 35 kg / m) with ldlc in the 25th to 90th percentile (nhanes ; 105 to 194 mg / dl for males ; 98 to 190 mg / dl for females) and normal blood pressure (140/90 mm hg) or well controlled with blood pressurelowering medication were recruited. all participants were nonsmokers, free of cvd, diabetes, liver, or renal disease, and not taking lipidlowering medications or supplements. nine participants were excluded during the 2week runin diet due to a schedule conflict, personal health issue, or inability to comply. five participants dropped out during the study but completed as least one diet treatment (figure 2). study participants were predominantly caucasian and nonhispanic (n=41) with 3 asians and 1 african american. baseline characteristics of study participants (n=45, 27 males and 18 females) are shown in table 3. participants atherosclerotic cardiovascular disease (ascvd) risk and metabolic syndrome (mets) criteria at baseline are shown in table 4. baseline characteristics of study participants (n=45) bmi indicates body mass index ; dbp, diastolic blood pressure ; hdlc, highdensity lipoprotein cholesterol ; hscrp, high sensitivity creactive protein ; ldlc, lowdensity lipoprotein cholesterol ; sbp, systolic blood pressure ; tc, total cholesterol. baseline values were obtained before randomization, at the end of 2 weeks runin aad. participants ascvd risk and mets criteria at baseline ascvd risk estimate is calculated using the web based risk estimator tools. avcsd indicates atherosclerotic cardiovascular disease ; mets, metabolic syndrome. the ascvd 10year risk is only calculated for the 40 to 79 year range. endpoint visits took place in the clinical research center (crc) at pennsylvania state university. serum lipid, lipoprotein subclasses cholesterol, and apolipoproteins were assayed using vertical auto profile (vap ii) (atherotech diagnostics lab, birmingham, al), which directly measures cholesterol in all lipoprotein classes. plasma lipoprotein particle number and size were assessed by a proton magnetic resonance spectroscopy assay (nmr lipoprofile iii ; liposcience, raleigh, nc), which measures the particle concentrations of lipoprotein subclasses and average particle size of lipoproteins. serum high sensitivity creactive protein (hscrp) was determined by a nephelometric method utilizing latex particles coated with monoclonal antibodies ; the analytical sensitivity was 0.2 mg / dl (quest diagnostics ; assay cv < 8%). plasma fasting insulin was measured by immunoassay ; fasting glucose was measured by spectrophotometry (quest diagnostics ; assay cv was < 8% for both). blood pressure was measured 3 times by a standard mercury stadiometer (w. a. baum co, copiague, ny) after sitting for 5 minutes (before the blood draw) and the average of the second and third measurements was used for the final value. statistical analyses were performed with sas (version 9.2 ; sas institute inc) and minitab software (version 16 ; minitab inc). the mixed models procedure (proc mixed) was used comparing the effects of 3 diets on the change value (from baseline) of all outcome variables. potential carryover effects were assessed by including diet sequence, period and dietperiod interaction as a fixed effect in the model ; age, bmi, sex, dietsex interaction were included as covariates. fisher 's ztransformation was used to compare the correlations between lipid biomarkers in different diets. ascvd 10year and lifetime risk was calculated using the online ascvd risk estimator tool (http://tools.cardiosource.org/ascvd-risk-estimator/) for every participant at baseline and at the end of each diet period. the change in risk score for each diet was estimated as the median with 95% ci by the wilcoxon signed rank test. the ascvd risk estimate is based on each participant 's age, gender, race, smoking status, diabetes status, hypertension treatment, blood pressure, total cholesterol, and hdlc. due to the large variation in previous avocado studies, we determined sample size based on a pistachio study that used a similar diet design (similar difference in macronutrients between the lowfat diet and the moderatefat, pistachio diet). using the sas procedure procpower with pairedmeans statement, a final sample size of 37 was needed to provide 95% power to detect a 10% decrease in ldlc by the avocado diet versus the lowerfat diet with a 2tailed =0.05. considering a 10% to 20% dropout rate diet compliance was very good. based on selfreported monitoring, adherence for all participants was 90% as assessed by the number of days participants consumed all study foods and no nonstudy foods. all 3 diets significantly decreased ldlc and total cholesterol (tc) versus baseline (table 5, figure 3). compared with the runin aad diet, the lf and mf diets reduced ldlc (lf : 5.3%, p=0.004 ; mf : 5.8% ; p=0.0004) and tc (lf : 4% ; mf : 4.7% ; p<0.01 for both) similarly. however, the reduction in ldlc and tc by the av diet (10% and 8% ; p<0.0001 for both) was significantly greater (p<0.05) than the lf and mf diets. hdlc decreased less (p<0.05) on the mf diet and av diet versus the lf diet. also, the lf diet significantly increased tg and vldlc by 17.6% and 10.9%, respectively (p<0.001 for both), while the mf and av diets did not. the mf and av diets decreased nonhdlc (5.1% and 9.3%, p<0.01 for both), but the lf diet did not. furthermore, the av diet elicited a greater (p=0.01) reduction in nonhdlc (9.3%, p<0.0001) versus the mf diet (5.1%, p=0.01). tc / hdlc and ldlc / hdlc significantly decreased after the av diet (4.9% and 6.6%) and were significantly lower (p=0.04 and p<0.0001) than after the mf and lf diets. the lf increased tg / hdlc by 27.3% (p<0.0001) from baseline, whereas the mf and av diets had no effect on tg / hdlc and were significantly lower (p<0.01) than the lf diet. the av and mf diets decreased apob100 from baseline, whereas the lf diet did not. the ratio of apob / apoa1 was decreased by the av diet but was not affected by the mf and lf diets (table 5, figure 3). effect of diets on lipids, lipoproteins, apolipoproteins, glucose, insulin, crp, and blood pressure all values are meanssems. aad indicates average american diet ; av, avocado diet ; dbp, diastolic blood pressure ; hscrp, high sensitive creactive protein ; hdlc, highdensity lipoprotein cholesterol ; homa, homeostatic model assessment ; idlc, intermediatedensity lipoprotein cholesterol ; ldlc, lowdensity lipoprotein cholesterol ; lf, lowerfat diet ; lp(a), lipoprotein(a) ; mf, moderatefat diet ; sbp, systolic blood pressure ; tc, total cholesterol ; vldlc, verylowdensity lipoprotein cholesterol. values in diet treatments with different superscript letters (a, b, and c) are significantly different (tukey posthoc test by sas, p<0.05). values with different letters (a, b, and c) are significantly different (tukey posthoc test by sas, p<0.05). av indicates avocado diet ; apoa1, apolipoprotein a1 ; apob, apolipoprotein b ; hdlc, highdensity lipoprotein cholesterol ; idlc, intermediatedensity lipoprotein cholesterol ; ldlc, lowdensity lipoprotein cholesterol ; lf, lowerfat diet ; mf, moderatefat diet ; tc, total cholesterol ; tg, triglycerides ; vldl, verylowdensity lipoprotein. although ldlc was reduced on all three diets, the number of ldl particles (ldlp) did not decrease significantly after the lf and mf diets (figure 4, table 6). there was a trend for a reduction in ldlp by the mf diet (38 nmol / l, p=0.07), however, this was significantly less (p=0.05) than the reduction by the av diet (80.1 nmol / l, p=0.001). the difference was due to the increase in small ldlp by the lf and mf diets (table 6). similar reductions from baseline (p<0.001 for all) in large ldlp were observed on the lf, mf, and av diets. mean ldl particle size decreased on all 3 diets : lf (0.24 nm, p<0.0001), mf (0.21 nm, p<0.0001), and av (0.12 nm, p=0.008). ldl particle size was significantly larger (p=0.03) after the av diet compared with the lf diet (table 6). the change in ldl particle size was highly correlated with the change in large ldlp (r=0.68, p<0.0001), and the change in total ldlp was highly correlated with the change in small ldlp (r=0.62, p<0.0001) (figure 5). effect of diets on lipoprotein particle size and subclasses (particle number from nmr lipoprofile) all values are meanssems. aad indicates average american diet ; av, avocado diet ; hdlp, highdensity lipoprotein particle number ; idlp, intermediatedensity lipoprotein particle number ; ldlp, lowdensity lipoprotein particle number ; lf, lowerfat diet ; mf, moderatefat diet ; vldlp, verylowdensity lipoprotein particle number. significant change compared to baseline aad, p<0.05 ; values in diet treatments with different superscript letters (a, b, and c) are significantly different (tukey posthoc test by sas, p<0.05). (a) change in ldl subclasses cholesterol ; (b) change in total, large and small ldl particle numbers. values with different letters (a, b, and c) are significantly different (tukey posthoc test by sas, p<0.05). av indicates avocado diet ; ldlc, lowdensity lipoprotein cholesterol ; ldlp, lowdensity lipoprotein number ; lf, lowerfat diet ; mf, moderatefat diet. (a) correlation between the change in small ldlp and the change in total ldlp ; (b) correlation between the change in large ldlp and the change in total ldlp ; (c) correlation between the change in small ldlp and the change in ldl particle size ; (d) correlation between the change in large ldlp and the change in ldl particle size. av indicates avocado diet ; ldlp, lowdensity lipoprotein particle number ; lf, lowerfat diet ; mf, moderatefat diet. the ldl subclass results from nmr and vap profile generally concurred : we observed similar reductions in the large, buoyant ldl cholesterol (ldl1 + 2) on the lf, mf, and av diets compared with baseline (p<0.0001 for all) (table 7, figure 4). however, only the av diet reduced small, dense ldl cholesterol (ldl3 : 4.0 mg / dl, p=0.01 ; ldl3 + 4 : 4.1 mg / dl, p=0.04). the mf diet did not affect ldl3 and ldl4 whereas the lf diet increased ldl4 from baseline (table 7). vap ldl peak max time (lower value represents higher density of ldl particles) also decreased from baseline by the lf and mf diet, but not with the av diet. the levels of ldl1 + 2 and large ldlp, ldl3 + 4 and small ldlp were highly correlated at baseline (r=0.71 and 0.70, p<0.0001 for both) ; their change values on the diets also were correlated (r=0.42 and 0.36, p<0.0001 for both) (figure 6). effect of diets on lipoprotein subclasses (cholesterol concentration from vap test) all values are meanssems. aad indicates average american diet ; av, avocado diet ; hdl, highdensity lipoprotein ; idlc, intermediatedensity lipoprotein cholesterol ; ldl, lowdensity lipoprotein ; lf, lowerfat diet ; mf, moderatefat diet ; remnant lp, lipoprotein remnants ; vap, vertical auto profile ; vldlc, verylowdensity lipoprotein cholesterol. significant change compared to baseline aad, p<0.05 ; values in diet treatments with different superscript letters (a / b) are significantly different (tukey posthoc test by sas, p<0.05). (a) correlation between the change in small ldlp and the change in ldl3 + 4 ; (b) correlation between the change in large ldlp and the change in ldl1 + 2. av indicates avocado diet ; ldl, lowdensity lipoprotein ; ldlp, lowdensity lipoprotein particle number ; lf, lowerfat diet ; mf, moderatefat diet. the lf diet increased cholesterol in vldl1 + 2 and vldl3, whereas the mf and av diets had no affect (table 7, figure 7). lipoprotein remnants (vldl3+idl) were decreased by the av diet (2.4 mg / dl, p=0.008) and increased by the lf diet (1.5 mg / dl, p=0.02). the change in vldl subclass particle number (table 6) was mostly consistent with vap results except for the decrease in small vldlp by the lf diet (in contrast to the increase in vldl3), which may be due to small vldl particles that were cholesterol enriched after the lf diet (table 7, figure 7). similar to the change in vldl1 + 2, large and medium vldlp were increased by the lf diet, however, they were not affected by the mf and av diets. the av diet but not mf diet decreased small vldlp from baseline (5.8 nmol / l, p=0.001). vldl particle size was increased by the lf diet and was significantly larger than by the mf diet (p=0.01) but not different from the av diet (table 6). (a) change in vldl and idl subclasses cholesterol concentration ; lipoprotein remnant particles consist of vldl3 and idl. values with different letters are significantly different (tukey posthoc test by sas, p<0.05). av indicates avocado diet ; idl, intermediatedensitylipoprotein ; idlp, intermediatedensitylipoprotein particle number ; lf, lowerfat diet ; mf, moderatefat diet ; vldlc, verylowdensity lipoprotein cholesterol ; vldlp, verylowdensity lipoprotein particle number. however, hdl3 was significantly decreased after the lf diet but not after the mf and av diets (table 7). unlike hdlc, the total hdlp was not affected by all diets (table 6). after the lf diet, there was a trend for small hdlp to be lower than the mf (p=0.06) and av diets (p=0.08), which is consistent with the hdl3 change (tables 6 and 7). hdl particle size was slightly decreased by the lf (0.07 nm), mf (0.1 nm), and av (0.01 nm) diets versus baseline (p<0.01 for all) (table 6). fasting hscrp, insulin, glucose, homa (homeostatic model assessment) score, systolic and diastolic blood pressure were not affected by any diet (table 5). this study is the first randomized controlled feeding trial to our knowledge to evaluate the additional ldlc lowering effect of avocados beyond their mufa content. this also is the first feeding study to use 2 advanced lipidtesting methods to evaluate the effects of lf versus mf diets on lipoprotein subclass changes. our results demonstrate greater benefits on lipid / lipoprotein profiles when sfa is replaced by mufa than cho, which is consistent with current dietary recommendations. an important finding we observed is that the av diet lowered ldlc, tc, ldlp, nonhdlc, tc / hdlc, and ldl / hdlc significantly more than the mf diet. this has important implications for future research that needs to be conducted to determine the functional effects of fruits and vegetables on lipid / lipoprotein risk factors that are independent of the substitution of macronutrients. in our study, both vap and nmr analyses demonstrated that the lf, av, and mf diets decreased large, buoyant ldl (ldl1 + 2, large ldlp) similarly ; however, only the av diet significantly decreased cholesterol in small, dense ldl (sdldl) and lipoprotein remnants from baseline, and lowered small ldlp concentration compared to the mf and lf diets. the mf diet did not have the same benefit as the av diet, which clearly indicates that other nutrients / bioactives beyond fatty acids contribute to the health benefits of avocados. we hasten to add that the mf diet did have beneficial effects but, importantly, the nutrientdense food source of mufa (ie, an avocado) had superior effects. of note, is that the mufa literature is variable with respect to cardiovascular effects which may reflect the food source(s) of mufa used. identifying other nutrientdense foods that contain mufa that confer additional cardiovascular benefits, or other nutrientdense foods (ie, fruits and vegetables) that can be consumed with high mufa oils will be important for evolving future dietary guidance. the effects of the av diet on sdldl may be due to the combined effects of mufa and other bioactives, especially phytosterols and fiber. avocados are the richest fruit source of sitosterol ; one hass avocado (136 g) provides 114 mg of plant sterols, and 100 g provides 2.11 g soluble fiber and 2.7 g insoluble fiber. avocados also contain a unique sevencarbon (c7) ketosugar mannoheptulose and its polyol form perseitol (about 4 g per fruit), which may suppress insulin secretion and promote calorie restriction. sialvera reported that plant sterol supplementation (4 g per day for 2 months) decreased sdldl cholesterol in mets patients on a western diet ; lamarche also reported that including plant sterols (1 g per 1000 kcals), viscous fiber (9 g per 1000 kcals), soybean protein and almonds in a lowsfa diet for 4 weeks decreased sdldl cholesterol (21%, p<0.01) in patients with mildly elevated ldlc. daily consumption of highfiber oat cereal providing 14 g dietary fiber / day for 12 weeks decreased sdldl cholesterol (17.3%, p<0.05) in overweight men. although the combination of fiber and plant sterols in avocados may contribute to the effect, there may be other bioactives and nutrients that also contribute because of the relatively lower dose of fiber and plant sterols delivered by one avocado compared to other supplementation studies. dietary mufa has been associated with lower sdldl when substituted for sfa, although the results reported are inconsistent. gill reported that ldl3 was 25% lower after a highmufa diet (13.7% energy) versus a lowmufa diet (7.8% energy) in hypercholesterolemic participants. kratz reported that a high mufa diet (38.7% energy from fat, 23.2% energy from mufa) provided by refined olive oil significantly reduced ldl peak particle diameter (0.36 nm, p=0.012) versus a high sfa diet (16% energy from sfa). in another study there was no effect of replacing sfa with mufa on ldl particle size. in the predimed study, only one study showed that the addition of walnuts to freeliving subjects habitual diets elicited a 13% reduction in the number of sdldl particles. lamarche showed that a dietary portfolio that incorporated plant sterols, soy protein, viscous fiber, and almonds decreased sdldl by 21% (p<0.01). holligan reported a significant decrease in sdldl cholesterol after consumption of a moderate fat diet including 2 servings / day of pistachios in healthy adults with high ldlc. one explanation for the inconsistent results is that there might be some synergistic effects of the dietary fiber, plant sterols, mufa, and other bioactive compounds or nutrients in specific foods and different background diets. further studies are needed to characterize the bioactives in avocados and investigate the mechanisms by which avocados affect sdldl production. the prediction of cvd risk by the 2013 prevention guidelines ascvd risk estimator showed significant improvement for overall 10 year and lifetime risk by the mf and av diets but not the lf diet (table 8). the predicted reduction in chd risk by the av diet could be greater due to changes in nonhdlc, apob, ldlp, sdldl, lipoprotein remnants, and lipid / lipoprotein ratios. recent clinical studies have found that the ldlattributable atherosclerotic risk is better predicted by ldlp and sdldl than by using standard lipid / lipoprotein measurements in patients at high risk for mets and/or diabetes. furthermore, sdldl and total ldlp have potential importance for comprehensively monitoring the efficacy of lifestyle interventions on cvd risk. in the present study, sdldl concentration was correlated with an atherogenic lipid / lipoprotein profile, which tracks with atherogenic dyslipidemia (figure 8). importantly, in our study, larger ldl particles and ldlc decreased similarly by the lf, mf, and av diets, which indicate all 3 diets would decrease risk of cardiovascular disease. however, because of the effects of the lf diet on tg and hdlc, there was no benefit on 10year and lifetime risk of ascvd. although the longterm benefit of lf diet versus aad was not tested in our study, this is another illustration of the importance of replacing saturated fat with unsaturated fat, ie, mufa as reported herein. predicted changes in ascvd risk by the three diets aad indicates average american diet ; av, avocado diet ; avcsd, atherosclerotic cardiovascular disease ; lf, lowerfat diet ; mf, moderatefat diet. the change in risk from baseline is the estimated median with 95% ci by the wilcoxon signed rank test. (a) correlations between ldl3 + 4 and tg, ldl1 + 2 and tg ; (b) correlations between ldl3 + 4 and vldlc, ldl1 + 2 and vldlc ; (c) correlations between ldl3 + 4 and hdlc, ldl1 + 2 and hdlc. aad indicates average american diet ; av, avocado diet ; hdlc, highdensity lipoprotein cholesterol ; ldlc, lowdensity lipoprotein cholesterol ; lf, lowerfat diet ; mf, moderatefat diet ; tg, triglycerides ; vldlc, verylowdensity lipoprotein cholesterol. the change in ldl particle size and subclasses due to the mf and av diets we observed differs from some mediterranean diet studies. in predimed study, the mediterranean diet supplemented with nuts significantly increased ldl size (0.2 nmol / l) and large ldlp (53.8 nmol / l), and decreased very small ldlp (111 nmol / l) in a subgroup of subjects (n=156) at high cvd risk. richard reported that consumption of a mediterranean diet for 5 weeks decreased small ldl particles (11.7%), and increased ldl peak particle size (0.18 nm) compared with an isoenergetic north american control diet (5 weeks). in our study, both the mf and av diets significantly decreased large ldlp and ldl particle size. these differences may reflect the higher fat content and other foods / nutrients in the mediterranean diet. also, unlike the mediterranean diet studies, our participants were at lower risk for mets and chd (table 4), thereby demonstrating benefits of the diets we studied for the primary prevention of cvd. several studies have demonstrated that nonhdlc is a better predictor of cvd risk compared with ldlc because it takes into account the atherogenic vldl and lp remnant particles. in our study, the av and mf diets significantly decreased nonhdlc and apob compared to baseline while the lf diet did not. vldl and idl, especially the small, dense lp remnant particles, are independently associated with the prediction, progression, and residual risk of cvd. also, the reduction of tg / hdlc by the mf and av diet (compared to the lf diet) indicated a beneficial effect of mufa on insulin sensitivity. it must be appreciated that most participants in our study did not have mets (even though they were at risk because they were overweight or obese), and the prediction value of advanced lipid measures versus standard lipid / lipoprotein needs to be further evaluated in different populations in longerterm intervention studies. hdlc was decreased by all diets in our study, but total hdlp did not change. the mesa study (multiethnic study of atherosclerosis) reported that hdlp but not hdlc was independently associated with chd after adjusting for ldlp and other confounding factors. in our study, the lf diet decreased hdl3, which is important for cholesterol efflux and may be upregulated by mufa. moreover, small, dense hdl3 particles may protect ldl against oxidationand attenuate apoptosis in endothelial cells as shown in recent studies. studies are needed to determine if hdl functionality is improved by a moderate fat diet with avocados. it was a wellcontrolled clinical trial and we achieved a high level of diet compliance, weight maintenance, and had a low dropout rate. the latter is attributable to the runin diet period, which familiarized participants with the study. moreover, our study was designed to differentiate the effects of bioactive compounds in avocados beyond fatty acids. one limitation of our study design is that one avocado per day contributed a different percentage of energy over different calorie levels (7% to 13% over the 6 calorie levels). another limitation is that our study participants did not lose weight, which is the first line of treatment for overweight / obesity. clearly, weight loss would elicit beneficial effects on lipids / lipoproteins and cvd risk status. nonetheless, for individuals who do not lose weight, we have shown that a moderate fat diet high in mufa, especially from one avocado per day has beneficial effects on lipids / lipoproteins and cvd risk status. finally, our participants did not represent the ethnic diversity of the u.s. population. our study has shown that one hass avocado per day has beneficial effects beyond their fatty acid profile on decreasing ldlc and other emerging cvd risk factors. herein, we present new information that a moderate fat diet low in sfa and high in mufa from an avocado daily achieved greater reductions in ldlc, sdldlc, ldlp, nonhdlc, ldl / hdlc, and tc / hdlc than a high mufa diet with a similar macronutrient and fatty acid profile. thus, inclusion of a food source rich in mufa and bioactives confers additional cvd benefits compared to a mufamatched, low sfa diet. wang, fleming, hill, and krisetherton : designed the research and wrote the manuscript ; wang, bordi, fleming, and krisetherton : conducted the clinical trial ; wang : collected samples, analyzed data and performed statistical analysis ; krisetherton and wang : had primary responsibility for the final content of the manuscript ; and all authors : read and approved the final manuscript. wang, bordi, fleming, hill, and krisetherton reported no conflict of interest in this study. the authors sincerely thank devon bordi, marcella smith, tracey banks, dr mosuk chow, and ningtao wang, who provided excellent assistance in the clinical trial implementation and statistical analyses.	backgroundavocados are a nutrientdense source of monounsaturated fatty acids (mufa) that can be used to replace saturated fatty acids (sfa) in a diet to lower low density lipoprotein cholesterol (ldlc). wellcontrolled studies are lacking on the effect of avocado consumption on cardiovascular disease (cvd) risk factors.methods and resultsa randomized, crossover, controlled feeding trial was conducted with 45 overweight or obese participants with baseline ldlc in the 25th to 90th percentile. three cholesterollowering diets (6% to 7% sfa) were fed (5 weeks each) : a lowerfat diet (lf : 24% fat) ; 2 moderatefat diets (34% fat) provided similar foods and were matched for macronutrients and fatty acids : the avocado diet (av) included one fresh hass avocado (136 g) per day, and the moderatefat diet (mf) mainly used high oleic acid oils to match the fatty acid content of one avocado. compared with baseline, the reduction in ldlc and nonhighdensity lipoprotein (hdl) cholesterol on the av diet (13.5 mg / dl, 14.6 mg / dl) was greater (p<0.05) than the mf (8.3 mg / dl, 8.7 mg / dl) and lf (7.4 mg / dl, 4.8 mg / dl) diets. furthermore, only the av diet significantly decreased ldl particle number (ldlp, 80.1 nmol / l, p=0.0001), small dense ldl cholesterol (ldl3 + 4, 4.1 mg / dl, p=0.04), and the ratio of ldl / hdl (6.6%, p<0.0001) from baseline.conclusionsinclusion of one avocado per day as part of a moderatefat, cholesterollowering diet has additional ldlc, ldlp, and nonhdlc lowering effects, especially for small, dense ldl. our results demonstrate that avocados have beneficial effects on cardiometabolic risk factors that extend beyond their hearthealthy fatty acid profile.clinical trial registrationurl : http://www.clinicaltrials.gov. unique identifier : nct01235832.
in longitudinal clinical studies, where the change over time of an outcome is of interest, it is sometimes seen that the change is dependent on the initial value. the change over time may be significantly larger in subjects who had really higher (or lower) initial values as compared to subjects whose initial values were closer to the population mean. this differential effect may partly be due to a phenomenon known as regression to the mean. in other words, since there is always within - subject variability, the initial value may have been high (or low) for certain subjects just by random chance, and upon remeasurement the outcome values for these subjects would have just regressed back to the true mean. for example, this phenomenon may be illustrated via the analysis results of data in a registry based on cystic fibrosis (cf) patients that was conducted by investigators at children 's hospital of philadelphia. one of the goals of the study was to determine the rate of change of the pulmonary function as measured by percent of predicted forced expiratory volume in 1 second (fev1%), over a 4-year period in a large cohort of children with cystic fibrosis. cf foundation 's national cf patient registry data collected from 1991 to 1995 for 968 children (461 male) aged 5 to 8 years with pancreatic insufficiency and fev1% between 60% and 140% were analyzed longitudinally. the significant decline in fev1% was found to be dependent on baseline fev1% ; children with initial fev1% 90 declined 2.6 u / y more than those with initial fev1% 90 and for subjects with initial measurement 90. we assess the regression to the mean in subjects with initial measurements greater than 90 via the two methods described in this paper. for the method in the section 3, we used the gaussian kernel along with the plug - in bandwidth selection method described in to estimate g. this was done via proc kde in sas. the regression to the mean via this method was estimated to be -4.78 with bootstrap standard error of 0.85. since the percent predicted fev1 measurements is a non - negative variable, we were also able to estimate the regression to the mean via the method described in section 4. we used the varying kernel, varying bandwidth method suggested in with the epanechnikov boundary kernel to estimate u. this was done using the muhaz r package. (see section 4 for more details.) the regression to mean estimate via this method was seen to be -5.21 with bootstrap standard error of 1.02. the regression to the mean estimate by both the methods are comparable in this case. via the first method we see that 55% of the change in percent predicted fev1 between 1991 and 1992 is due to the regression to the mean effect and by the second method we see that 60% of the change is attributable to regression to the mean. we propose two approaches for estimating das and mulder 's expression of regression to the mean for non - normal population. our first approach is based on a kernel density estimation method and our second approach is based on kernel estimation approaches for hazard rate function. the estimates in both the methods may be easily obtained via the state of the art techniques available in popular statistical software such as sas and r. we also calculate the bootstrap standard error for estimates. applying our methods to fev % data from cystic fibrosis foundation 's national patient registry showed that both the regression to the mean estimates are within the margin of bootstrap standard error estimates. as in das and mulder 's original paper, our methods are restricted to calculating the regression to the mean when there are only two time points. extension of these methods to longitudinal studies with more than two time points is an area of future research.	background : part of the change over time of a response in longitudinal studies may be attributed to the re - gression to the mean. the component of change due to regression to the mean is more pronounced in the subjects with extreme initial values. das and mulder proposed a nonparametric approach to estimate the regression to the mean.aim:in this paper, das and mulder 's method is made data - adaptive for empirical distributions via kernel estimation approaches, while retaining the orig - inal assumptions made by them.results:we use the best approaches for kernel density and hazard function estimation in our methods. this makes our approach extremely user friendly for a practitioner via the state of the art procedures and packages available in statistical softwares such as sas and r for kernel density and hazard function estimation. we also estimate the standard error of our estimates of regression to the mean via nonparametric bootstrap methods. finally, our methods are illustrated by analyzing the percent predicted fev1 measurements available from the cystic fibrosis foundation 's national patient registry.conclusion:the kernel based approach presented in this article is a user - friendly method to assess the regression to the mean in non - normal populations.
williams - campbell syndrome (wcs) is a rare congenital syndrome characterized by defective or completely absent bronchial wall cartilage in subsegmental bronchi, leading to distal airway collapse, producing a mechanical abnormality that may contribute to the formation of bronchiectasis distal to the collapsed bronchi. the defect usually is between the fourth and sixth order bronchial divisions, but it may extend between first to eight generations of proximal bronchi. the deficiency in cartilage occurs early in life when the lungs are still developing and growing. the symptoms and prognosis ultimately depend on the extent of cartilage maldevelopment of the bronchi. although the syndrome has been best described in children with recurrent pneumonia and broncho - obstructive symptoms such as coughing and wheezing, there have been case reports in adults diagnosed late, mainly because of misdiagnosis with other more common pathologies. wcs was first described by williams and campbell in 1960 as a rare form of congenital bronchiectasis. it was proposed that the abnormal development of cartilage in bronchial tree was responsible for this presentation. other anatomic features of the syndrome include absence or destruction of other (non - cartilaginous) bronchial wall structures by inflammation and a relatively uniform, bilateral distribution of the process. there is also early development of clubbing because of recurrent suppurative infections and extensive radiological changes of saccular bronchiectasis. in 1976, the first report of the occurrence of familial bronchiectasis in siblings was published and supported the theory that wcs was congenital, based on the uniformity of the cartilaginous defect. it may be possibly the result of an autosomal recessive mechanism, but genetic studies have not identified the specific gene so far. although most described cases presented sporadically in early childhood, subclinical cases maybe diagnosed as late as in adulthood. some authors have classified wcs in congenital and acquired forms. the congenital form is usually found in children and is associated with congenital anomalies such as polysplenia, malrotation of the abdominal viscera, thorax piriformis, congenital cardiac diseases and bronchial isomerism. other authors support the acquired hypothesis, in which bronchiectasis is secondary to adenovirus infections that cause bronchomalacia. diagnosis requires an appropriate clinical history, the characteristic expiratory airway collapse on radiological investigation, and exclusion of other causes of congenital and acquired bronchiectasis. pathology of the affected bronchi by bronchoscopy showing the deficiency of cartilaginous plates in the bronchial wall is the confirmatory test. early reports relied on bronchography and fluoroscopic visualization, which did not offer good quality images. fiberoptic bronchoscopy on the other hand is often non - diagnostic and not all patients are good candidates for these procedures. during the 1990s, ct became the radiological investigation of choice. in 2006, di scioscio. described a case of wcs using inspiratory and expiratory ct imaging, which showed inspiratory ballooning of bilateral cylindrical / cystic bronchiectasis distal to the third - generation bronchi with hyperinflation of the lung. on expiration phase, there was a complete collapse of the bronchiectasis, suggesting the absence of cartilaginous plates in the subsegmental bronchi. used in addition to conventional ct, a three - dimensional reconstruction of the bronchial tree. described by them showed collapsed subsegmental bronchi on conventional ct, but on ct bronchoscopy, images appeared deficient of cartilage rings (absence of ring impressions) from the main stem to the subsegmental level, which could be of great significance, especially if lung transplant is considered as a treatment option. therefore, considering its non - invasive methodology, facility of execution, and good patient tolerance, multi - slice spiral ct or ct bronchoscopy should be the test of choice to study cystic lung diseases in particular wcs. prophylaxis can be achieved if an oral or intravenous antibiotic is given for 7 - 10 days or until sputum production decreases. for severe cases, several different antibiotics may be used sequentially in a continuous regimen to minimize bacterial resistance. in 2011, hacken. conducted a systematic review, aiming to answer what are the effects of treatments in people with bronchiectasis but without cystic fibrosis. the following treatment recommendations were given : exercise or inspiratory muscle training may improve quality of life and exercise endurance in people with bronchiectasis but without cystic fibrosis. prolonged use of antibiotics improves clinical response rates and may improve quality of life and reduce time to first exacerbation, but it may not reduce exacerbation rates or improve lung function. inhaled corticosteroids may reduce sputum volume and improve dyspnea, but have not been shown to reduce exacerbations. there are not enough studies to prove if bronchopulmonary hygiene, physical therapy, mucolytics, inhaled hyperosmolar agents, oral corticosteroids, leukotriene receptor antagonists, short - acting beta 2 agonists, long - acting beta 2 agonists, or anticholinergic therapy is beneficial. there are case reports that domiciliary non - invasive positive pressure ventilation (nppv) may have an advantage in adult patients with wcs who have severe respiratory failure and recurrent pulmonary infections. wcs is an obstructive disorder that shares some similarities with chronic obstructive pulmonary disorder (copd). nppv combined with long - term home oxygen therapy decreases carbon dioxide retention and improves dyspnea in hypercapnic copd. surgery is often considered for people with extreme damage to one or two lobes of the lung who are at risk of severe infection or bleeding. however, surgery in two patients, one given a triple lobectomy and the other a right upper lobectomy, resulted in severe respiratory failure. transplantation has been reported in a patient with severe respiratory symptoms from wcs, but the patient died a year later. upon postmortem examination, it was observed that the main bronchi had bronchomalacia, which was attributed to a respiratory infection during the post - surgery period. given the rarity of the disease, 2006 guidelines from the international society for heart and lung transplantation give no specific recommendation on selection of patients for lung transplantation, but state that with non - cf bronchiectasis the lung transplant community has generally followed the guidelines used for cf patients. as per these recommendations, the transplant window can be established when fev1 reaches 30% of predicted or with rapid deterioration of fev1, with increasing frequency of exacerbations or an icu stay, refractory / recurrent pneumothorax and hemoptysis not controlled by embolization. bronchiectasis is characterized by irreversible widening of medium - sized airways, with inflammation, chronic bacterial infection and destruction of bronchial walls. the diagnosis of wcs requires an appropriate clinical history, characteristic expiratory collapse of airways, and exclusion of other causes of congenital or acquired bronchiectasis. there are numerous etiologies that can induce or contribute to the pathophysiologic processes that result in bronchiectasis. they include airway obstruction (e.g., foreign body aspiration), defective host defenses, cystic fibrosis, young 's syndrome, rheumatic and systemic diseases, dyskinetic cilia, pulmonary infections, allergic bronchopulmonary aspergillosis and cigarette smoking. the initial evaluation of a patient with bronchiectasis should include a complete blood count with differential, immunoglobulin quantization (igg, igm and iga), and sputum culture and smear for bacteria, mycobacteria and fungi. a central distribution is suggestive of allergic bronchopulmonary aspergillosis ; predominant upper lobe distribution is characteristic of cystic fibrosis ; middle and lower lobe distribution is consistent with primary ciliary dyskinesia ; middle lobe and lingular segment of the left upper lobe involvement is characteristic of non - tuberculous mycobacteria ; and lower lobe involvement is typical of idiopathic bronchiectasis. diagnosis requires an appropriate clinical history, the characteristic expiratory airway collapse on radiological investigation, and exclusion of other causes of congenital and acquired bronchiectasis. pathology of the affected bronchi by bronchoscopy showing the deficiency of cartilaginous plates in the bronchial wall is the confirmatory test. early reports relied on bronchography and fluoroscopic visualization, which did not offer good quality images. fiberoptic bronchoscopy on the other hand is often non - diagnostic and not all patients are good candidates for these procedures. during the 1990s, ct became the radiological investigation of choice. in 2006, di scioscio. described a case of wcs using inspiratory and expiratory ct imaging, which showed inspiratory ballooning of bilateral cylindrical / cystic bronchiectasis distal to the third - generation bronchi with hyperinflation of the lung. on expiration phase, there was a complete collapse of the bronchiectasis, suggesting the absence of cartilaginous plates in the subsegmental bronchi. used in addition to conventional ct, a three - dimensional reconstruction of the bronchial tree. described by them showed collapsed subsegmental bronchi on conventional ct, but on ct bronchoscopy, images appeared deficient of cartilage rings (absence of ring impressions) from the main stem to the subsegmental level, which could be of great significance, especially if lung transplant is considered as a treatment option. therefore, considering its non - invasive methodology, facility of execution, and good patient tolerance, multi - slice spiral ct or ct bronchoscopy should be the test of choice to study cystic lung diseases in particular wcs. prophylaxis can be achieved if an oral or intravenous antibiotic is given for 7 - 10 days or until sputum production decreases. for severe cases, several different antibiotics may be used sequentially in a continuous regimen to minimize bacterial resistance. in 2011, conducted a systematic review, aiming to answer what are the effects of treatments in people with bronchiectasis but without cystic fibrosis. the following treatment recommendations were given : exercise or inspiratory muscle training may improve quality of life and exercise endurance in people with bronchiectasis but without cystic fibrosis. prolonged use of antibiotics improves clinical response rates and may improve quality of life and reduce time to first exacerbation, but it may not reduce exacerbation rates or improve lung function. inhaled corticosteroids may reduce sputum volume and improve dyspnea, but have not been shown to reduce exacerbations. there are not enough studies to prove if bronchopulmonary hygiene, physical therapy, mucolytics, inhaled hyperosmolar agents, oral corticosteroids, leukotriene receptor antagonists, short - acting beta 2 agonists, long - acting beta 2 agonists, or anticholinergic therapy is beneficial. there are case reports that domiciliary non - invasive positive pressure ventilation (nppv) may have an advantage in adult patients with wcs who have severe respiratory failure and recurrent pulmonary infections. wcs is an obstructive disorder that shares some similarities with chronic obstructive pulmonary disorder (copd). nppv combined with long - term home oxygen therapy decreases carbon dioxide retention and improves dyspnea in hypercapnic copd. surgery is often considered for people with extreme damage to one or two lobes of the lung who are at risk of severe infection or bleeding. however, surgery in two patients, one given a triple lobectomy and the other a right upper lobectomy, resulted in severe respiratory failure. transplantation has been reported in a patient with severe respiratory symptoms from wcs, but the patient died a year later. upon postmortem examination, it was observed that the main bronchi had bronchomalacia, which was attributed to a respiratory infection during the post - surgery period. given the rarity of the disease, 2006 guidelines from the international society for heart and lung transplantation give no specific recommendation on selection of patients for lung transplantation, but state that with non - cf bronchiectasis the lung transplant community has generally followed the guidelines used for cf patients. as per these recommendations, the transplant window can be established when fev1 reaches 30% of predicted or with rapid deterioration of fev1, with increasing frequency of exacerbations or an icu stay, refractory / recurrent pneumothorax and hemoptysis not controlled by embolization. bronchiectasis is characterized by irreversible widening of medium - sized airways, with inflammation, chronic bacterial infection and destruction of bronchial walls. the diagnosis of wcs requires an appropriate clinical history, characteristic expiratory collapse of airways, and exclusion of other causes of congenital or acquired bronchiectasis. there are numerous etiologies that can induce or contribute to the pathophysiologic processes that result in bronchiectasis. they include airway obstruction (e.g., foreign body aspiration), defective host defenses, cystic fibrosis, young 's syndrome, rheumatic and systemic diseases, dyskinetic cilia, pulmonary infections, allergic bronchopulmonary aspergillosis and cigarette smoking. the initial evaluation of a patient with bronchiectasis should include a complete blood count with differential, immunoglobulin quantization (igg, igm and iga), and sputum culture and smear for bacteria, mycobacteria and fungi. a central distribution is suggestive of allergic bronchopulmonary aspergillosis ; predominant upper lobe distribution is characteristic of cystic fibrosis ; middle and lower lobe distribution is consistent with primary ciliary dyskinesia ; middle lobe and lingular segment of the left upper lobe involvement is characteristic of non - tuberculous mycobacteria ; and lower lobe involvement is typical of idiopathic bronchiectasis. the diagnosis of wcs is made by exclusion, after detailed work - up and exclusion of the other common causes of bronchiectasis. when patients ' signs and symptoms include recurrent respiratory infections and diffuse bronchiectasis, wcs should be included in the differential diagnosis.	williams - campbell syndrome is a rare congenital syndrome characterized by the absence of cartilage in subsegmental bronchi leading to formation of bronchiectasis distal to the affected bronchi. the differential diagnosis of bronchiectasis is broad and the rarity of the disease poses a diagnostic and management challenge for clinicians. this present review aims to help the understanding of the clinical manifestations, pathophysiological features, diagnostic modalities, management and differential diagnosis of williams - campbell syndrome. a medline / pubmed search was performed identifying all relevant articles. no restrictions were used for publication dates. the author used the keywords williams - campbell syndrome, non - cystic fibrosis bronchiectasis and congenital bronchiectasis finding 503, 195 and 489 articles, respectively.
a neonatal intensive care unit (nicu) is a specially equipped nursery where critically ill and unstable infants receive diagnostic, therapeutic and life support care for a wide range of illnesses and conditions. a nicu is for those infants who are preterm, have low birth weight, or perinatal problems, or congenital abnormalities, respiratory disorders, neuromuscular disorders and for those who have undergone thoraco - abdominal surgery. physiotherapy is a part of the services delivered by the interdisciplinary team in the nicu. chest physiotherapy commonly includes techniques like percussion, vibration, positioning for postural drainage and airway suctioning. it may be useful for the maintenance of a clear airway, as also to re - expand collapsed segments of the lungs, maintain adequate levels of oxygenation, facilitate early weaning, and reduce the probabilities of re - intubation. for neuromuscular dysfunction, common therapeutic strategies for neuromuscular physiotherapy include positioning, skin to skin holding (kangaroo care), therapeutic handling, orofacial stimulation, taping, range of motion exercise, soft tissue mobilization (surgical scar release), hydrotherapy and parent education (feeding, dressing, positioning of infants for sleep, interaction / play). these developmental strategies are beneficial for the promotion of posture and movement appropriate to gestational age and medical stability, to modulate sensory stimulation in the infant 's nicu environment, to promote behavioral organization and physiological stability, to foster infant - parent attachment, and to provide direct intervention for neonatal feeding dysfunction and oral motor deficits. previous studies carried out in icus did not explore the practice pattern of physiotherapists in neonatal icus. a study done in india to identify the role of the physiotherapists in icus demonstrated that physiotherapists were involved in chest physiotherapy and mobilization, but the role of the physiotherapist specific to a nicu was not clear. therefore, there was a need to identify the current practice patterns of physiotherapists in nicus. the aim of the study is to determine the practice patterns of physiotherapists in nicus in india with regard to cardiopulmonary and neuromuscular physiotherapy. a total of 10 physiotherapists working in nicus, experts in the field of neonatal physiotherapy were given the practice patterns of physiotherapists in neonatal intensive care units questionnaire for content validation and accordingly the final questionnaire was prepared [appendix 1 ]. these covered two primary roles of physiotherapy in a neonatal icu : chest and neuromuscular physiotherapy. chest physiotherapy mainly focused on assessment and treatment whereas neuromuscular physiotherapy primarily focused on treatment. answers had to fall into the grades : always, frequently, sometimes, rarely or never. a cross - sectional survey was conducted across india, in which 285 questionnaires were emailed to physiotherapists working in nicus. list of hospitals was obtained from the nabh [national accreditation board of hospital and healthcare providers ] and mci [medical council of india ] websites. responses were numerically coded to allow for descriptive summaries and frequency analyses of the data. data were analyzed via spss version 17 (spss inc, chicago, illinois, usa). frequency variables regarding chest and neuromuscular physiotherapy were merged in order to create three responses ; always or frequently, sometimes and rarely or never. a total of 10 physiotherapists working in nicus, experts in the field of neonatal physiotherapy were given the practice patterns of physiotherapists in neonatal intensive care units questionnaire for content validation and accordingly the final questionnaire was prepared [appendix 1 ]. these covered two primary roles of physiotherapy in a neonatal icu : chest and neuromuscular physiotherapy. chest physiotherapy mainly focused on assessment and treatment whereas neuromuscular physiotherapy primarily focused on treatment. answers had to fall into the grades : always, frequently, sometimes, rarely or never. a cross - sectional survey was conducted across india, in which 285 questionnaires were emailed to physiotherapists working in nicus. list of hospitals was obtained from the nabh [national accreditation board of hospital and healthcare providers ] and mci [medical council of india ] websites. responses were numerically coded to allow for descriptive summaries and frequency analyses of the data. data were analyzed via spss version 17 (spss inc, chicago, illinois, usa). frequency variables regarding chest and neuromuscular physiotherapy were merged in order to create three responses ; always or frequently, sometimes and rarely or never. a total of 10 physiotherapists working in nicus, experts in the field of neonatal physiotherapy were given the practice patterns of physiotherapists in neonatal intensive care units questionnaire for content validation and accordingly the final questionnaire was prepared [appendix 1 ]. these covered two primary roles of physiotherapy in a neonatal icu : chest and neuromuscular physiotherapy. chest physiotherapy mainly focused on assessment and treatment whereas neuromuscular physiotherapy primarily focused on treatment. answers had to fall into the grades : always, frequently, sometimes, rarely or never. a cross - sectional survey was conducted across india, in which 285 questionnaires were emailed to physiotherapists working in nicus. list of hospitals was obtained from the nabh [national accreditation board of hospital and healthcare providers ] and mci [medical council of india ] websites. responses were numerically coded to allow for descriptive summaries and frequency analyses of the data. data were analyzed via spss version 17 (spss inc, chicago, illinois, usa). frequency variables regarding chest and neuromuscular physiotherapy were merged in order to create three responses ; always or frequently, sometimes and rarely or never. a total of 285 questionnaires were e - mailed to physiotherapists across india, with a total of 139 completed questionnaires returned. the responses were received from 12 states including andhra pradesh, delhi, gujarat, haryana, karnataka, kerala, madhya pradesh, maharashtra, orissa, tamil nadu, uttar pradesh and west bengal. the majority of responders were from karnataka (n = 40 [28.7% ]), maharashtra (n = 32 [23.0% ]) and gujarat (n = 28 [20.1% ]) [figure 1 ]. response rate from different states of india (n= 139) the frequency with which different chest physiotherapy assessment measures were used in the neonatal icu is given in table 1 and figure 2. a total of 94.9% responders performed chest physiotherapy in nicus ; of these more than 30% of the responders marked always or frequently for chest physiotherapy assessment. this included pre - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], ventilator parameter setting [n = 9 (6.4%) ], post - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], suction during extubation [n = 16 (11.5%) ], opinion for weaning [n = 9 (6.4%) ], and decision - making in extubation [n = 10 (7.1%) ]. chest physiotherapy assessment in a neonatal intensive care unit chest physiotherapy assessment in a neonatal intensive care unit the frequency with which chest physiotherapy treatment was used in the nicus is given in table 2 and figure 3. more than 50% of physiotherapists marked always or frequently for chest physiotherapy treatment. these included percussion [n = 103 (74.1%) ], vibration [n = 105 (75.5%) ], chest manipulation (percussion + vibration) [n = 102 (73.3%) ], postural drainage [n = 94 (67.6%) ], head down position during the use of mechanical ventilator [n = 50 (35.9%) ], only suctioning [n = 82 (58.%) ], chest manipulation followed by suctioning [n = 91 (65.4%) ], prone positioning in ventilated neonates [n = 31 (22.3%) ], pre - treatment nebulization [n = 48 (34.5%) ], artificial manual breathing unit (ambu) [n = 64 (46.0%) ], saline [n = 32 (23.0%) ], post - treatment nebulization [n = 22 (15.8%) ], and use of mucolytic agent [n = 12 (8.6%) ]. chest physiotherapy treatment in a neonatal intensive care unit chest physiotherapy treatment in a neonatal intensive care unit. ambu - artificial manual breathing unit the frequency with which neuromuscular physiotherapy was used in the nicus is given in table 3 and figure 4. a total of 80.5% physiotherapists performed neuromuscular physiotherapy in the nicu. among them, 30% of physiotherapists marked always or frequently. these methods included passive range of motion [n = 69 (49.6%) ], therapeutic handling [n = 68 (48.9%), multimodal sensory stimulation [n = 43 (30.9%) ], orofacial stimulation [n = 47 (33.8% ], kangaroo care [n = 43 (30.9%) ], neonatal massage [n = 20 (14.3%) ], taping [n = 11 (7.9%) ], scar tissue mobilization [n = 10 (7.1%) ], positioning [n = 101 (72.6%) ], and parent education [n = 84 (60.4%) ]. hydrotherapy was not frequently performed by the physiotherapists in india [n = 0 ]. neuromuscular physiotherapy treatment in a neonatal intensive care unit neuromuscular physiotherapy treatment in a neonatal intensive care unit a total of 285 questionnaires were e - mailed to physiotherapists across india, with a total of 139 completed questionnaires returned. the responses were received from 12 states including andhra pradesh, delhi, gujarat, haryana, karnataka, kerala, madhya pradesh, maharashtra, orissa, tamil nadu, uttar pradesh and west bengal. the majority of responders were from karnataka (n = 40 [28.7% ]), maharashtra (n = 32 [23.0% ]) and gujarat (n = 28 [20.1% ]) [figure 1 ]. response rate from different states of india (n= 139) the frequency with which different chest physiotherapy assessment measures were used in the neonatal icu is given in table 1 and figure 2. a total of 94.9% responders performed chest physiotherapy in nicus ; of these more than 30% of the responders marked always or frequently for chest physiotherapy assessment. this included pre - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], ventilator parameter setting [n = 9 (6.4%) ], post - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], suction during extubation [n = 16 (11.5%) ], opinion for weaning [n = 9 (6.4%) ], and decision - making in extubation [n = 10 (7.1%) ]. chest physiotherapy assessment in a neonatal intensive care unit chest physiotherapy assessment in a neonatal intensive care unit the frequency with which chest physiotherapy treatment was used in the nicus is given in table 2 and figure 3. more than 50% of physiotherapists marked always or frequently for chest physiotherapy treatment. these included percussion [n = 103 (74.1%) ], vibration [n = 105 (75.5%) ], chest manipulation (percussion + vibration) [n = 102 (73.3%) ], postural drainage [n = 94 (67.6%) ], head down position during the use of mechanical ventilator [n = 50 (35.9%) ], only suctioning [n = 82 (58.%) ], chest manipulation followed by suctioning [n = 91 (65.4%) ], prone positioning in ventilated neonates [n = 31 (22.3%) ], pre - treatment nebulization [n = 48 (34.5%) ], artificial manual breathing unit (ambu) [n = 64 (46.0%) ], saline [n = 32 (23.0%) ], post - treatment nebulization [n = 22 (15.8%) ], and use of mucolytic agent [n = 12 (8.6%) ]. chest physiotherapy treatment in a neonatal intensive care unit chest physiotherapy treatment in a neonatal intensive care unit. ambu - artificial manual breathing unit the frequency with which neuromuscular physiotherapy was used in the nicus is given in table 3 and figure 4. a total of 80.5% physiotherapists performed neuromuscular physiotherapy in the nicu. among them, 30% of physiotherapists marked always or frequently. these methods included passive range of motion [n = 69 (49.6%) ], therapeutic handling [n = 68 (48.9%), multimodal sensory stimulation [n = 43 (30.9%) ], orofacial stimulation [n = 47 (33.8% ], kangaroo care [n = 43 (30.9%) ], neonatal massage [n = 20 (14.3%) ], taping [n = 11 (7.9%) ], scar tissue mobilization [n = 10 (7.1%) ], positioning [n = 101 (72.6%) ], and parent education [n = 84 (60.4%) ]. hydrotherapy was not frequently performed by the physiotherapists in india [n = 0 ]. neuromuscular physiotherapy treatment in a neonatal intensive care unit neuromuscular physiotherapy treatment in a neonatal intensive care unit a total of 285 questionnaires were e - mailed to physiotherapists across india, with a total of 139 completed questionnaires returned. the responses were received from 12 states including andhra pradesh, delhi, gujarat, haryana, karnataka, kerala, madhya pradesh, maharashtra, orissa, tamil nadu, uttar pradesh and west bengal. the majority of responders were from karnataka (n = 40 [28.7% ]), maharashtra (n = 32 [23.0% ]) and gujarat (n = 28 [20.1% ]) [figure 1 ]. response rate from different states of india (n= 139) the frequency with which different chest physiotherapy assessment measures were used in the neonatal icu is given in table 1 and figure 2. a total of 94.9% responders performed chest physiotherapy in nicus ; of these more than 30% of the responders marked always or frequently for chest physiotherapy assessment. this included pre - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], ventilator parameter setting [n = 9 (6.4%) ], post - treatment vital parameter assessment (heart rate, respiratory rate, and spo2) [n = 120 (86.3%) ], suction during extubation [n = 16 (11.5%) ], opinion for weaning [n = 9 (6.4%) ], and decision - making in extubation [n = 10 (7.1%) ]. chest physiotherapy assessment in a neonatal intensive care unit chest physiotherapy assessment in a neonatal intensive care unit the frequency with which chest physiotherapy treatment was used in the nicus is given in table 2 and figure 3. more than 50% of physiotherapists marked always or frequently for chest physiotherapy treatment. these included percussion [n = 103 (74.1%) ], vibration [n = 105 (75.5%) ], chest manipulation (percussion + vibration) [n = 102 (73.3%) ], postural drainage [n = 94 (67.6%) ], head down position during the use of mechanical ventilator [n = 50 (35.9%) ], only suctioning [n = 82 (58.%) ], chest manipulation followed by suctioning [n = 91 (65.4%) ], prone positioning in ventilated neonates [n = 31 (22.3%) ], pre - treatment nebulization [n = 48 (34.5%) ], artificial manual breathing unit (ambu) [n = 64 (46.0%) ], saline [n = 32 (23.0%) ], post - treatment nebulization [n = 22 (15.8%) ], and use of mucolytic agent [n = 12 (8.6%) ]. chest physiotherapy treatment in a neonatal intensive care unit chest physiotherapy treatment in a neonatal intensive care unit. the frequency with which neuromuscular physiotherapy was used in the nicus is given in table 3 and figure 4. a total of 80.5% physiotherapists performed neuromuscular physiotherapy in the nicu. among them, 30% of physiotherapists marked always or frequently. these methods included passive range of motion [n = 69 (49.6%) ], therapeutic handling [n = 68 (48.9%), multimodal sensory stimulation [n = 43 (30.9%) ], orofacial stimulation [n = 47 (33.8% ], kangaroo care [n = 43 (30.9%) ], neonatal massage [n = 20 (14.3%) ], taping [n = 11 (7.9%) ], scar tissue mobilization [n = 10 (7.1%) ], positioning [n = 101 (72.6%) ], and parent education [n = 84 (60.4%) ]. hydrotherapy was not frequently performed by the physiotherapists in india [n = 0 ]. neuromuscular physiotherapy treatment in a neonatal intensive care unit neuromuscular physiotherapy treatment in a neonatal intensive care unit. to the best of our knowledge, this is the first study reporting practice patterns of indian physiotherapists working in nicus. according to the results of our survey, the novel findings of our study are as follows : chest physiotherapy assessment predominantly focused on vital parameters which includes heart rate, respiratory rate, and spo2the most common technique used in chest physiotherapy treatment was chest manipulation which involves percussion, vibration, postural drainage and suctionfor neuromuscular physiotherapy, positioning, parent education, passive range of motion exercise, and therapeutic handling were predominantly used by physiotherapists. chest physiotherapy assessment predominantly focused on vital parameters which includes heart rate, respiratory rate, and spo2 the most common technique used in chest physiotherapy treatment was chest manipulation which involves percussion, vibration, postural drainage and suction for neuromuscular physiotherapy, positioning, parent education, passive range of motion exercise, and therapeutic handling were predominantly used by physiotherapists. in our survey 86% of physiotherapists noted their patients vital parameters pre- and post treatment, in order to determine the patient 's physiological status. an indian study on physiotherapy practice patterns in adult icus showed that the response rate for vital parameter assessment was 98%. this suggests that regular vital parameter monitoring is a standard practice pattern among physiotherapists working in neonatal and adult icus in india. our survey showed that 6% of physiotherapists were involved in ventilator parameter setting, as against 10% in adult icus in india and 12% in europe. our survey showed that 7% physiotherapists were involved in decision - making during extubation compared to 25% in european icus. the current study showed that 6% physiotherapists were involved in weaning patients off the ventilator, compared to 18% for adult icu patients in india and 22% in europe. percussion is used to augment mobilization of secretions by mechanically dislodging viscous or adherent mucus from the airway. it was shown that 74% of respondents used percussion as a chest physiotherapy treatment of choice in neonates. a similar study on adult icus in india showed that the response rate for percussion was 93.6% whereas it was 98% in europe and 79% in australia. vibration is used in conjunction with percussion to help move secretions to the larger airway. according to our survey, 75% of respondents used vibration for neonates. for adult icu patients it was 96.2%, 98% in europe, and 87% in australia. postural drainage is an intervention for airway clearance by mobilizing secretions in one or more lung segments to the central airways by placing the patient in various positions so that gravity assists in the drainage process. as per our survey 67.3% of physiotherapists working with neonates in an icu used postural drainage. for physiotherapists working with adult icu patients, the response rate was 95% in india, and 98% in europe. according to our survey 58.9% of the physiotherapists used airway suction for neonates. for adult icu patients the response rate for airway suction was 94.2% in india, 70% in europe and 82% in australia. evidence showed that prone position is used to facilitate extremity flexion toward the midline, limit uncontrolled flailing extremity movement, and encourage more stable vital signs. according to our survey as per our survey the manual resuscitator was made use of by 46% physiotherapists for both mobilizing secretion and hyperinflation of lungs during treatment. the importance of nebulization lies in the humidification of inspired gas, the delivery of a medication like bronchodilator or in aiding bronchial hygiene. our survey showed that 35% of respondents used pre - treatment nebulization and 16% used post - treatment nebulization. in adult normal saline (0.9% nacl) was used for lavage during suction by 23.0% physiotherapists whereas a mucolytic agent was used by 8.6% to reduce the thickness or viscosity of bronchial secretion. most common neuromuscular physiotherapy techniques used by indian physiotherapists for neonates are positioning, therapeutic handling, passive range of motion exercise, and parent education. evidence suggests that infants born preterm have immature musculoskeletal systems that are influenced by positioning. exposure to prolonged atypical positioning in the nicu has been associated with torticollis, positional plagiocephaly, reduced movement quality, and lower extremity malalignment. supportive positioning may enhance the development of normal skeletal alignment and provide opportunities for normal movement patterns, like to promote more optimal cranial molding and prevent plagiocephaly and torticollis. in our survey 72.6% research on educating parents to interpret the meaning of their infant 's behavioral cues and developmental status has been shown to reduce parental stress and improve parental mental health, as well parent - infant bonding. in our survey 60.4% respondents were involved with parent education. a passive range of motion is mainly given to the infant who has congenital restriction of joint mobility, and evidence also suggests that passive range of motion in the extremities has been advocated as an intervention to increase bone mineral density. as per our survey 49.6% therapeutic handling improves motor development of infants ; 48.9% of physiotherapists used therapeutic handling for neonates. feeding is a functional activity of the highest priority for infants and caregivers and as such is an integral part of neonatal physiotherapy practice. evidence suggests that orofacial stimulation hastens feeding progression in neonates and decreases the transition time of oral feeding. evidence for multimodal sensory stimulation suggests that sensory stimuli may enhance state regulation, speed transition to full nipple feedings, mitigate stressful environmental stimuli and shorten length of hospital stay, whereas tactile stimulation alone may improve short - term growth and reduce length of stay. adjunct interventions like kangaroo care or skin to skin holding showed improvement in physiological stability, fostered infant - parent attachment, and promoted soft flexion of the infant 's arms, legs, trunk, and neck. the immediate benefits for self - regulation may include temperature regulation, improved oxygen saturation, and decreased respiratory rate. neonatal massage was shown to produce favorable effects to reduce stress levels and improve infant - parent attachment in neonatal icu environment. we found that a very small percentage of physiotherapists (7.9%) used taping in india. hydrotherapy with a swaddled infant may be a useful adjunct intervention for facilitating self - regulation. our survey showed that hydrotherapy is rarely used by indian physiotherapists in the nicu (n = 5). the limitations of this study include the possibility that participants responded to the survey questionnaire with perceived ideal answers, thereby giving what they considered to be the best or more appropriate answer to each question. future research can be focused on more specific assessment and treatment measures used by physiotherapists in the nicus and to identify factors such as inter - professional needs and type of specialized care that may influence practice patterns. practice patterns of physiotherapists working in nicus involve both chest physiotherapy as well neuromuscular physiotherapy. chest physiotherapy assessment has been found to focus mainly on vital parameters which involves an assessment of heart rate, respiratory rate and spo2. treatment is found to predominantly focus on airway clearance techniques like percussion, vibration, postural drainage and airway suction. for neuromuscular physiotherapy strategies preferred by most physiotherapists were parent education, passive range of motion exercise, therapeutic handling and positioning.	objective : to determine practice pattern of physiotherapists in the neonatal intensive care units (icus) in india with regards to cardiopulmonary and neuromuscular physiotherapy.materials and methods : a cross - sectional survey was conducted across india, in which 285 questionnaires were sent via e - mail to physiotherapists working in neonatal intensive care units.results:a total of 139 completed questionnaires were returned with a response rate of 48.7%, with a majority of responses from karnataka, maharashtra and gujarat. more than 90% of physiotherapists performed chest physiotherapy in neonatal icus. chest physiotherapy assessment predominantly focused on vital parameter assessment (86%) and in treatment predominantly focused on percussion (74.1%), vibration (75.5%), chest manipulation (73.3%), postural drainage (67.6%) and suction (65.4%). in neuromuscular physiotherapy more than 60% of physiotherapists used positioning, and parent education, whereas more than 45% focused on passive range of motion exercise and therapeutic handling.conclusion:the practice pattern of physiotherapists for neonates in neonatal intensive care units involves both chest physiotherapy as well neuromuscular physiotherapy. chest physiotherapy assessment focused mainly on vital parameter assessment (heart rate, respiratory rate and partial pressure of oxygen saturation spo2). treatment focused on airway clearance techniques including percussion, vibration, postural drainage and airway suction. in neuromuscular physiotherapy most physiotherapists focused on parent education and passive range of motion exercise, therapeutic handling, as well as positioning.
chordoid glioma of the third ventricle, a rare neuroepithelial tumor of the central nervous system, mostly originates from the anterior part of the third ventricle. chordoid glioma is classified as grade ii tumor according to 2007 world health organization (who) classification of brain tumors (1). approximately 85 cases of chordoid glioma have been reported in english literatures since its first report in 1998 (2). although chordoid glioma is located within the ventricular system, intraventricular seeding or metachronous tumors along the ventricular wall has not been reported. herein, we present the first case of chordoid glioma arising from the third ventricle with multifocal intraventricular seeding in the lateral and fourth ventricles. we also described perfusion mr imaging features of this case different from a previous report. a head ct scan revealed a large high attenuated mass with lobulated margins and small calcifications in the anterior 3rd ventricle (fig. additional small hyper - attenuated masses along the ependymal surface of both lateral ventricles were also found. a large area of peritumoral edema was noted in the left frontal periventricular white matter. a brain mri revealed a 3.1 4.2 4.5 cm well defined multi - lobulated tumor in the anterior third ventricle and suprasellar region displacing the right lateral ventricle superiorly (fig. the mass showed iso - signal intensity on both t1- and t2-weighted images relative to the brain cortex. postcontrast t1-weighted imaging also demonstrated additional multiple enhancing masses along the ependymal surface of lateral ventricles, the fourth ventricle, and in the bilateral foramen of luschka. perfusion mri showed markedly elevated cerebral blood volume (cbv) within the tumors on cbv map (fig. the ratio of maximum tumor cbv relative to white matter was calculated using the following equation : maximum rcbv = maximum cbv tumor / maximum cbv white matter. the maximum cbv was obtained using a circular region of interest with a diameter of 10 mm from each main tumor and the adjacent right frontal white matter. the patient first underwent a tumor removal surgery for the daughter mass in the left lateral ventricle through a left frontal transcortical approach. at surgery, a hard multi - lobulated and encapsulated mass was found to be attached to the ependymal wall. two months following the first surgery, a second operation was performed to remove the main tumor in the third ventricle. the patient underwent tumor removal surgery via a bifrontal craniotomy and a transcallosal interhemispheric approach. at surgery, a hard whitish mass filling the anterior part of the third ventricle was found to be firmly attached to the hypothalamus. on histology, these tumors consisted of clusters and cords of oval - to - polygonal epithelioid cells with eosinophilic cytoplasm in a mucinous stroma (fig. immunohistochemical analysis revealed that these tumor cells were diffusely positive for glial - fibrillary acid protein (gfap), cd 34, and vimentin. in addition, they were focally positive for epithelial membrane antigen (ema) and s-100 protein (fig. 1e, f) with a ki-67 labeling index of less than 0.5%. follow - up, the patient was doing well without any evidence of tumor recurrence in the third or lateral ventricles on follow - up mri. follow - up mri also showed no changes in the size of small enhancing tumors in the fourth ventricle or foramen of luschka. chordoid glioma is a rare low grade neuroepithelial tumor with both glial and chordoid features. the tumor tends to occur in adult women (a female to male ratio of 2 to 1) at mean age of 45 years (range, 5 - 72 years) (3). clinical symptoms including headache, nausea, vomiting, visual disturbances, confusion, lethargy, urinary incontinence, ataxia, hypothyroidism, diabetes insipidus, weight loss or weight gain, and behavioral disturbances due to intracranial hypertension, hydrocephalus, compression of the hypophysis, optic chiasm, and hypothalamus by the tumor (123). chordoid glioma is typically arising in the region of the anterior third ventricle / hypothalamus. there have been a few reports regarding the unusual locations of chordoid glioma such as juxtaventricular white matter and thalamus (4). in contrast to previous reports, our case of chordoid glioma had a large main tumor in the third ventricle with multiple smaller masses in the lateral and fourth ventricles, suggesting intraventricular dissemination of the third ventricular tumor rather than concomitant tumor occurrence. given the location of the tumor, there is a possibility of intraventricular seeding of choroid glioma which has not been previously reported. therefore, the true biological behavior of this tumor remains to be established (4). our case supports the idea that chordoid glioma might be derived from ependymal cells or tanycytes. several studies have shown that tumor cells of chordoid glioma have features of ependymal differentiation on electron microscopy such as basal lamina, microvilli, and intermediate filaments (5). tanycytes are believed to be the primitive progenitor cells in the phylogenetic development of ependymal cells and glial cells. they are primarily present in the circumventricular organs with predominance in the anterior portion of the third ventricle (6). brain tumors arising from the ependymal lining of the ventricles are likely to have cerebrospinal fluid (csf) seeding. for example, csf seeding has been seen in about 8% to 33% of patients with ependymoma at presentation (7). although intraventricular seeding of diffuse who grade ii glioma is rare, it can occur during follow - up period after incomplete resections of primary tumor (8). on imaging studies, chordoid gliomas are solid, ovoid in shape, well circumscribed, and located in the anterior third ventricle and/or suprasellar region. at ct, most tumors are hyperdense on unenhanced scan and enhanced homogenously after injection of contrast agent. at mri, chordoid gliomas have isosignal intensity on t1-weighted images and slightly hypersignal intensity on t2-weighted images. there has been only one case report describing the perfusion mr imaging features of chordoid glioma. grand. (9) have reported that the cbv of the tumor is not elevated compared to that of white matter. the relative maximum cbv ratio was 6.95 in our case, suggesting that this tumor entity might be composed of heterogeneous subgroups. however, t1-weighted leakage and t2- and/or t2 -weighted imaging residual effects were not corrected in our case. on histopathology, chordoid gliomas had characteristic appearance consisting of cords and clusters of epithelioid cells with eosinophilic cytoplasm and relatively uniform round to oval nuclei embedded in mucinous stroma (123). immunohistochemically, chordoid gliomas were strongly and diffusely positive for gfap, vimentin, cd 34, and focally positive for ema and cytokeratin. therefore, cd 34 can be used to differentiate chordoid glioma from chordoid meningioma, pilocytic astrocytoma, and ependymoma (3). our case was also diffusely positive for gfap, cd 34, vimentin, and focally positive for ema and s-100 protein. however, gross total resection is often difficult given its location in the deep structure of brain and its proximity to critical neurovascular structures. therefore, a more conservative treatment approach other than surgical resection is warranted (10). in our case in addition, there was no change in remained tumor in the fourth ventricle on follow - up mri after 45 months of gross total resection, confirming the low grade nature of this tumor. using chemotherapy for chordoid glioma has not been reported. in conclusion, we described the first case of chordoid glioma in the third ventricular with intraventricular dissemination in the lateral and fourth ventricles. although chordoid glioma is a low grade tumor, the tumor can have elevated cbv on perfusion mr imaging.	chordoid glioma is a rare low grade tumor typically located in the third ventricle. although a chordoid glioma can arise from ventricle with tumor cells having features of ependymal differentiation, intraventricular dissemination has not been reported. here we report a case of a patient with third ventricular chordoid glioma and intraventricular dissemination in the lateral and fourth ventricles. we described the perfusion mr imaging features of our case different from a previous report.
this work examines a simultaneous acquisition method for combined single photon emission computed tomography (spect) and x - ray computed tomography (ct) imaging systems. currently all commercially available spect - ct systems for preclinical imaging use a sequential scan mode that results in undesirably long acquisition times that limit the potential throughput of the imaging system. acquisition of simultaneous multi - modality data may improve temporal and physiological correlation between each modality and improve image registration. additionally, reducing scan times minimizes the time any given animal must remain under anesthesia. although spect scans typically demand the greatest time requirements, complementary ct scans in a preclinical system can add roughly 2 to 15 minutes or more depending upon the imaging system and the selected protocol. these scan times are significantly higher than those found on clinical gantries as most preclinical imaging systems lack slip ring technology that enables high speed rotation and true helical acquisition. here, we look at the possibility of modifying a commercially available inveon preclinical spect - ct system (siemens medical solutions usa, inc., knoxville, tn) such that both modalities acquire data simultaneously, thus reducing the overall acquisition time. whereas previous work by nagarkar. worked to develop a custom - designed system with the capability of performing simultaneous spect - ct, the system was only available as a resource to a single research group. in contrast, this work sought to use a commercially available system with minor modifications to the system in order to develop new functionality that may be accessible to a larger number of institutions worldwide. in this work, we address key considerations that may allow similar principles as those described for our available imaging equipment to be applied to a number of other commercial platforms both preclinically and clinically.. simultaneous acquisition of spect and ct modalities enables improved temporal matching and the potential for more accurate image registration that may impact the application of attenuation and scatter correction. simultaneous imaging with respiratory or cardiac monitoring hardware would enable the use of the same physiological signal for each modality for use in correcting this motion. in addition to improving temporal matching, the ability to simultaneously acquire both spect and ct modalities may provide modest improvements in efficiency with regard to image acquisitions. this is achieved by the elimination of typical movements required to move and reset the gantry and bed between modalities. although the primary time limiting factor for any spect - ct study is generally the spect data collection, eliminating unnecessary inefficiencies in image acquisition can potentially reduce times significantly for animal studies involving a large number of subjects. most in vivo preclinical micro - ct systems have an x - ray tube and detector mounted to a rotating stage as shown in figure 1(a). the x - ray tube and detector are rotated about the object positioned in the center of the field of view (fov) while projections are acquired at specified angles for a length of time determined by the exposure time. this method of acquisition is known as step and shoot and enables the high resolution imaging capabilities required of preclinical imaging systems at the expense of increased scan times. spect imaging is performed in a similar fashion with detector heads mounted on a rotating stage and photons (or counts) from a radioactive source collected at specified angles around the object in the fov. preclinical spect detectors are often fitted with various collimator designs, such as multi - pinhole collimators, that enable higher resolution imaging while maintaining sensitivity performance. more in - depth information regarding preclinical spect and ct systems can be found in small animal imaging and molecular imaging : principles and practice. this work examines the feasibility of modifying a commercially available small animal spect / ct platform and assesses key performance parameters and limitations related to such modifications. modification of existing scanner systems to offer such technology may provide a cost effective way of improving small animal spect / ct imaging workflows without the need to buy additional specialized hardware. the spect - ct system modified for this work consisted of two spect detectors : an x - ray detector and an x - ray source mounted coaxially on a rotating gantry. each of these components is mounted on high - precision, computer - controlled motion stages for automatic fov adjustment. the x - ray source operates in a voltage range of 30 to 80 kvp, while the spect detectors operate within an energy range of 30 to 300 kev. the spect data were acquired in listmode format that enabled the energy window to be selected after acquisition rather than having to know the optimum energy window prior to beginning the scan. an inveon spect - ct system (siemens medical solutions usa, inc.) was modified to support simultaneous acquisitions with the intent of performing this task with minimal hardware or software modification. this particular unit has a number of motion sensors that detect the movement of the x - ray and spect detector components in order to prevent possible collisions as the components move towards the center of the gantry to reach their default imaging positions. manufacturer precautions do not allow the ct and spect subsystems to be used simultaneously at their default imaging positions as the components would potentially collide as they meet towards the center of the gantry. disabling these precautionary mechanisms enables the spect and ct components to be simultaneously moved in towards the center of rotation while also allowing custom positioning of each of the components so that the fields of view of each subsystem can be optimized for simultaneous imaging without colliding. ideally, the spect and ct fields of view (fovs) would be identical in simultaneous acquisition modes ; however, this system has two x - ray detector options of an 8.4 cm 5.5 cm or 10 cm 10 cm detector. the active ct fov depends on the detector size and the magnification setting (source to detector relative position). the spect fov varies as well depending on the collimator chosen and the magnification settings. in order to best match the simultaneous acquisition fovs, the x - ray ct components were placed in a medium magnification mode that provided a magnification factor of approximately 1.9. in this configuration, the achievable spatial resolution of the ct is 40 to 200 microns, depending on the x - ray detector binning factor. with the larger area detector (10 cm 10 cm) installed in this gantry, the maximum axial fov that could be matched to the spect system was 6.7 cm. however, the axial fov can be extended by scanning with a helical orbit during acquisition. before starting fully 3d spect / ct imaging, we tested the simultaneous acquisition configuration to determine how the two modalities would interact using two - dimensional imaging techniques. radiographs and scintigraph images were taken using ct and spect subsystems, respectively, with a 1 mci co point source in the spect fov and the ct kvp varied from 60 to 80 kvp. this was performed for each kvp setting to examine potential effects of x - ray scatter and pulse pileup through the collimators of the spect subsystem during simultaneous acquisition. scintigraphs were acquired for each setting with a data acquisition time of 100 s per projection. the emission data were histogrammed using a wide open window with a range from 0 to 300 kev and also a 20% energy window around the co photopeak (122 kev) with a lower bound of 110 kev and an upper bound of 134 kev. figure 2 shows the spectrum acquired from the spect detector with varying x - ray voltage with a co source present. these projection data were used to create plots of measured counts versus x - ray tube voltage. the data were analyzed to find the x - ray tube setting where the measured counts with the x - ray tube on and no co source in the fov was approximately equal to the measured counts with only the co source in the fov. since the x - ray tube and spect detectors are orthogonally oriented with no x - ray photons directly incident on the spect detectors, this crossover point would be the maximum x - ray tube kvp setting that would provide the greatest possible x - ray flux for ct imaging with the least count contamination of the spect data. these results were used as the optimum configuration of the x - ray tube for all subsequent simultaneous spect / ct protocols. the spect system in this platform uses a helical acquisition orbit. the ct subsystem was only capable of circular orbits (no bed travel during gantry rotation) when using the software provided by the manufacturer. the ct subsystem was modified to also acquire data helically to match the primary spect acquisition mode and to enable acquisitions with ideal axial sampling, better matched fields of view and ultimately simultaneous acquisition of spect and ct data. in brief, the user was given the ability to specify a distance to move the bed pallet axially between each ct projection. an additional issue that arose with acquiring simultaneous spect - ct data in this gantry was that the center of the ct fov was mechanically positioned approximately 18 mm axially from the center of the spect fov. this issue could not be resolved without significant hardware modifications to the spect and ct subsystem mounting hardware, which was outside our constraints of minimal modification. this limitation simply meant that images acquired during simultaneous spect - ct scans covered slightly different regions along the axial direction of the phantom with the loss in coregistered fov being equal to the offset between the two imaging subsystems (18 mm). an embedded computer that is internal to the gantry controls all commands to the hardware while an external workstation, or host computer, sends requests to the embedded computer via a gigabit ethernet connection. this architecture was exploited in this study by forcing simultaneous acquisition of data by performing independent acquisitions on the two separate computers simultaneously. during tomographic scans, spect data was acquired by the embedded computer while the x - ray ct data was collected using the host computer, which defined the gantry and bed motion for the imaging protocol. ct only data acquired with circular gantry orbits were reconstructed using a dedicated workstation with cobra high speed reconstruction software (exxim computing corporation). helical ct data (acquired during simultaneous spect - ct scans) were reconstructed using an implementation of a 3d weighted fbp algorithm for spiral ct as described by [9, 10 ]. ct data acquisitions were performed using a voltage of 70 kvp (found from initial planar studies) and anode current of 500 a. ct only scans were acquired with 360 projections over a full 360-degree rotation. for each ct projection an exposure time of 0.25 s was used. for the derenzo phantom studies, contrast was enhanced by dissolving iodized salt into the water prior to imaging with ct. the radius of rotation for the spect detectors was set to 50 mm resulting in a 100 mm bore size and a transaxial fov of roughly 6.6 cm. spect data were acquired with 120 and 180 projections over 360 degrees with per projection acquisition times of 7 to 30 seconds, depending on the study and radioactivity in the phantom. this resulted in total scan times of 15 minutes for the uniformity phantoms and 3040 minutes for derenzo phantoms imaged using spect / ct. an additional 72-minute spect only scan was performed on the derenzo phantom to acquire a high count dataset for comparison. it is important to note that although scan times varied, the object was only exposed to incident radiation from the ct x - ray tube for no more than approximately 90 seconds during the course of the entire scan. the tc phantoms used for this study were a uniform water phantom with a 30 mm diameter and a derenzo phantom (micro hot spot phantom, data spectrum corp.) with rod sizes of 4.8, 4.0, 3.2, 2.4, 1.6, and 1.2 mm. the uniform syringe contained an aqueous solution of tc with a total activity of 1.8 mci at scan time. the derenzo phantom was injected with approximately 3 mci of tc. for each series of experiments, the number of total counts in each scan was kept as similar as possible for the scans lasting 3040 minutes by increasing the necessary per projection acquisition time to compensate for the natural decay of the tc isotope. for each of the tc phantom studies, data were acquired in the following modes : spect only, ct only, and simultaneous spect - ct. the ct data were examined to verify that the spect isotope in the fov during the ct did not cause any image artifacts or errors in quantitative measurements from the ct modality. the collimator used for spect acquisition of the tc phantom data was a 3-pinhole rat whole - body tungsten collimator with 1.2 mm diameter pinholes. this collimator was chosen because it provides spatial resolution sufficient for animal imaging with the largest fov possible on this spect platform, providing the greatest potential spect - ct coscan range. all spect data were reconstructed with an implementation of 3d - osem using a model of the system point - spread function with 8 iterations, 12 subsets, and a voxel size of 0.5 mm. regions of interest were drawn on the spect only, ct only, and spect - ct data and statistical difference between values assessed with p 0.05 were assumed to be a nonsignificant difference between samples. visual analysis of the derenzo phantom scans was performed using irw to determine whether there is significant degradation in the spect image quality between x - ray on and off images. using irw to draw line profiles along the y - axis of the central axial view and rois over the phantom rods, we examined peak - to - valley ratios, standard deviation to mean values, snr, and 95% ci within the rods to determine if and how much degradation in image quality might occur in simultaneous spect - ct measurements. figure 4 shows a photograph of the derenzo phantom loaded into the spect - ct system, and figure 5(a) shows a reconstructed simultaneous multimodal image of the derenzo phantom using spect with contrast - enhanced ct. figure 5(b) illustrates the rois drawn within the hot rods used for the quantitative analysis of each of the derenzo phantom scans. figure 6 shows the plot of the counts measured in the spect projection data as the x - ray tube voltage was swept from 80 to 60 kvp in the absence and presence of a co point source. at 70 kvp, the mean counts from the orthogonally oriented x - ray source are nearly equivalent to those from a point source located in the center of the fov. the 70 kvp x - ray tube setting was used for all subsequent simultaneous imaging tests with further proof of this optimization shown in figure 7. note that the curves do not sum linearly because when the co source is present in the fov, it scatters additional x - rays into the spect detectors. in figure 7(a), an image of a co point source in the central fov is shown with no x - rays present from the ct system and a wide open energy window of 30300 kev. in this figure, the single point source appears as five individual sources because of the 5-pinhole collimator used to acquire the projection data. data acquired using a wide open energy window with the co point source in the fov and with the x - ray source on and at full power (80 kvp, 500 a) showed obvious scatter and pulse pileup effects. in this image these effects on the spect projection image were severe, as seen in figure 7(b). adjusting the energy window to the standard 20% imaging window (110134 kev) used for co imaging showed a dramatic improvement in planar image quality even with full power x - rays present ; however, some significant artifacts remained in the project data (figure 7(c)). data using the standard 20% co imaging window and the optimized 70 kvp x - ray tube settings resulted in relatively clean projection images (figure 7(d)) acquired in a simultaneous manner with these fully optimized results used for subsequent testing. the results in table 1 show that the ct image uniformity was not adversely affected by the presence of radioactive tc with no statistical difference between mean hu values (p > 0.05). the uniformity phantom snr of the syringe filled with water was 3.88, while the uniformity of the syringe filled with tc solution was 3.84, a minimal 1.03% decrease in uniformity. the measurements from the spect only scan (no x - rays) and the simultaneous spect - ct scan (with x - rays) are shown in table 2. these measurements also yielded no significant difference in mean count values with p > 0.05 and a difference in counts of 28%. snr also showed a minimal 12% difference which was nearly 47% less than the standard deviation to mean ratio for mean counts. visual inspection of the spect / ct derenzo phantom data, shown in figure 8, showed little degradation in image quality and spatial resolution. figure 8(a) shows a 37-minute spect only acquisition. figure 8(b) is a spect only study with an acquisition time of 72 minutes, representing a best - case scenario. finally, figure 8(c) shows the derenzo phantom imaged by a 30-minute simultaneous spect - ct workflow. a slight negative impact on image quality and resolution appeared to occur in the simultaneously acquired spect image. the peak - to - valley ratios for each profile drawn on the various rod sizes show consistent ratios for the 37-minute spect, 72-minute spect, and the spect - ct scans, with percentage difference from the average values never reaching more than 8% for the spect - ct studies. additionally, the ratios of peak - to - valley measurements between decreasing rod sizes also show consistent reductions indicating that the simultaneous spect - ct acquisitions are not significantly degraded compared to the spect only workflows. figure 9 shows a plot of the line profile drawn across the 2.4 mm rods for the spect only and spect - ct acquisitions. tables 3 and 4 present the average peak - to - valley ratio measurements for each rod size as well as calculations of the ratio of peak - to - valley between rod sizes. in regions of interest drawn on the hot rods of the derenzo phantom, signal to noise ratio in the rods improved with simultaneous imaging contrary to the initial visual assessment. the mean values for the 37-minute and 72-minute spect acquisitions are consistent as the mean number of counts in the segmented regions increases proportionally with scan time (approximately 2x). the simultaneous spect - ct acquisition also shows agreement with this expected result as the ratio of mean counts in identical regions is less by a factor of 1.2. table 5 shows the full roi statistics as well as the signal to noise ratio and standard deviation to mean calculations. the data indicate that the inveon spect - ct can be used for simultaneous spect - ct imaging. good results were obtained both for a uniform phantom with known activity concentrations and the derenzo phantom used for image quality assessments. for each series of measurements using the empirically derived parameters, the results indicated minimal degradation of image quality when simultaneous spect and ct acquisition was performed for the isotopes examined. although work in the area of simultaneous detection and cross - talk correction methods have been performed [12, 13 ], none of this research looked at the possibility of converting a commercially available unit for the purposes of simultaneous imaging. the ability to convert a readily available imaging system means a greater likelihood that other researchers could use this work where previous research systems are only accessible by those labs that created them. our work can potentially be used to improve synchronization between modalities when performing complex imaging workflows such as those required for cardiac and respiratory gated acquisitions. with sequential acquisition, simultaneous acquisition reduces this variation as each projection acquired for spect and ct were acquired during the same general time in the animal 's physiology. improvements in gating capabilities using low - dose detector technology may also improve gating applications with significantly shorter exposure times. testing indicated that the highest useful x - ray setting that could be used simultaneously with spect imaging on this platform was 70 kvp. projection data acquired with x - ray energies above 70 kvp showed significant artifacts that are most likely due to pileup effects and some septal penetration of the collimator. although not directly visualized, x - ray photons falling outside the energy window settings could result in dead time issues if the photon flux was sufficient. because of the orthogonality of the ct and spect components, the estimated counts per second were not substantial enough to cause deadtime issues on this system. if the subject moves in one modality then it also has moved in the other. many studies have been performed to assess the effects of misregistration of correction data which result in incorrect voxel values in the regions of movement. during this work only a small number of slip ring ct systems exist for small animal imaging which limits the gantry rotation speed of small animal spect / ct platforms. this results in fairly large percentages of the total ct acquisition time being the physical movement of components. simultaneous acquisition can improve this as the gantry potentially needs only to rotate once to complete the acquisition of both modalities as shown by austin. in the supplementary materials. additional time savings were realized by implementing helical ct. on this step and shoot platform, extending the axial range may result in multiple bed positions being acquired. this increases the scan time linearly with the number of bed positions. by using a helical ct to match the spect acquisition this removes the necessity for multiple bed imaging leading to although simultaneous imaging has some unique benefits, it is not without compromise. on this particular imaging platform, the primary limitation is the maximum achievable spect resolution. because of the hardware positioning within the gantry, the minimum distance between the spect detector and the subject is increased compared to the standard hardware configuration resulting in a reduced maximum resolution. the collimators used to image the derenzo phantom in this study are specified by the manufacturer to achieve a maximum spatial resolution of 1.4 mm fwhm. derenzo phantom images presented in this work showed that even in this reduced resolution configuration rod diameters between 1.6 and 2.4 mm were clearly visible. this slight decrease in resolution shows that rat imaging applications would not be significantly affected nor would mouse imaging protocols, such as screening, where this decrease in resolution would only minimally impact the data. protocols requiring high resolution imaging would certainly be degraded using a simultaneous technique on this particular commercial platform. there appeared to be a slight reduction in image quality and image resolution for simultaneously acquired data. this was most likely caused by the introduction of scattered x - ray photons and pileup effects. resolution reductions were seen as a reduction in the separation between the smallest visible rods in the derenzo phantom. this loss may have been caused by reduction in overall contrast during the simultaneous spect - ct scan. the wide energy gap between tc photon emission (140 kev) and the average x - ray photon energy (2535 kev) make separation of the counts easier. for isotopes with emissions that overlap the range of x - ray energies, such as i (2536 kev), separation of the individual contributions could be more challenging. although this is a limitation for this technique, low energy spect isotopes are not used as frequently as higher energy isotopes such as tc and i. future work in simultaneous acquisition methods could include development of novel ways to perform simultaneous imaging with i and to assess any specific sensitivities that might exist with this isotope. the primary issue would be to develop a method by which the gammas emitted from i could be separated from any ct x - rays. one approach would be to use a monte carlo model that would enable a statistical method of removing the x - rays from the i spect projection data, such as the gate model created for this platform by lee. animal acquisition was not possible for this study because of facility limitations where this research occurred. this work does provide the necessary tools for this to be repeated at a facility where access to animals for in vivo animal studies could be performed.	multi - modality imaging provides coregistered pet - ct and spect - ct images ; however such multi - modality workflows usually consist of sequential scans from the individual imaging components for each modality. this typical workflow may result in long scan times limiting throughput of the imaging system. conversely, acquiring multi - modality data simultaneously may improve correlation and registration of images, improve temporal alignment of the acquired data, increase imaging throughput, and benefit the scanned subject by minimizing time under anesthetic. in this work, we demonstrate the feasibility and procedure for modifying a commercially available preclinical spect - ct platform to enable simultaneous spect - ct acquisition. we also evaluate the performance of simultaneous spect - ct tomographic imaging with this modified system. performance was accessed using a 57co source and image quality was evaluated with 99mtc phantoms in a series of simultaneous spect - ct scans.
the incidence of renal infarction in an autopsy study was 1.4%, whereas clinical diagnosis was made in only 0.014% of the studied patients. the clinical manifestations of renal artery thrombosis vary, depending on whether the occlusion affects both renal arteries or not. delay of the correct diagnosis may prove disastrous for the patient. our case is probably the first one in english literature describing a patient with unilateral renal artery thrombosis after lobectomy for lung cancer. a 53-year - old female patient was admitted to our hospital with the diagnosis of lung cancer. her medical history was clear, except that she was a social drinker and smoker (3 - 4 glasses of wine / week, 5 cigarettes / day for 25 years). physical examination and blood test analysis were normal, and cardiac function was found to be excellent. the preoperative transbronchial needle aspiration cytology revealed an adenocarcinoma in the left upper lobe. computed tomography (ct) of the thorax, abdomen and brain revealed nothing apart from a mass located in the left upper lobe, 2 cm in diameter. the patient received a formal left upper lobectomy in combination with mediastinal lymph node sampling. no complications were observed during the operation, and the immediate postoperative period was straightforward until the third postoperative day, when she began to complain of a flank pain located at the lower side of the left hemithorax and the nearby lumbar area. the pain was not affected by respiratory movements and position changes, and it was continuous without radiation. laboratory data showed a minor elevation of white blood cell count (12,500 cells / mm) and lactate dehydrogenase (371 consequently, the patient was treated with painkillers and anti - inflammatory agents. despite the medication given, pain persisted for the next 24 h without any changes. an upper abdomen ultrasound scan was performed combined with a chest x - ray revealing nothing that could explain the condition. an expert nephrologist was called in order to manage the problem. however, precious time was already lost and further therapy with thrombolytics did not offer significant improvement, since the left kidney was considered already nonfunctioning. the levels of blood urea nitrogen and creatinine did not show remarkable elevation compared with the preoperative ones (urea : 33 41 mg / dl, creatinine : 0.71 0.83 mg / dl). the patient was discharged two weeks after the operation and anticoagulation therapy with warfarin was given. six months later, renal function remains satisfying and the patient is free of any symptoms. it is of crucial importance for acute renal artery thrombosis, although it is an uncommon situation, to be diagnosed early. in the vast majority of the cases, however, significant delay is the rule, causing irreversible damage to the kidney. the clinical presentations of renal arterial thrombosis are not specific for the disease and usually include flank pain originating from the ipsilateral lumbar area, accompanied by fever, nausea and vomiting anuria or oliguria combined with proteinuria or hematuria are findings indicating bilateral renal artery occlusion. blood tests are not apt to establish an accurate diagnosis because of lack in sensitivity and specificity. common causes of renal artery occlusion include congestive heart failure, atrial fibrillation, and valvular or ischemic heart disease. moreover, trauma, clotting disorders, cancer and some rare hereditary diseases are also mentioned. plain x - ray and ultrasonography can be significantly helpful, but the final diagnosis is usually determined by contrast - enhanced ct. invasive diagnostic tools such as renal artery angiography are not without complications. in our case, the diagnosis was delayed and as a result the left kidney lost its function. given that the patient was free of cardiac diseases and clotting disorders, lung cancer could have been the cause for renal arterial thrombosis. although the patient was receiving subcutaneous low molecular weight heparin 3,500 iu from the first postoperative day, this very serious complication did happen. diagnosis was reached incidentally with the help of contrast - enhanced ct, which seems to be the gold standard for renal artery occlusion diagnosis. therapy in acute renal thrombosis typically consists of low molecular weight heparin in the acute phase, followed by warfarin. the value of antiplatelets is still under study. because of the high risk for more thromboembolic complications in these patients it is advisable that the anticoagulation treatment should be continued, and a careful follow - up is mandatory in conclusion, renal arterial thrombosis is a rare and serious condition, which should be diagnosed early. nonspecific symptomatology complicates the differential diagnosis and a high level of suspicion is required in order to not lose precious time. if lumbar flank pain appears and all other possible diagnoses are excluded, contrast - enhanced ct should be considered. apart from the usual causes, the clinician should keep in mind this extremely uncommon case, which may be the first one described in english literature.	acute renal arterial thrombosis is a rare but very urgent situation demanding immediate treatment. it is characterized by unspecific symptomatology which often misleads the clinicians. as a result, precious time can be lost until the correct diagnosis is reached. the case of a 53-year - old female who underwent a left upper lobectomy for lung cancer is presented. on the third postoperative day, the patient began to complain of a flank pain located at the lower side of the left hemithorax and the nearby lumbar area. a renal arterial thrombosis was finally diagnosed and subcutaneous low molecular weight heparin was started immediately. the patient was discharged two weeks later and anticoagulation therapy with warfarin was given. six months later, renal function remains satisfying and the patient is free of any symptoms. this is probably the first case in english literature of renal arterial thrombosis following lobectomy for lung cancer.
perineal hernias following abdomino - perineal excision of rectum (aper), requiring repair, have been reported to occur in < 1% [1, 2 ] of people. patients may exhibit some perineal bulging following an increase in intra - abdominal pressure, but are rarely symptomatic and, therefore, do not require operative intervention. the literature to date has reported that perineal hernias occur primarily following aper ; however, there is very little data with regard to incidence and the management of perineal hernias. a 79-year - old gentleman who initially underwent a laparoscopic assisted extralevator abdomino - perineal excision (elape) of the rectum for extensive circumferential low rectal cancer after neo - adjuvant long course chemoradiotherapy developed a symptomatic perineal hernia. initial closure of the perineal wound was performed using a double layer of interrupted mattress sutures with a 2/0 absorbable braided suture. the patient 's only co - morbidity was intermittent asthma, for which he was prescribed a salbutamol inhaler. given the nature of the disease, it was felt that he would benefit from adjuvant chemotherapy. subsequent radiological surveillance did not show any evidence of recurrence and cea levels remained low. however, magnetic resonance imaging (mri) carried out at 19 months demonstrated that the small bowel had herniated through the levator sling and the patient had started to experience severe discomfort in the perineal region. repair of the defect was carried out in conjunction with the plastic surgery team, using a pedicled gracilis flap. again, despite an uneventful recovery, 5 months later the patient experienced lower abdominal pain and nausea and noted a distinct swelling of his left upper thigh. a repeat mri confirmed a further recurrence of the perineal hernia in the form of uncompromised bowel, which extended beneath the gracilis flap, which then continued into the adductor compartment of the left thigh (fig. figure 1:(a and b) coronal and transverse magnetic resonance imaging scans demonstrating perineal hernia extending into the left thigh. (demonstrated by white arrows.) (a and b) coronal and transverse magnetic resonance imaging scans demonstrating perineal hernia extending into the left thigh. given the recurrent nature of the hernia, a rectus flap repair was performed and after 15 months he remains hernia free. although the majority of patients after aper will demonstrate an element of perineal herniation, they do not exhibit any symptoms and, therefore, do not require operative repair. commonly reported symptoms secondary to perineal hernias include perineal discomfort, a dragging sensation, skin breakdown, urinary dysfunction, bowel obstruction and evisceration of pelvic contents [14 ]. given the paucity of available literature, it is difficult to identify definitive predisposing factors ; however, reported factors include smoking, chemoradiotherapy, sacrectomy / coccygectomy, excessive length of small bowel, lack of peritoneal closure and excision of the levators [1, 2, 4 ]. with conventional aper, involvement of circumferential resection margins (crm) has been identified to occur most commonly at the floor of the pelvis. the aim of elape is to reduce this crm positivity, along with rates of intra - operative perforation. tissue morphometry of elape specimens showed that this reduction in crm positivity occurs as a result of removing more tissue from around the tumour than standard aper, and thus improving rates of local recurrence and overall survival. despite these superior oncological outcomes, elape has also been associated with an increase in perineal wound complications, which may be attributed to the increased size of the perineal defect. there has been an increase in the uptake of laparoscopic aper in recent years, which may be attributed to an increase in the incidence of perineal hernias ; however, actual figures have not been reported. a possible reason is that a laparoscopic technique may result in fewer adhesions and thus less restriction of small bowel herniation. our patient had undergone both neo - adjuvant and adjuvant therapy, along with a laparoscopic elape. 's systematic review on the outcome of primary perineal reconstruction following elape demonstrated lower rates of perineal complications compared with primary closure. there has been debate surrounding reconstructive technique, but no significant differences in perineal complication rates have been demonstrated between biological mesh and myocutanous flap reconstruction. primary reconstruction using biological mesh may be preferable due to shorter operating times, absence of donor site morbidity and the option of operating independent of a plastic surgeon. however, the tendency for biological mesh to thin with time, leading to possible recurrence and the risk of infection, may make some opt for myocutanous flap reconstruction. cost benefits have been suggested when using a biological mesh over a rectus abdominis flap due to their association with a reduced length of post - operative stay. due to the variety of flap reconstructions reported and due to relatively low numbers in each study, preference is still yet to be determined. the use of a transabdominal technique allows of better visualisation of the defect and allows identification of tumour recurrence, while a transperineal approach minimises the potential risks associated with entering the peritoneal cavity. laparoscopic repair has been shown to have results comparable with those of open repair, but is dependent on patient selection (body habitus, comorbidities, intraperitoneal adhesions). perineal hernia repair can be a challenging procedure, with a recurrence rate of up to 30% ; foster. demonstrated that primary reconstruction at the time of elape may be preferable to potentially prevent perineal hernias. presently, it is thought that a high - quality prospective trial is needed to help answer the many questions that arise between surgeons when discussing the management options for perineal hernias. repair after elape poses a significant challenge ; hence, further research is needed to determine the best method of managing and preventing this difficult complication. however, we advocate that management of the perineal defect at the time of initial surgery should be discussed and carried out with the input of the plastic surgeons.	symptomatic perineal hernias following abdomino - perineal excision of rectum have been reported to occur uncommonly. we present the case of a 79-year - old gentleman who developed a perineal hernia after laparoscopic - assisted extralevator abdomino - perineal excision (elape) of the rectum. despite initial myocutaneous flap repair, there was further symptomatic recurrence. magnetic resonance imaging demonstrated non - compromised bowel extending beneath the gracilis flap with extension into the adductor compartment of the left thigh. given the recurrent nature, a rectus flap repair was performed and after 15 months, he remains hernia free. there is currently no consensus as to the optimal operative technique in the prevention and management of these hernias ; however, primary reconstruction at the time of elape may be preferable. symptomatic perineal hernias can be severely debilitating and require operative repair. we suggest that surgical options should be discussed and carried out with the input of a plastic surgeon.
a 70-year - old japanese man presented at our hospital with an asymptomatic, 20 30 mm irregularly shaped blackish, flat elevated plaque with a gray nodule in the periphery on his left lower leg (fig. the lesion had been present for 10 years and had recently enlarged, associated with bleeding. dermoscopy revealed leaf - like areas, large blue - gray ovoid nests and multiple blue - gray globules (fig. basal cell carcinoma and malignant melanoma were listed as suspected diagnoses, and we performed an excisional biopsy of the tumor. histopathological examination of the tumor revealed infundibular structures of the outer hair sheath in the center of the plaque, which consisted of three distinct parts (fig. 2a). the first part showed circumscribed massive aggregation of basophilic basaloid cells containing abundant melanin granules with peripheral palisading and retraction spaces (fig. this part of the tumor exhibited trichilemmal keratinization with squamous eddies, which were surrounded by a fibrous stroma (fig. the third part showed reticular aggregation of basaloid cells with small infundibular cystic structures in the papillary dermis (fig. the stromal part showed no fibrosis and abundant mucin deposition staining positive with alcian blue (fig. immunohistochemical staining revealed diffuse positivity for anti - bcl-2 in the first part (fig. 3b), positivity only at the periphery of tumor nests in the second part (fig. microscopic examination of the tumor showed that it consisted of three distinct histological types : massive aggregation of basophilic cells was diagnosed as solid - type basal cell carcinoma, massive aggregation of clear cells with trichilemmal keratinization was diagnosed as proliferating trichilemmal tumor, and reticular small aggregations of basophilic basaloid cells in the papillary dermis were diagnosed as infundibulocystic basal cell carcinoma. basal cell carcinoma with follicular differentiation in 1987 by tozawa and ackerman, and later in 1990 the tumor was proposed as a variant of basal cell carcinoma showing differentiation to infundibular cyst - like structures by walsh and ackerman. clinically, the tumor presents as small papules / nodules, most commonly on the face. characteristic histopathological features of the tumor are small symmetrical, circumscribed aggregations of basaloid cells containing numerous infundibular cyst - like structures. infundibulocystic basal cell carcinoma is different from trichoepithelioma in that there are no follicular bulbs and papillae, no stromal fibrosis around the tumor, reticulated pattern of neoplastic cells, and abundant epithelial mucin. in the present case, anti - bcl-2 immunohistochemical staining was diffusely positive in portions of typical basal cell carcinoma and infundibulocystic basal cell carcinoma., bcl-2 is present within basal keratinocytes, mesenchymal cells of follicular papilla, clear cells of eccrine glands, melanocytes and lymphocytes. anti - bcl-2 staining is useful for distinguishing between trichoepitheliomas and basal cell carcinoma, the former staining positively in the periphery of tumor nests, whereas the latter shows diffuse and widespread staining in all tumor nests. basal cell carcinoma is a neoplasm proposed to be derived from epithelial germ cells, and infundibulocystic basal cell carcinoma is derived from the infundibulum of the hair follicle, whereas proliferating trichilemmal tumor is derived from the hair follicle isthmus. there have been four reports of basal cell carcinoma arising with trichilemmoma without a pre - existing sebaceous nevus. taken together, the present findings suggest that basal cell carcinoma may be derived from hair germ cells, including the outer hair sheath and hair follicular infundibulum. the authors report no conflicts of interest. there were no funding sources for this work.	a 70-year - old japanese man presented at our hospital with an asymptomatic, blackish, irregularly shaped plaque with a gray nodule in the periphery on his left lower leg. the lesion had been present for 10 years and had recently enlarged, associated with bleeding. histopathologically, the tumor consisted of three distinct parts : the first part showed massive aggregation of basophilic basaloid cells with peripheral palisading and abundant melanin granules, and was diagnosed as solid - type basal cell carcinoma. the second part showed aggregation of clear cells with squamous eddies, and was diagnosed as proliferating trichilemmal tumor. the third part showed reticular aggregation of basaloid cells with infundibular cysts in the papillary dermis, and was diagnosed as infundibulocystic basal cell carcinoma. we diagnosed this tumor as basal cell carcinoma with various forms of hair follicle differentiation, including differentiation into the outer root sheath.
neuropathic pain is a chronic condition caused by a primary lesion or dysfunction of the nervous system. the pain can be spontaneous, a painful response to innocuous stimuli (allodynia), or an exaggerated pain in response to noxious stimuli (hyperalgesia).1 approximately 65%85% of spinal cord injury (sci) patients report experiencing neuropathic pain, and approximately one - third of them have severe neuropathic pain.2 neuropathic pain does not resolve over time and in some cases worsens over time.3 current pharmacological treatments provide incomplete and often insufficient relief. therefore, neuropathic pain has increasingly been recognized as an important contributor to poorer physical, psychological, and social functioning in people with sci.46 alternative, nonpharmacological interventions for neuropathic pain management are in great need for this population. various neurostimulation techniques have been developed and used for neuropathic pain management after sci, with varying degrees of success,7 including transcutaneous electrical nerve stimulation (tens),8 electroacupuncture,9 spinal cord stimulation,10 deep brain stimulation,11 and transcranial direct current stimulation.1214 breathing - controlled electrical stimulation (breestim) is a recently developed, novel, nonpharmacological intervention for neuropathic pain management.15 briefly, in the breestim treatment, single - pulse electrical stimulation (estim) is delivered transcutaneously to a peripheral nerve. we have shown that breestim is effective in reducing chronic neuropathic pain in people with paraplegia and above - the - knee amputation. in these cases, breestim was applied only to the median nerve in the arm. as such, it was not likely that breestim modified the source of pain at the periphery. these preliminary observations suggest that breestim could modify the affective component of neuropathic pain, ie, increased tolerance to pain. we further systematically compared the analgesic effects between breestim and conventional estim to a peripheral nerve, or voluntary breathing only on experimentally induced pain in healthy subjects. we observed that breestim resulted in a general desensitization effect (measured by elevated electrical pain thresholds in both treated and nontreated hands), while there was no such effect, or even a general sensitization effect after estim or voluntary breathing only.1719 the findings support preliminary clinical observations15,16 that breestim - induced analgesia is likely related to modification of the affective component, ie, central effect. this effect has been attributed to internal pain - coping mechanisms activated by estim during the specific window of voluntary breathing.16 mechanisms of neuropathic pain in sci are not well understood. these components are processed in parallel and were thought to be inseparable.20,21 for example, when superficial tactile stimulation is applied to the hand area where acupuncture points are located, activation is seen only in somatosensory cortices. however, when the acupuncture points are stimulated, and pain is experienced by the subjects, activation of additional cortical areas, such as anterior cingulate cortex and insula, is observed.22 it is thus theoretically possible that the sensory and affective components of neuropathic pain could be modulated separately for therapeutic purposes. for example, breestim is shown to relieve pain by selectively affecting the affective component of neuropathic pain.15,16 the primary aim of this study was to examine the effectiveness of breestim for neuropathic pain management in people with sci. given the analgesic effect of breestim was observed in healthy subjects,1719 it was hypothesized that breestim is able to produce greater analgesic effects than estim in sci subjects. this information (degree and duration of analgesic effect) is important for future dosing studies. thirteen sci subjects (6 males and 7 females and history of sci : 58.2 months, from 7 to 150 months, impairments ranging from c4 ais b to l1 ais b) participated in this study. inclusion criteria were as follows : 1) neuropathic pain after sci ; 2) chronic pain lasting > 3 months ; 3) between 18 and 75 years of age ; and 4) stable oral pain medication regimen for at least 2 weeks. patients were explicitly instructed to continue their pain medications regardless of experimental outcome, including the parameters for their intrathecal drug delivery pumps and spinal cord stimulation during the course of the study. exclusion criteria were as follows : 1) currently adjusting pain medications for neuropathic pain ; 2) pain which was not neuropathic or not related to sci, eg, pain from carpal tunnel syndrome ; 3) presence of a pacemaker ; 4) inability to follow commands or to give consent ; 5) presence of asthma or other pulmonary disease ; and 6) medical instability. the pain syndromes were classified according to the international spinal cord injury pain classification.2325 to ensure that the pain was neuropathic in origin, only subjects with a leeds assessment of neuropathic symptoms and signs (lanss) score of 12 were enrolled.2628 the average lanss score was 16.7, ranging from 12 to 23 (table 1). this study was approved by the committee for the protection of human subjects at the university of texas health science center at houston and tirr memorial hermann hospital. there were two experiments. in experiment (exp) 1, a total of 120 electrical stimuli during either breestim (breestim120) or estim (estim120) were delivered to all subjects in a randomized order with at least 3 days apart. in exp 2, a subset of 7 subjects received an additional, higher dose of breestim (a total of 240 stimuli, breestim240) on a different day to explore the dose response effect of breestim. note that both breestim and estim only performed once in each experiment. in exp 1, subjects were seated comfortably with two hands rested on the experimental table. a pair of trimmed surface electrodes ~22 cm was placed over the medial aspect of the distal forearm along the path of the median nerve from forearm to hand on the dominant hand side. two electrodes were separated by ~10 mm. during breestim120, subjects wore a leak - proof face mask that was connected to a pneumotach system (hans rodulph, inc., shawnee, ks, usa) to record the airflow rate. a single - pulse (0.1 ms square wave) estim was delivered to the median nerve transcutaneously when the airflow rate reached the preset threshold level at 40% of its peak value. this usually occurred when subjects took a fast, strong, and deep inhalation. during estim120 estim pulses were randomly delivered every 1.52 seconds. in both interventions, the same amount of estim (120 stimuli) was delivered, and the intensity of estim was controlled by subjects themselves. during both interventions, subjects were encouraged to gradually increase the intensity of estim to a painful, yet tolerable level. subjects were explicitly instructed that aversiveness of estim was important for both breestim and estim interventions. the intensity of estim was recorded at the 1st, 20th, 40th, 60th, 80th, 100th, and 120th stimuli. in exp 2, a convenient subset of patient subjects (4 females and 3 males) was recruited to receive an additional session of breestim at a higher dose 240 stimuli (breestim240). breestim240 and breestim120 were given on 2 different days with 7 days between (breestim120 vs breestim240) in a randomized order. the same procedure was used for breestim 240 as in exp 1. prior to each intervention in both experiments, subjects had a familiarization session to ensure that they understood the procedure and requirements. during each treatment session, breaks were encouraged and allowed upon request to ensure sufficient rest. all subjects tolerated treatment sessions well. these settings and protocols were similar to our recent series of breestim experiments.1719 technical details of breestim are available online in a methodology video article : http://www.jove.com/video/50077/.15 the primary outcome measure was vas score. vas has been extensively used and validated.29 specifically, vas (010 point scale) score is recommended as an outcome measure for pain intensity after sci.27 vas score was assessed before and immediately after the intervention. postintervention vas score was also assessed through follow - up phone calls every 4 hours except nighttime (10 pm7 am). it is important to emphasize that subjects were explicitly instructed to maintain the same pain medication regimen even if they felt that treatment had helped in pain reduction. the analgesic effect after breestim or estim was based on subjective reports of the vas score. the degree of analgesic effect, ie, reduction in vas score or the difference between pre- and postintervention vas scores was then calculated. it is known that sci patients usually have different levels of pain at different locations of the body.30 to compare the overall analgesic effect between breestim and estim, a global pain intensity was estimated from vas scores averaged across different locations in this study.31 two - way repeated measures anovas with factors of intervention (pre vs post) and stim (breestim120 vs estim120, or breestim120 vs breestim240) were used to compare the analgesic effects using vas scores. two - way repeated measures anovas were also used to compare the intensity of electrical current between two interventions (stim) at different trials (7 levels, at 1st, 20th, 40th, 60th, 80th, 100th, and 120th). paired t - tests thirteen sci subjects (6 males and 7 females and history of sci : 58.2 months, from 7 to 150 months, impairments ranging from c4 ais b to l1 ais b) participated in this study. inclusion criteria were as follows : 1) neuropathic pain after sci ; 2) chronic pain lasting > 3 months ; 3) between 18 and 75 years of age ; and 4) stable oral pain medication regimen for at least 2 weeks. patients were explicitly instructed to continue their pain medications regardless of experimental outcome, including the parameters for their intrathecal drug delivery pumps and spinal cord stimulation during the course of the study. exclusion criteria were as follows : 1) currently adjusting pain medications for neuropathic pain ; 2) pain which was not neuropathic or not related to sci, eg, pain from carpal tunnel syndrome ; 3) presence of a pacemaker ; 4) inability to follow commands or to give consent ; 5) presence of asthma or other pulmonary disease ; and 6) medical instability. the pain syndromes were classified according to the international spinal cord injury pain classification.2325 to ensure that the pain was neuropathic in origin, only subjects with a leeds assessment of neuropathic symptoms and signs (lanss) score of 12 were enrolled.2628 the average lanss score was 16.7, ranging from 12 to 23 (table 1). this study was approved by the committee for the protection of human subjects at the university of texas health science center at houston and tirr memorial hermann hospital. there were two experiments. in experiment (exp) 1, a total of 120 electrical stimuli during either breestim (breestim120) or estim (estim120) were delivered to all subjects in a randomized order with at least 3 days apart. in exp 2, a subset of 7 subjects received an additional, higher dose of breestim (a total of 240 stimuli, breestim240) on a different day to explore the dose response effect of breestim. note that both breestim and estim only performed once in each experiment. in exp 1, subjects were seated comfortably with two hands rested on the experimental table. a pair of trimmed surface electrodes ~22 cm was placed over the medial aspect of the distal forearm along the path of the median nerve from forearm to hand on the dominant hand side. two electrodes were separated by ~10 mm. during breestim120, subjects wore a leak - proof face mask that was connected to a pneumotach system (hans rodulph, inc., shawnee, ks, usa) to record the airflow rate. a single - pulse (0.1 ms square wave) estim was delivered to the median nerve transcutaneously when the airflow rate reached the preset threshold level at 40% of its peak value. this usually occurred when subjects took a fast, strong, and deep inhalation. during estim120 estim pulses were randomly delivered every 1.52 seconds. in both interventions, the same amount of estim (120 stimuli) was delivered, and the intensity of estim was controlled by subjects themselves. during both interventions, subjects were encouraged to gradually increase the intensity of estim to a painful, yet tolerable level. subjects were explicitly instructed that aversiveness of estim was important for both breestim and estim interventions. the intensity of estim was recorded at the 1st, 20th, 40th, 60th, 80th, 100th, and 120th stimuli. in exp 2, a convenient subset of patient subjects (4 females and 3 males) was recruited to receive an additional session of breestim at a higher dose 240 stimuli (breestim240). breestim240 and breestim120 were given on 2 different days with 7 days between (breestim120 vs breestim240) in a randomized order. the same procedure was used for breestim 240 as in exp 1. prior to each intervention in both experiments, subjects had a familiarization session to ensure that they understood the procedure and requirements. during each treatment session, breaks were encouraged and allowed upon request to ensure sufficient rest. all subjects tolerated treatment sessions well. these settings and protocols were similar to our recent series of breestim experiments.1719 technical details of breestim are available online in a methodology video article : http://www.jove.com/video/50077/.15 vas has been extensively used and validated.29 specifically, vas (010 point scale) score is recommended as an outcome measure for pain intensity after sci.27 vas score was assessed before and immediately after the intervention. postintervention vas score was also assessed through follow - up phone calls every 4 hours except nighttime (10 pm7 am). it is important to emphasize that subjects were explicitly instructed to maintain the same pain medication regimen even if they felt that treatment had helped in pain reduction. the analgesic effect after breestim or estim was based on subjective reports of the vas score. the degree of analgesic effect, ie, reduction in vas score or the difference between pre- and postintervention vas scores was then calculated. it is known that sci patients usually have different levels of pain at different locations of the body.30 to compare the overall analgesic effect between breestim and estim, a global pain intensity was estimated from vas scores averaged across different locations in this study.31 two - way repeated measures anovas with factors of intervention (pre vs post) and stim (breestim120 vs estim120, or breestim120 vs breestim240) were used to compare the analgesic effects using vas scores. two - way repeated measures anovas were also used to compare the intensity of electrical current between two interventions (stim) at different trials (7 levels, at 1st, 20th, 40th, 60th, 80th, 100th, and 120th). paired t - tests were used to compare the duration and degree of analgesic effects between two interventions. in exp 1, both breestim120 and estim120 had significant analgesic effects. averaged across 13 patients, vas scores decreased from 6.3 to 3.7 after breestim120 and from 5.2 to 4.4 after estim120 (figure 1). a repeated measures two - way anova revealed a main effect of intervention (f=31.38, p<0.001). no main effect of stim was found. there was a significant intervention stim interaction (f=27.19, p<0.001). post hoc turkey tests showed that pre - breestim120 vas score (6.30.4) was greater than all other vas scores, post - breestim120 vas score (3.70.5) was smaller than other vas scores, and pre - estim120 vas score (5.20.4) was greater than post - estim120 vas score (4.40.5). reduction in vas score was significantly greater after breestim120 (2.60.3) than after estim120 (0.80.3) (p<0.001). the duration of analgesic effect was significantly longer after breestim120 than after estim120 (p=0.04). on average, the analgesic effect lasted 14.26 hours after breestim120, but only 1.91 hours after estim120. as listed in table 1 all subjects had some effects after breestim120, while 5 out of 13 subjects did not have any response after estim120. the intensity of electrical current was significantly greater during estim120 than during breestim120 (f=7.34, p=0.0189) (figure 2). in exp 2, vas score decreased from 6.80.50 to 3.80.57 after breestim120 and from 5.60.55 to 3.40.41 after breestim240. repeated measures two - way anovas showed a main effect of intervention (f=62.84, p<0.001). reduction of vas score was also similar between breestim120 (2.70.59) and breestim240 (2.90.29). among the tested subjects, there were large variations in the duration of analgesic effects. on average, there was no significant difference in the duration between breestim120 (29.928.6 hours) and breestim240 (6035 hours). in the present study, the main novel finding was that breestim120 produced greater and longer analgesic effects for sci patients suffering from chronic neuropathic pain, as compared to estim120. this is the first study suggesting that breestim - induced analgesia observed in healthy subjects1719 could be translated and applied to sci patients. as mentioned earlier, pharmacological treatment is often inadequate and has considerable side effects. patients with physical disabilities and pain, including sci patients, have reported preference for nonpharmacological alternative treatments.32,33 these results are promising that breestim could serve as an alternative treatment for sci patients for neuropathic pain management. a cochrane review article showed variable results of tens in the management of chronic pain,34 even when different parameters were tried, such as different frequencies of estim in tens.35 similarly, variable responses to estim were also observed in the present study. overall, breestim had greater and longer analgesic effect than estim in sci with chronic neuropathic pain. this observation was consistent with previous findings of better analgesic effects of breestim on experimentally induced pain in healthy subjects.1719 we noticed that there was a significant difference in preintervention baseline pain levels. these results suggest that patients have had fluctuations in their baseline pain levels prior to the interventions, but breestim has had significant analgesic effects. the findings of better analgesic effects after breestim than after estim suggest that additional pain - coping mechanisms are likely integrated during breestim. in exp 1, the subjects received the same amount of electrical stimuli (120 stimuli in total), but the intensity of estim was greater during estim than during breestim. though we can not comment whether these patients would benefit from estim with more stimulation (eg, 1,000 stimuli), estim alone evidently did not play a critical role in analgesic effect during breestim. it has also been shown that estim is reported to be effective in patients with peripheral neuropathic pain36 but less effective in patients with central neuropathic pain.37 furthermore, pain - related brain activity was found to be reduced only during the fast inspiration period, as compared to other patterns of breathing. however, this decreased pain - related activity was dissociated from spinal nociceptive transmission.38 taken together, our results suggest that central mechanisms play an important role in neuropathic pain in sci patients. possible central pain - coping mechanisms during breestim are related to unique features of voluntary breathing, as suggested in previous studies.1719 there are limitations in this study. it is clinically meaningful and has been validated and recommended for sci patients.27,29 however, report of vas score is subjective. it could be potentially biased by subjects, particularly in the follow - up assessment, even though we explicitly instructed subjects not to do so. in this study with a cross - over design, the bias of subjective reports is likely to be minimized by within - subject comparisons. physiological biomarkers, such as heart rate variability (hrv), can be a valuable tool to assess analgesic effects objectively.39,40 hrv can be included as an objective outcome variable in future studies. no significant difference in vas score reduction and duration of analgesic effects was observed between breestim120 and breestim240 in the 7 subjects in exp 2. in this preliminary study, alternatively, no difference in analgesic effects could be due to the fact that analgesic effects have reached the plateau with breestim120, and more stimulation would not make any further difference. we were not able to associate any particular components with a positive response or greater duration of effect, such as age, time since injury, level of injury, completeness or cause of injury, or presence of certain medication class (eg, opiates). it is clinically meaningful and has been validated and recommended for sci patients.27,29 however, report of vas score is subjective. it could be potentially biased by subjects, particularly in the follow - up assessment, even though we explicitly instructed subjects not to do so. in this study with a cross - over design, the bias of subjective reports is likely to be minimized by within - subject comparisons. physiological biomarkers, such as heart rate variability (hrv), can be a valuable tool to assess analgesic effects objectively.39,40 hrv can be included as an objective outcome variable in future studies. no significant difference in vas score reduction and duration of analgesic effects was observed between breestim120 and breestim240 in the 7 subjects in exp 2. in this preliminary study, alternatively, no difference in analgesic effects could be due to the fact that analgesic effects have reached the plateau with breestim120, and more stimulation would not make any further difference. we were not able to associate any particular components with a positive response or greater duration of effect, such as age, time since injury, level of injury, completeness or cause of injury, or presence of certain medication class (eg, opiates). these findings in this preliminary study suggest that breestim is an effective alternative nonpharmacological treatment for chronic neuropathic pain in patients suffering from sci. breestim may be tried as a complement to the pharmacological treatment of neuropathic pain in sci patients.	objectivethe objective of this study was to examine the effectiveness of a novel nonpharmacological intervention breathing - controlled electrical stimulation (breestim) for neuropathic pain management in spinal cord injury (sci) patients.subjects and methodsthere were two experiments : 1) to compare the effectiveness between breestim and conventional electrical stimulation (estim) in experiment (exp) 1 and 2) to examine the dose response effect of breestim in exp 2. in exp 1, 13 sci subjects (6 males and 7 females, history of sci : 58.2 months, from 7 to 150 months, impairments ranging from c4 ais b to l1 ais b) received both breestim and estim in a randomized order with at least 3 days apart. a total of 120 electrical stimuli to the median nerve transcutaneously were triggered by voluntary inhalation during breestim or were randomly delivered during estim. in exp 2, a subset of 7 subjects received breestim120 and 240 stimuli randomly on two different days with 7 days apart (breestim120 vs breestim240). the primary outcome variable was the visual analog scale (vas) score.resultsin exp 1, both breestim and estim showed significant analgesic effects. reduction in vas score was significantly greater after breestim (2.60.3) than after estim (0.80.3) (p<0.001). the duration of analgesic effect was significantly longer after breestim (14.26 hours) than after estim (1.91 hours) (p=0.04). in exp 2, breestim120 and breestim240 had similar degree and duration of analgesic effects.conclusionthe findings from this preliminary study suggest that breestim is an effective alternative nonpharmacological treatment for chronic neuropathic pain in patients suffering from sci.
cilioretinal artery (cra) occlusions are rare in young patients. in these cases, the most commonly associated causes are considered to be the same as those implicated in central retina artery occlusions, such as vasculitic processes, migraine, cardiac disorder, and coagulation abnormality. the aim of this article was to report for the first time the medical records and investigational results of an unusual case of simultaneous occlusion of three cras after scleral buckling surgery under local anesthesia. a complete ophthalmic examination, including color fundus image, fundus fluorescein angiography, optical coherence tomography, visual field, as well as systemic and laboratory assessments, was performed. a case of contemporaneous blockage of three cras after ab externo surgery for retinal detachment in a 29-year - old caucasian woman was reported. the interdisciplinary approach and the imaging results have allowed us the clinical definition of such a very rare case. here, we reported that optical coherence tomography is an indispensable tool to better delineate the pathological process and follow atrophic changes in the macula, especially in cases in which fundus fluorescein angiography and systemic tests may be poorly informative. the causes of cilioretinal artery (cra) occlusion are considered to be the same as those implicated in central retina artery occlusion, such as coagulation abnormality, vasculitic processes, migraine, and cardiac disorder. clinically, the occlusion of the cra appears as an area of retinal whitening, representing ischemic cloudy swelling of the inner layers of retina. in general, fundus fluorescein angiography (ffa) shows a delayed fluorescein filling and emptying of the irrigation area of these arteries. although recovery of retinal circulation is possible, the inner retinal layer usually shows atrophic changes in the late stage of evolution. in these cases relatively recently, optical coherence tomography (oct) has improved the diagnostic ability to delineate different eye diseases in both the anterior and posterior segments, including acute and longstanding retinal artery occlusions.13 here we report an unusual case of occlusion of three cras after scleral buckling surgery under local anesthesia. a 29-year - old caucasian woman presented at our eye clinic after a left eye accidental contusion. the best - corrected visual acuity (bcva) was 20/20 in the right eye and light perception in the contralateral eye. slit lamp examination was unremarkable in the right eye, while external ocular findings and anterior segment abnormalities in the left eye included eyelid ecchymosis, a subconjunctival hemorrhage, a corneal abrasion, and a total hyphema. intraocular pressure (iop), as assessed by means of goldmann applanation tonometry, was 14 mmhg in the right eye and 15 mmhg in the left eye. two days after, therapy with tranexamic acid (500 mg twice per day) was started. a retinal examination revealed a retinal dialysis and a retinal detachment in the inferotemporal sector, with macula on. retinal cryotherapy, scleral buckling (14 mm behind the limbus), and evacuative puncture were performed in local anesthesia. at the end of the surgery, the retina had flattened, and the central retinal artery was normally perfused. in the first postoperative day, the left eye visual acuity decreased again to counting finger. moreover, the assessment of the direct photomotor reflex revealed a poor response to light of the left pupil. the iop was 14 mmhg. at the dilated fundus examination, a flattened retina with subtle pale elevation within the macular region extending from the temporal edge of the optic disk to the fovea all these clinical and fundus features were compatible with the simultaneous occlusion of three cras emerging from the temporal edge of the optic disk (figure 1). at the following day, ffa evidenced only a vertical choroidal watershed centered on the optic disk, a faint screen effect in the perifoveal area due to intraretinal edema, and a normal cra perfusion (figure 2). an oct (zeiss cirrus hd - oct ; carl zeiss meditec ag, jena, germany) examination revealed an increased reflectivity and a diffuse thickening of the inner retinal layers (figure 3). at visual field examination (sita - fast central 30 - 2 strategy ; carl zeiss meditec ag), central scotoma and generalized retinal sensitivity reduction, with a foveal threshold of 5 db, mean defect of 13.68 db, and pattern standard deviation of 10.35 db, were diagnosed (figure 4a). laboratory tests showed a white cell count of 13.310/l (normal range : 4.0010.0010/l), a hematocrit of 35.7% (normal range : 38.0%48.0%), and a total serum protein of 5.6 g / dl (normal range : 6.68.3 mg / dl). all other clinical examinations, including cardiology workup, carotid ultrasound, and immunological and coagulation tests, were negative. no atrophy or focal pallor of the optic disk was observed, and retinal whitening was completely resolved. oct imaging evidenced macular atrophy subsequent to the acute edematous change in the eye and diffuse macular thinning (figure 5).4 the visual field showed the reduction of central scotoma with a foveal threshold of 29 db, mean defect of 10.36 db, and pattern standard deviation of 11.88 db (figure 4b). written informed consent to publish this case report and the accompanying images was obtained from the patient. although this simple method of repair is associated with a high anatomic success rate, well - known postoperative complications, some of which are rare, may occur. however, no case of simultaneous occlusion of cras after scleral buckling under local anesthesia has been, to our knowledge, reported before in the literature. as previously reported, intraoperative manipulations of the globes may cause iops greater than the central retinal artery perfusion pressures. since cilioretinal arteries arise from the posterior ciliary arteries, a significant peak in iop might result in their occlusion. however, in our case, there is no direct or indirect sign of compression of the central retinal vein due to scleral buckling or encirclement.5,6 here, scleral buckling was performed after local anesthesia ; therefore, localized vasoconstriction from the anesthetic or mechanical pressure from the bolus of the anesthetic solution may have occurred during the surgery, thus determining an obstruction of the cra and an ischemic injury in the cilioretinal field.7,8 in fact, although the exact pathomechanism of this complication is unknown, retrobulbar injections have been associated with postoperative visual loss because of ischemia to the visual pathways.9 also, retinal vascular occlusion after orbital locoregional anesthesia has been described after phacoemulsification, but is rare.10 generally, in patients younger than 40 years, different clinical examinations are suggested to evaluate whether a hypercoagulable state or a vasculitic process is implied in the occlusive event. furthermore, retinal neurons may be damaged after reperfusion by means of mechanisms of chemical oxidation, which may explain the late atrophy of the inner retinal layers. with regard to the final visual restoration, in our case report, it may be explained by foveal perfusion from the temporal side by branches of superior branch retinal artery. in fact, as evidenced by the visual field at the third month, the patient revealed a normal foveal sensitivity despite a central scotoma in the temporal half (with sita - fast central 30 - 2 strategy) and a restored inferotemporal central area corresponding to the aforementioned superior temporal artery. moreover, quantitative oct at the third month detected thinning along the distribution of three cilioretinal arteries and an intact central subfield. ophthalmologists who examine patients with areas of retinal whitening, especially with the support of oct, should consider paracentral acute middle maculopathy in the differential diagnosis, which is an isolated or plurifocal (as in case of central retinal vein occlusion) ischemic event of the deep capillary plexus of the retina.11 clearly, in our case report, the area of retinal whitening is noticeably distinct, appearing more opaque and white in color (neither gray - white nor wedge - shaped), more superficial in the retina following the distribution of the nerve fiber layer (as assessed by oct) and the cras, and more extensive than that potentially related to the focal areas of retinal capillary ischemia. moreover, the dark aspect of retinal layers underneath hyperreflectivity due to the shadowing of the upper layers is absent in our case. therefore, the aforementioned ocular findings are more compatible with occlusion of cras rather than paracentral acute middle maculopathy. the interdisciplinary approach and the imaging results have allowed us the clinical definition of such an unusual case. clearly, when this complication occurs, carotid disease, and local or systemic factors, should be evaluated. however, although cardiovascular investigations and laboratory workup are useful to assess potential risk factors, oct is an indispensable tool to better delineate the pathological process and follow atrophic changes in the macula (in our report, oct imaging after 3 months shows thinning of the inner retina in the previously thickened nasal retina, which indicates permanent damage of this area), especially in cases in which ffa and systemic tests are poorly informative.	backgroundcilioretinal artery (cra) occlusions are rare in young patients. in these cases, the most commonly associated causes are considered to be the same as those implicated in central retina artery occlusions, such as vasculitic processes, migraine, cardiac disorder, and coagulation abnormality. the aim of this article was to report for the first time the medical records and investigational results of an unusual case of simultaneous occlusion of three cras after scleral buckling surgery under local anesthesia.methodsa complete ophthalmic examination, including color fundus image, fundus fluorescein angiography, optical coherence tomography, visual field, as well as systemic and laboratory assessments, was performed.resultsa case of contemporaneous blockage of three cras after ab externo surgery for retinal detachment in a 29-year - old caucasian woman was reported. the interdisciplinary approach and the imaging results have allowed us the clinical definition of such a very rare case.conclusionhere, we reported that optical coherence tomography is an indispensable tool to better delineate the pathological process and follow atrophic changes in the macula, especially in cases in which fundus fluorescein angiography and systemic tests may be poorly informative.

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